Journal Book Terpene

Phytochemistry Vol. 67, No. 5, 2006

Reports on Structure Elucidation

Contents

TERPENOIDS

Salvidorol, a nor-abietane diterpene with a rare carbon skeleton andtwo abietane diterpene derivatives from Salvia dorrii

pp 424–428

Ahmed A. Ahmed *, Abou El-Hamd H. Mohamed, Joe Karchesy,Yoshinori Asakawa

Salvidorol (1), a irregular abietane-type diterpene and two epimeric diterpenes were isolated

from the aerial parts of Salvia dorrii. The structures were established by high-field NMR

techniques (1H–1H COSY, DEPT, HMQC, HMBC, NOESY, HRMS) and X-ray analysis.

O

H

H

OH

OHC

OH

H

HH

CO

O

R

HO

OH

1

2

34

5

6

7

8

910

15

16

1714

13

12

11

1819

1

2

3 45

6

7

89

10

1517

14

13

1211

1819

16

20

2 R =α OMe

3 R =β OMe

1

Iridoid glucosides from Kickxia abhaica D.A. Sutton from Scrophulariaceae pp 429–432

Adnan J. Al-Rehaily *, Maged S. Abdel-Kader, Mohammad S. Ahmad,Jaber S. Mossa

From Kickxia abhaica two iridoid glucosides (1–2), were isolated. Their structures were

established by spectral analysis, including 2D NMR data.OO

R1O OH

HOR2

1- R1 = OCOCH3 ; R2 = Glc

2- R1 = H ; R2 = Glc-6-OHbenzoyl'

Five labdane diterpenoids from the seeds of Aframomum zambesiacum pp 433–438

Marguerite Kenmogne, Elise Prost, Dominique Harakat,Marie-Jose Jacquier, Michel Frederich, Lucas B. Sondengam,Monique Zeches, Pierre Waffo-Teguo *

Five labdane diterpenoids were isolated from the seeds of Aframomum zambesiacum along

with the known labdanes, aframodial, aulacocarpin A and B, galanal A, and galanolactone

and a linear sesquiterpene, nerolidol. Their structures were elucidated by spectroscopic

analysis. Antiplasmodial activity against Plasmodium falciparum for some of the isolated

compounds was evaluated.

O

O

O

7 : R = H, 8 : R = OHR

HO

OH

PHYTOCHEMISTRY

www.elsevier.com/locate/phytochem

Hydroxylation of the sesterterpene leucosceptrine by the fungus Rhizopus stolonifer pp 439–443

Muhammad Iqbal Choudhary *, Rosa Ranjit, Atta-ur-Rahman,Krishna Prasad Devkota, Syed Ghulam Musharraf, Tirtha Maiya Shrestha

The microbial transformation of leucosceptrine (1) by Rhizopus stolonifer, afforded two

leucosesterpenes, 1a-hydroxyleucosceptrine (2), and 8a-hydroxyleucosceptrine (3).

O

O

H3C

CH3

CH3O

H3C

O

OH

HO

HO

H

H

H3C

H

OH

O

O

H3C

CH3

CH3O

H3C

O

OH

OH

HO

H

H

H3C

HO

H

2 3

Clerodane and labdane diterpenoids from Nuxia sphaerocephala pp 444–451

Lengo Mambu *, Philippe Grellier, Loic Florent, Roger Joyeau,David Ramanitrahasimbola, Philippe Rasoanaivo,Francois Frappier

Four clerodane and three labdane diterpenoids (1–7) were isolated from the leaves of Nuxia

sphaerocephala. Their structures have been elucidated on the basis of NMR and MS data.

The antiplasmodial activity of the compounds has been evaluated.R1

COOHH

R

R2R R1 R2 R3

1. H,H H H2 OH3 O H4. H,H E-caffeoyloxy

R3

HH

H

OH

OH

Rings B,D-seco limonoids from the leaves of Swietenia mahogani pp 452–458

Samir A.M. Abdelgaleil, Matsumi Doe, Yoshiki Morimoto,Munehiro Nakatani *

Three types of rings B,D-seco limonoids were isolated and structures of nine compounds

were elucidated by spectroscopic methods.

O

OOH

MeO2C

OR

OR

OTig

O

O

O

RH

1

2

3

PHENOLICS

Flavones and isoflavones from the west African Fabaceae Erythrina vogelii pp 459–463

Alain F. Kamdem Waffo, Philip H. Coombes, Dulcie A. Mulholland *,Augustin E. Nkengfack, Zacharias T. Fomum

The stem bark of Erythrina vogelii collected in Nigeria has yielded two isoflavones vogelins

H (1) and I (2), a flavone, vogelin J (3), and eight known flavonoids.

vogelin H (1)

O

O

HO

OOH

HO

420 Contents / Phytochemistry 67 (2006) 419–423

Phenolic compounds from the flowers of Garcinia dulcis pp 464–469

S. Deachathai, W. Mahabusarakam *, S. Phongpaichit, W.C. Taylor, Y.-J. Zhang,C.-R. Yang

Dulcisxanthones C–F and dulcinone together with 22 known compounds were isolated from

the flowers of Garcinia dulcis. The radical scavenging and antibacterial activities were

investigated.O

O OHOMe

OMeOMe

MeO

Xanthone derivatives from Cratoxylum cochinchinense roots pp 470–474

W. Mahabusarakam *, W. Nuangnaowarat, W.C. Taylor

Xanthones and caged-prenylated xanthones, named cochinchinones A–D, a synthetic

known caged-prenylated xathone and seven known xanthones were isolated from the roots

of Cratoxylum cochinchinense. Some of the compounds exhibited effective antioxidative

properties. O

O OH

O

H3CO

O

ALKALOIDS

Alkaloids from Oriciopsis glaberrima Engl. (Rutaceae) pp 475–480

Jean Duplex Wansi *, Jean Wandji, Alain Francois Kamdem Waffo,Happi Emmanuel Ngeufa, Jean Claude Ndom, Serge Fotso,Rajendra Prasad Maskey, Dieudonne Njamen, Tanee Zacharias Fomum,Harmut Laatsch

Alkaloid derivatives, oriciacridone A (1) and B (2), were isolated from the stems bark of

Oriciopsis glaberrima Engl., and their structures determined spectroscopically. The extract

exhibited in vitro significant antimicrobial activity against a range of micro-organisms.

N O

O O

OH

OH

O

OH

O OH

N

H

HH

R

1 R = H

2 R = OH

GENERAL CHEMISTRY

Terpenoids and phenol derivatives from Malva silvestris pp 481–485

Francesca Cutillo, Brigida D�Abrosca, Marina DellaGreca *, Antonio Fiorentino,Armando Zarrelli

A sesquiterpene and a tetrahydroxylated acyclic diterpene were isolated from Malva

silvestris. The structures of the compounds were determined by spectroscopic NMR and MS

analyses. Their effects on germination and growth of Lactuca sativa L. have been studied in

the concentration range 10)4–10)7 M.

OMe

OH

O

Contents / Phytochemistry 67 (2006) 419–423 421

Hydroquinone diglycoside acyl esters from the stems of Glycosmis pentaphylla pp 486–491

Junsong Wang, Yingtong Di, Xianwen Yang, Shunlin Li, Yuehu Wang,Xiaojiang Hao *

From the stems of Glycosmis pentaphylla, three hydroquinone diglycoside acyl esters and

one known one were isolated.

OH

OMe

OH

O

HOOH

OH

O

O

OH

OH OH

O

O

O

OHOH

O

O

O

OH

OMe

OH OH

O

O

O

HO

MeO

MeO

OMe

OH

O

O

HO

MeO

OMe

OH

OH

O

O

OH

OH OH

O

O

O

OH

Unusual chromenes from Peperomia blanda pp 492–496

Leosvaldo S.M. Velozo, Marcelo J.P. Ferreira, Maria Isabel S. Santos,Davyson L. Moreira, Vicente P. Emerenciano *, Maria Auxiliadora C. Kaplan

Two chromenes were isolated and identified from the methanol extract of the aerial parts of

Peperomia blanda in addition to stigmasterol, sitosterol and campesterol. Their structures

were established as 2S-(4-methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7-dimethyl-2H-

chromene and 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7-dimethyl-2H-chromene

through spectroscopic methods.

O

O

R2

R14

28

9

10

1'

1''

4'

1- R1=H; R2=OH; 2- R1=OH; R2=H;

Cytotoxic and aromatic constituents from Salvia miltiorrhiza pp 497–503

Ming-Jaw Don, Chien-Chang Shen, Wan-Jr Syu, Yi-Huei Ding,Chang-Ming Sun *

Five naturally occurring products along with 13 known constituents were isolated from the

root of Salvia miltiorrhiza. Selected compounds were evaluated for their biological activity.OO

OOH O

O

OH

O

O

HOO

O

OHO

O

CH3(CH2)14

O

O

O

O

Oligomeric secoiridoid glucosides from Jasminum abyssinicum pp 504–510

Francesca Romana Gallo *, Giovanna Palazzino, Elena Federici, Raffaella Iurilli,Franco Delle Monache, Kusamba Chifundera, Corrado Galeffi

From the root bark of Jasminum abyssinicum, three oligomeric secoiridoid glucosides,

craigosides A–C, were isolated and their structures established.

CH2H2C

H2C

CH3

OH

O

R2O

OR1 R3

1" 5"

4"3"

2"

8"

9" 10"

7"

6"

O10

COOMe

O

HOOC

OH

OHO

OH

OH

1'

2'

3'4'

5' 6'

3

1

4

56

7

8

9

1

422 Contents / Phytochemistry 67 (2006) 419–423

Lignan, phenolic and iridoid glycosides from Stereospermum cylindricum pp 516–520

Tripetch Kanchanapoom *, Pawadee Noiarsa, Hideaki Otsuka,Somsak Ruchirawat

Lignan, phenolic and iridoid glycosides were isolated from the leaves and branches of

Stereospermum cylindricum

MeO

HO

O-Glc

OMe

OH

OH

OH

(+)-cycloolivil 4'-O-β-D-glucopyranoside

Acetylated flavonol diglucosides from Meconopsis quintuplinervia pp 511–515

Xiao-Ya Shang, Ying-Hong Wang, Chong Li, Cheng-Zhong Zhang,Yong-Chun Yang, Jian-Gong Shi *

Four acetylated flavonal diglucosides 1–4, together with five known flavonol

glycosides, have been isolated from Meconopsis quintuplinervia.

O

OH

OR1

OH

HO

OO

O

HOHO

OHOR3

O

HOOR2

R5

1 R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OH2 R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OAc3 R1 = Me, R2 = Ac, R3 = OH, R4 = H, R5 = CH2OH4 R1 = R3 = H, R2 = Ac, R4 = OH, R5 = H

R4

OTHER CONTENTS

Corrigendum p 521

Announcement p 522

The Phytochemical Society of Europe–Pierre-Fabre 2006 Award for Phytochemistry

Author Index p I

Guide for Authors pp II–III

* Corresponding author

INDEXEDNDEXED/ABSTRACTEDABSTRACTED ININ: Current Awareness in Biological Sciences (CABS), Curr Cont ASCA. Chem. Abstr. BIOSIS Data, PASCAL-CNRS Data, CAB Inter, Cam Sci Abstr, Curr Cont/Agri Bio Env Sci, Curr Cont/Life Sci, Curr Cont Sci Cit Ind, Curr Cont SCISEARCHData, Bio Agri Ind

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ISSN 0031-9422

Contents / Phytochemistry 67 (2006) 419–423 423

Salvidorol, a nor-abietane diterpene with a rare carbon skeletonand two abietane diterpene derivatives from Salvia dorrii

Ahmed A. Ahmed a,*, Abou El-Hamd H. Mohamed b, Joe Karchesy c, Yoshinori Asakawa d

a Department of Chemistry, Faculty of Science, El-Minia University, El-Minia 91516, Egyptb Department of Chemistry, Aswan-Faculty of Science, South Valley University, Aswan, Egypt

c Department of Wood Science and Engineering, Oregon State University, Corvallis, OR 97331, USAd Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan

Received 21 October 2005; received in revised form 4 December 2005Available online 3 February 2006

Abstract

Salvidorol (1), a irregular nor-abietane-type diterpene, was isolated from the aerial parts of Salvia dorrii, in addition to two epimericabietane diterpenes (2 and 3). This is the first report of a nor-diterpene with an irregular skeleton. The structures were established byhigh-field NMR techniques (1H–1H COSY, DEPT, HMQC, HMBC, NOESY and HRMS) and in case of 2 was confirmed by X-rayanalysis.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Salvia dorrii; Lamiaceae; Nor-abietane diterpene; Salvidorol; 7a- and 7b-Methoxyrosmanol

1. Introduction

The genus Salvia, a member of the family Lamiaceae,consists of about 500 species distributed throughout theworld. Some species of this genus have held a place ofimportance from ancient times, due to their medicinalproperties (Penso, 1980). They are rich in flavonoids (Barb-eran, 1986), monoterpenes (Emboden et al., 1967) andditerpenes with abietane and clerodane skeletons (Luis,1991; Rodriguez-Hahn et al., 1992). Many diterpenes werereported from Salvia have shown antioxidant (Nakanati,1994) and antibacterial activities (Sosa et al., 1994).Recently, several nitrogen containing compounds were iso-lated from S. miltiorrhiza and were examined for cytotoxicand antimicrobial properties (Ming-Jaw et al., 2005). Theflavonoid constituent of S. dorrii has been studied before(Wollenweber et al., 1992). In this paper we describe fromS. dorrii (Kellog) Abrams the isolation and structural elu-

cidation of salvidorol (1), a novel carbon skeletal nor-abie-tane diterpene and two diterpenes type abietane.

2. Results and discussion

The methylene chloride extract of the air-dried aerialparts of S. dorrii was chromatographed on silica gel andSephadex LH-20 columns to give a novel nor-diterpene(1), for which the name salvidorol was given, and two epi-meric abietane diterpenes (2 and 3) (new). Compound 1,yellowish oil, ½a�20

D þ 1:53� (c 0.98, CHCl3), its IR spectrumshowed absorption bands at 3409 cm�1 (OH) and1719 cm�1 (C@O). The low resolution EIMS showed amolecular ion peak [M]+ at m/z 318 (100%), followed bya fragment at m/z 300 [M � H2O]+. The high resolutionmass spectrum exhibited a molecular ion peak [M]+ at m/z318.1824 (calcd. 318.1817), in accord with the molecularformula of C19H26O4. The structure of salvidorol (1) wasdetermined from careful investigation of the 1D and 2DNMR measurements. The 1H NMR spectrum revealed

0031-9422/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.phytochem.2005.12.009

* Corresponding author. Tel.: +208 634 5267; fax: +208 634 2601.E-mail address: [email protected] (A.A. Ahmed).


Phytochemistry 67 (2006) 424–428

PHYTOCHEMISTRY

mailto:[email protected]

the presence of two singlet signals at dH 1.12 (3H, H-18)and 1.11 (3H, H-19), an isopropyl group at dH 1.25 (3H,H-16), 1.26 (3H, H-17) and 3.27 (1H, H-15), a broad singletat dH 5.80 (H-6) and a formyl proton at d 10.0 (s, H-7). Themost characteristic and important signal being a one-pro-ton signal at dH 3.45 (1H, ddd, J = 12.0, 12.0, 3.0 Hz),which correlated in 1H–1H COSY with three signals atdH 1.15 (H-1a), 2.30 (H-1b) and 1.68 (H-5a). Therefore,this proton was assigned for H-10 and suggested theabsence of H-20, which supported the presence of anor-diterpene skeleton. The 13C NMR spectrum showed 19carbon signals were classified by DEPT experiments as fol-lows: four methyl carbon signals at dC 22.1 (C-16), 22.2(C-17), 20.8 (C-18) and 30.1 (C-19), three methylene car-bon signals at dC 36.8 (C-1), 22.8 (C-2) and 42.6 (C-3), fourmethine carbon signals at dC 52.6 (C-5), 91.9 (C-6), 28.5 (C-10) and 27.0 (C-15). The formyl carbon signal appeared atd 191.1, while the protonated aromatic carbon signalappeared at dC 125.3 (C-14). The downfield shift of C-6at dC 91.9 in the 13C NMR spectrum suggested the exis-tence of a hemiacetal moiety in the structure. Moreover,all proton and carbon signals were determined by 1H–1HCOSY, HMQC and HMBC (Table 1). The HMBC spec-trum (Fig. 1) was used to place the aldehydic group atC-8 on the basis of the correlation between the aromaticproton at dH 7.37 (H-14) with the aldehydic carbon signalat dC 191.9 (C-7). Other important correlations wereobserved, namely, H-5 with C-10, H-6 with C-10 and C-11, H-10 with C-8 and C-11, H-18 and H-19 with C-3

and C-5 and H-15 with C-12, C-13 and C-14. The couplingconstant between H-5 and H-6 was consistent with the b-configuration of hydroxyl group at C-6 (Gonzalez et al.,1989). Dreiding models demonstrated the angle betweenH-5 and H-6 was about 90, which was in agreement withthe broad singlet observed for H-6. This stereochemistrywas supported by a NOESY spectrum that exhibited effectsbetween H-6 (d 5.80) with H-1a (d 1.15) and H-19 (d 1.11),H-10 (d 3.45) with H-1b (d 2.30), H-2b (d 1.78) and H-18 (d1.12). Also, it showed a clear effect between the formaylproton with H-10 (at d 3.45) and H-1b (at d 2.30).Although, few regular abietane diterpenes lacking the 20-methyl group were reported from the genus Salvia (Leeet al., 1987), this is the first irregular abietane diterpenewhich lacking the 20-methyl group.

O

H

H

OH

OHC

OH

H

HH

CO

O

R

HO

OH

1

2

34

5

6

7

8

910

15

16

1714

13

12

11

1819

1

2

3 45

6

7

89

10

1517

14

13

1211

1819

16

20

2 R =α OMe

3 R =β OMe

1

Compound 2 was isolated as colorless crystal. Its 1HNMR spectrum showed an isopropyl moiety as one-protonseptet at d 3.07 (J = 7 Hz) and two geminal methyls dou-blets at d 1.21 and 1.22 (J = 7 Hz). A singlet signal at d6.79 was assigned to an aromatic proton. Moreover, itrevealed two doublets at d 4.26 and 4.71 (J = 3.0 Hz)assigned for H-7 and H-6, respectively, while H-5 appearedas singlet signal at d 2.24. Also, its 1H NMR spectrumshowed a sharp three-proton singlet at d 3.66 in accordwith 2 being a methoxylated derivative of rosmanol(Ahmed et al., 1995). The 13C NMR and the multiplicities

Table 1NMR data of 1 (600 MHz, CDCl3, d-values)

Position dC dH HBMC (H–C)

1a 36.8 1.15 m

1b 2.30 dd (12.0, 3.0)

2a 22.8 1.65 m

2b 1.78 dt (13.8, 4.0)

3a 42.6 1.46 m

3b 1.30 dd (13.8, 4.0)

4 32.8 s

5a 52.6 1.68 dd (12.0, 1.2) C-4, C-106 91.9 5.80 br s C-10, C-117 191.1 10.0 s

8 127.09 126.8

10 28.5 3.45 ddd (12.0, 12.0, 3) C-8, C-1111 137.812 148.0

13 132.014 125.3 7.37 s C-7, C-8, C-12

15 27.0 3.27 septet (7.2) C-12, C-13, C-1416 22.1 1.25 d (7.2) C-1317 22.2 1.26 d (7.2) C-1318 20.8 1.12 s C-3, C-519 30.1 1.11 s C-3, C-5

Fig. 1. Selective HMBC correlations of compound 1.

A.A. Ahmed et al. / Phytochemistry 67 (2006) 424–428 425

of the individual signals were determined using DEPT asfollows: five methyls (one oxygenated, d 58.14), three meth-ylenes, five methines (one aromatic, d 120.81 and two oxy-genated, d 74.99 and 77.61) and eight quaternary carbons(one carbonyl and five aromatics). The relative stereochem-istry of 2 could be deduced from NOESY experiment,where H-5, H-6, OMe and H-19 correlated with each other,indicating the a-orientation of these protons. Also, itshowed correlations between the aromatic proton with H-7 and H-15. Additionally, the stereochemistry of 2 was con-firmed by X-ray analysis (Fig. 2). Therefore, compound 2

was established to be 7a-methoxyrosmanol. Although,the NMR spectral data of 2 were identical with the previ-ously reported data for 7a-methoxyrosmanol (Takenakaet al., 1997), compound 2 showed opposite optical rotationsign ½a�22

D þ 6� (c = 0.35, CHCl3), while the previouslyreported optical rotation was ½a�22

D � 24:5� (c = 0.42,CHCl3), (Takenaka et al., 1997). Therefore, compound 2

could be enantiomer of the previously reported compound.Compound 3 was isolated as yellow oil, its CIMS exhib-

ited a molecular ion peak [M + H]+ at m/z 361 and exactmass at m/z 361.20119 (calcd. 361.20150), established theelemental composition as C21H30O5. Its IR spectrumshowed absorption bands indicative of a c-lactone group(1754 cm�1) and aromatic hydroxyl groups (3360 cm�1).The 1H NMR and 13C NMR spectral data of 3 were verysimilar to those of 2, except the optical rotation sign whichwas opposite, ½a�22

D � 52 (c = 1.2, CHCl3), suggesting that 3

was an epimer of 2. Comparison of the 1H and 13C NMRspectra of 3 with those of 2 showed some differences. Thesignals of H-6 (d 4.92) and H-7 (d 4.40) of 3 were detectedat downfield shift (Dd + 0.21 and Dd + 0.14, respectively)in comparison with those of 2. Moreover, the carbon signalat position 5 (dC 55.39) was shifted downfield. The posi-tions of the methoxyl group, isopropyl group and lactone

moiety were determined by HMBC spectrum. In this spec-trum, H-C connectivity between the aromatic proton andC-7 (dC 78.2), C-9 (dC 123.5), C-11 (dC 142.5) and C-15(dC 27.2); between the methoxyl and C-7 (dC 78.2), sup-ported the location of the methoxyl group at C-7 and theisopropyl at C-12. Moreover, it displayed correlationsbetween H-5 and C-7 (dC 78.2), C-9 (dC 123.5), C-10 (dC

47.9), C-18 (dC 21.9), C-19 (dC 31.8) and C-20 (dC 178.9);between H-6 and C-8 (dC 126.5) and C-20 (dC 178.9);between H-16, H-17 and C-15 (dC 27.2); between H-18,H-19 and C-3 (dC 37.9) and C-4 (dC 31.6). A NOESYexperiment of 3 showed a cross-peak between H-5 andH-6 with H-7, indicated the a-orientation of H-7. There-fore, compound 3 was assigned to be 7b-methoxyrosmanol,a new epimer of 2.

3. Experimental

3.1. General

NMR spectra were measured with a Bruker AMX-400spectrometer and Varian Unity 600 MHz NMR spectrom-etry, with TMS as an internal standard. The IR spectra[oily film, CHCl3] were taken on Perkin–Elmer FT-IR spec-trometer. Optical rotations were measured with a Perkin–Elmer 241 Polarimeter operating at sodium D line. MSwere recorded on a JEOL SX102A mass spectrometer(70 eV).

3.2. Plant material

Salvia dorrii (Kellog) Abrams (Lamiaceae) was collectedin the flowering stage near Mitchell, Oregon (voucher #195789 Oregon State University Herbarium).

Fig. 2. ORTEP diagram of the crystal structure of 2.

426 A.A. Ahmed et al. / Phytochemistry 67 (2006) 424–428

3.3. Extraction and isolation

The dichloromethane extract (20 mg) of the aerial parts(950 g) of S. dorrii was fractionated by flash column chro-matography (5 · 55 cm) over silica gel (1 kg) eluting with n-hexane with an increasing amount of CH2Cl2. The fraction(100%, n-hexane 1 L) contained hydrocarbons and waxes.The second fraction (n-hexane–CH2Cl2 3:1, 2 L) gave acrude material which was purified by a Sephadex LH-20(3 · 35 cm, n-hexane–CH2Cl2–MeOH 7:4:0.5, 300 mL) togive compounds 2 (14 mg), 3 (3 mg). The third fraction(CH2Cl2, 100%) was further purified by a Sephadex LH-20 (3 · 35 cm, n-hexane–CH2Cl2–MeOH 7:4:1, 500 mL)to afford compound 1 (2.5 mg).

3.3.1. Salvidorol (1)

Yellowish oil; ½a�20D þ 1:53� (c 0.98, CHCl3); IR

(mKBrmax cm�1): 3409, 3019, 2927, 1719, 1680, 1606, 1571,

1436; EIMS [M]+m/z 318 (100), [M � H2O] m/z 300 (30),285 (40), 257 (60), 231 (30), 205 (40), 181(15); HREIMSm/z 318.1824 (calc. for C19H26O4, 318.1817). 1H and 13CNMR (see Table 1).

3.3.2. 7a-Methoxyrosmanol (2)

Colorless crystal; ½a�22D þ 6� (c = 0.35, CHCl3); IR

(mKBrmax cm�1): 3590, 2960, 1730, 1200; EIMS [M]+m/z 360

(30), 316 (10), 285 (215); 1H NMR (400 MHz, CDCl3):d = 3.16 (1H, br. d, J = 14 Hz, H-1b), 2.00 (1H, m, H-1a), 1.55 (1H, m, H-2b), 1.68 (1H, m, H-2a), 1.19 (1H,m, H-3b), 1.46 (1H, br. d, J = 14 Hz, H-3a), 2.24 (1H, s,H-5), 4.71 (1H, d, J = 3.0 Hz, H-6a), 4.26 (1H, d,J = 3.0 Hz, H-7b), 6.79 (1H, s, H-14), 3.07 (1H, septet,J = 7 Hz, H-15), 1.21 (3H, d, J = 7 Hz, H-16), 1.22 (3H,d, J = 7 Hz, H-17), 0.93 (3H, s, H-18), 1.01 (3H, s, H-19),3.66 (3H, s, OMe); 13C NMR (100 MHz, CDCl3):d = 27.0 (t, C-1), 19.0 (t, C-2), 38.0 (t, C-3), 31.3 (s, C-4),51.1 (d, C-5), 75.0 (d, C-6), 77.6 (d, C-7), 125.9 (s, C-8),124.8 (s, C-9), 47.2 (s, C-10), 143.5 (s, C-11), 142.0 (s, C-12), 135.6 (s, C-13), 120.8 (d, C-14), 27.1 (d, C-15), 22.2(q, C-16), 22.5 (q, C-17), 22.0 (q, C-18), 31.5 (q, C-19),179.9 (s, C-20), 58.2 (q, OMe).

3.3.3. 7b-Methoxyrosmanol (3)

Yellow material; ½a�22D � 52� (c = 1.2, CHCl3); IR

(mKBrmax cm�1): 3360, 2957, 1754, 1682, 1556, 1454; CIMS

[M + H]+m/z 361 (100), 329 (60), 315 (8), 301 (12), 183(8); HRCIMS m/z 361.20119 (calc. for C21H28O5,361.20150). 1H NMR (400 MHz, CDCl3): d = 3.18 (1H,br. d, J = 14 Hz, H-1b), 1.91 (1H, m, H-1a), 1.51 (1H, m,H-2b), 1.61 (1H, m, H-2a), 1.18 (1H, m, H-3b), 1.42 (1H,br d, J = 14 Hz, H-3a), 1.90 (1H, s, H-5a), 4.92 (1H, d,J = 3.0 Hz, H-6a), 4.40 (1H, d, J = 3.0 Hz, H-7a), 6.77(1H, s, H-14), 3.00 (1H, septet, J = 7 Hz, H-15), 1.01(3H, d, J = 7 Hz, H-16), 1.12 (3H, d, J = 7 Hz, H-17),0.92 (3H, s, H-18), 0.97 (3H, s, H-19), 3.57 (3H, s, OMe);13C NMR (100 MHz, CDCl3): d 27.1 (t, C-1), 18.9 (t, C-2), 37.9 (t, C-3), 31.6 (s, C-4), 55.4 (d, C-5), 74.7 (d, C-6),

78.2 (d, C-7), 123.5 (s, C-8), 126.5 (s, C-9), 47.9 (s, C-10),142.5 (s, C-11), 142.1 (s, C-12), 135.5 (s, C-13), 118.9 (d,C-14), 27.2 (d, C-15), 22.1 (q, C-16), 22.7 (q, C-17), 21.9(q, C-18), 31.8 (q, C-19), 178.9 (s, C-20), 56.0 (q, OMe).

3.3.4. X-ray crystallography of compound 2Crystal data: C21H28O5, formula wt. 362.466, ortho-

rhombic, space group P212121, a = 8.7490 (3) A,b = 12.5470 (5) A, c = 17.1930 (9) A, V = 1887.34(14) A3, Z = 4, Dc = 1.276 Mg m�3. All diagrams and cal-culations were performed using maXus (Brucker Nonius,Delft & Mac Science, Japan), using graphite monochro-mated Mo Ka radiation (k = 0.71073 A). The structureswere refined by full-matrix least-squares on F2 using Bruc-ker SHELEXL-97 (Sheldrick, 1997). The final R and Rw were0.0504 and 0.1236, respectively. Crystallographic data forthe structural analysis have been deposited with the Cam-bridge crystallographic data center. These data can beobtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving html (or from the CCDC, 250572 union Road,Cambridge CB2 1EZ, UK; fax: +44 1223 336 033; e-mail:[email protected]).

Acknowledgements

This work was supported and financed by MatsumaeFoundation (Japanese Grant for Mr. Abou El-Hamd).We thank all members of the analytical center of Tuko-shima-Bunri University, Japan, for recording MS andNMR spectra. A.A.A. thanks the Alexander von HumboldtStiftung for financial support for the HPLC instrument.

References

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Gonzalez, A.G., Castro, Z.E.A., Luis, J.G., Ravelo, A.G., 1989. Newsecoditerpenes from Salvia texana. Transformations of 6,7-seco-abietanes in basic medium and their possible formation via oxygensinglet participation. J. Chem. Res. (S), 132–133.

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miltiorrhiza. J. Nat. Prod. 68, 1066–1070.Nakanati, N., 1994. In: Ho, C.T., Osawa, T., Huang, M.T., Rosen, R.T.

(Eds.), Food Phytochemicals for Cancer Prevention II: Teas, Spicesand Herbs, ACS Symposium Series, vol. 547. American ChemicalSociety, Washington, DC, p. 144.

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428 A.A. Ahmed et al. / Phytochemistry 67 (2006) 424–428

Iridoid glucosides from Kickxia abhaica D.A. Suttonfrom Scrophulariaceae

Adnan J. Al-Rehaily *, Maged S. Abdel-Kader, Mohammad S. Ahmad, Jaber S. Mossa

Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia

Received 17 September 2005; accepted 21 September 2005Available online 8 November 2005

Abstract

Two iridoid glucosides namely; 6-acetylantirrinoside (1), 6 0-O-p-hydroxybenzoylantirrinoside (2) were isolated from the aerial parts ofKickxia abhaica. Beside that, three known iridoid glucosides, antirrinoside (3), antirride (4) and mussaenosidic acid (5), one flavone gly-coside (6) and a hexitol, D-mannitol (7) were isolated. The structures of the iridoid glucosides 1–2 were established by 1D and 2D NMRspectral data, including COSY, HMQC and HMBC experiments, as well as HRMS.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Kickxia abhaica; Scrophulariaceae; Iridoid glucosides; 6-acetylantirrinoside; 6 0-O-p-hydroxybenzoylantirrinoside

1. Introduction

The genus Kickxia is comprised of about 47 speciesworldwide (Mabberley, 1997). In Saudi Arabia, the genusis represented by 10 species (Kickxia elatine, Kickxia aegyp-

tiaca, Kickxia acerbiana, Kickxia collenetteana, Kickxia

corallicola, Kickxia pseudoscoparia, Kickxia scalarum,Kickxia petiolata, Kickxia hastate and Kickxia abhaica),which are distributed in different parts of the country(Chaudhary, 2001). Most of these species are distributedin the South and West regions including K. abhaica. Onlyseven Kickxia species world wide were chemically investi-gated and resulted in the isolation of mainly flavonoidsand iridoid glycosides (Khan et al., 2001; Yuldashevet al., 1996; Handjieva et al., 1995; Amer, 1993; Kassem,1992; Khan et al., 1991; Singh and Prakash, 1987; Nicolettiet al., 1987; Toth et al., 1978a,b,c; Pinar, 1973). Up to thepresent time nothing has been reported about the chemistryof K. abhaica. Therefore, the present paper reports on theisolation and characterization of the two new iridoid gluco-sides, 6-acetylantirrinoside (1), 6 0-O-p-hydroxybenzoylan-

tirrinoside (2) from the aerial parts of K. abhaica. Inaddition, the plant also yielded three known iridoid gluco-sides, antirrinoside (3) (Scarpati et al., 1968; Chaudhuriet al., 1980), antirride (4) (Handjieva et al., 1993) and mus-saenosidic acid (5) (Damtoft et al., 1984), one flavone gly-coside, hispidulin 7-neohesperidoside (6) (Lee et al., 1994;Park et al., 1995) and a hexitol, D-mannitol (7) (Khanand Aqil, 1993).


Compound 1 was obtained as a gummy substance andits molecular formula C17H24O11 was determined byHRFABMS. The 17 carbons were resolved in the 13CNMR spectrum (Table 1). When compared to the spectrumof antirrhinoside (3), a very good correspondence could beseen for 15 of the signals, while the remaining two signalscould be assigned to an acetyl moiety. Compound 1 was,therefore, a monoacetate of 3, in agreement with the MSdata. The point of attachment was evident from the 1HNMR spectrum where the H-6 signal was seen at d 4.86,0.9 ppm downfield from that of 3. The position of the ace-tate group at C-6 was further confirm by 2D NMR 1H–13C


doi:10.1016/j.phytochem.2005.09.021

* Corresponding author. Tel.: +966 1 467 7258; fax: +966 1 467 7245.E-mail address: [email protected] (A.J. Al-Rehaily).



PHYTOCHEMISTRY


HMBC experiments. The HMBC spectrum showed 3J cor-relations between d 4.82 (H-4), dC-6 79.4, dC-9 53.3, andbetween d 4.86 (H-6) and dC-100 172.0, confirming theplacement of the acetate group at C-6. These findingsunambiguously established the structure of 1 as 6-acetylantirrhinoside.

Compound 2, analyzed for C22H26O12 by HRFABMS,was isolated as amorphous powder and its UV spectrumexhibited absorption bands at kmax 257 and 320 nm dueto the presence of a conjugated system. The 1H and 13CNMR spectra of 2 (Table 1) were diagnostic for antirrhin-soide esterified with an aromatic acid (Fauvel et al., 1995).In the 1H NMR of 2 the down field shift of H-6 0 protons tod 4.40 and 4.52, ca. 0.8 ppm from the usual positionstrongly support that C-6 0is the site of esterification. Fur-ther confirmation was made by HMBC experiment. Thatshowed 3J correlations between d 7.78(dC300/700132.9), dC-100

167.8 and dC-500 163.7, and between d 4.40, 4.52 (H-6 0a,H-6 0b) and dC-100 167.8, confirming the attachment of aro-matic acid moiety at C-6 0.The NMR spectra of 2 werefound to be similar with those reported (Dawidar et al.,1989) for 6 0-O-cinnamoylantirrhinoside but lacking the sig-nals for the a and b positions of cinnamoyl moiety. Basedon the foregoing data, the structure of 2 was established as6 0-O-p-hydroxybenzoylantirrinoside.

During the course of isolation of the above compounds,K. abhaica yielded three known iridoid glucosides 3–5, oneflavone glycoside (6) and one alditol (7). These compoundswere identified by comparison of their physical and spec-

troscopic data with those reported in the literatures. Thisis the first time that the iridoid glucosides 1–2 appearedin the literature and the first report of 6 from the familyscrophulariaceae. In addition, compounds 4 and 5 arereported for the second time from the genus Kickxia (Han-djieva et al., 1995).

O

R1OHO

HO

O

O

OHHOHO

R1 R2

O

OH

OHO

OGlc

O

OGlc

HO

COOHH

H

O

OH

OOH

H3CO

Rha- Glc- O

OH

CH

CHO H

CHO H

CH OH

CH OH

CH

OH

1: CH3CO H

2: H

3: H H

4

5

6 7

R2O

H

H

3. Experimental

3.1. General

Mp uncorr.; UV spectra were recorded on a Hewlett–Packard HP-845 UV–Vis spectrophotometer; FTIRspectra were obtained on a Nicolet Impact 410 spectropho-tometer; Specific rotation measurements were recorded ona Perkin–Elmer 242 MC polarimeter; NMR spectra wereacquired in CD3OD or DMSO on a Bruker AvanceDRX-500 instrument at 500 (1H) and 125 (13C) MHz usingthe residual solvent signal as internal standard. StandardBruker pulse programs were used for APT, DEPT, 2DNMR COSY, HMQC and HMBC spectra. HRFABMSwere obtained on a Bruker Bioapex-FTMS with electro-spray ionization; EIMS were measured using an E.I. Finn-igan model 4600 quadrupole system or a Shimadzu QP500GC/mass spectrometer; TLC: silica gel 60 F254 (Merck)plates; solvents: different concentration of MeOH–CHCl3and H2O–MeOH; CC: silica gel 60/230–400 mesh (EM

Table 11H and 13C NMR spectral data for compounds 1-2 in CD3OD (d values, J

in parenthesis in Hz)a

Proton 1 2

1H 13C 1H 13C

1 5.44 d (6.0) 94.6 4.99 d (8.0) 95.63 6.32 d (6.5) 143.4 6.24 d (6.0) 142.84 4.82 d (6.5) 107.5 4.77 d (6.0) 107.75 – 74.7 – 74.86 4.86 d (2.0) 79.4 3.69 d (1.0) 78.87 3.39 d (2.0) 64.2 3.15 br.s 66.08 – 64.5 – 63.09 2.36 d (6.0) 53.3 2.22 d (8.0) 53.310 1.39 s 17.4 1.24 s 17.6

10 4.57 d (8.0) 99.8 4.62 d (8.0) 99.920 3.14 m 74.7 3.16 m 74.830 3.30 m 77.7 3.49 m 75.740 3.15 m 71.8 3.32 m 71.850 3.30 m 78.6 3.33 m 77.760a 3.53 dd (11.75, 6.5) 63.0 4.40 dd (12.0, 7.0) 64.160b 3.83 dd (11.75, 2.5) 63.0 4.52 dd (12.0, 2.5) 64.1

100 – 172.0 – 167.8200 2.03 s 20.3 – 122.2300/700 7.78 d (9.0) 132.9400/600 6.73 d (9.0) 116.3500 – 163.7

a Assignments made by combination of COSY, DEPT, HMQC, HMBCdata and comparison with the literature.

430 A.J. Al-Rehaily et al. / Phytochemistry 67 (2006) 429–432

Science); RP C-18 silica gel. Centrifugal preparative TLC(CPTLC; using Chromatotron�, Harrison Research Inc.model 7924): 1–4 mm silica gel P254 disc. The isolated com-pounds were visualized under short- and long-wave UVlight, followed by spraying with p-anisaldehyde reagent.

3.2. Plant material

K. abhaica D.A. Sutton was collected in April, 2003from Baljurashi, Saudi Arabia and identified by Dr. M.Atiqur Rahman, College of Pharmacy, King Saud Univer-sity, Riyadh, Saudi Arabia. A voucher specimen (# 14716)was deposited at the herbarium of the College of Phar-macy, KSU.


The air-dried aerial parts (1.0 kg) of K. abhaica wereexhaustedly extracted with petroleum ether (12 g), followedby EtOH at room temperature to yield after evaporation120 g. The ethanol extract was dissolved in hot methanolto afford white precipitate (7 g) identified as D-mannitol(7). The soluble methanol fraction was concd., diluted withwater and successively extracted with CHCl3 (3 · 300 ml),EtOAc (3 · 200 ml) and butanol (2 · 200 ml). The ethylac-etate (2 g) and butanol (20 g) extracts were combinedtogether and subjected to flash chromatography on silicagel (600 g) using chloroform and then increasing concentra-tions of MeOH (20–50%) in CHCl3 to give 5 fractions; 1(4.7 g), 2 (3.3 g), 3 (2.8 g), 4 (1.1 g), 5 (4.1 g).

Fraction 1 (4.7 g) was rechromatographed on silica gel(60 g) using 10% CHCl3–MeOH to afford sub-fractionsA–E. Sub-fraction A (820 mg) was separated by RP-col-umn (30 g) using 40% H2O–MeOH as a solvent to givetwo fractions a and b. Fraction a (150 mg) was purifiedby CPTLC (1 mm silica gel disc) using 8% MeOH–EtOActo yelid 1 (10 mg). Fraction b (600 mg) was separated byCPTLC (4 mm silica gel disc) using 10% MeOH–EtOActo give three fractions I–III. Fraction I (39 mg) was purifiedby RP-column using 40% H2O–MeOH as a solvent toafford 2 (14 mg). Fraction C (800 mg) was subjected toCPTLC (4 mm silica gel disc) using 20% MeOH–CHCl3to give three sub-fractions i–iii. Sub-fraction ii (400 mg)was purified by CPTLC (2 mm silica gel disc) using 20%MeOH–CHCl3–NH3 to give 4 (15 mg). Portion of fractionD (250 mg) was separated by CPTLC (2 mm silica gel disc)using 20% MeOH–CHCl3–acetic acid, further purificationby LH-20 (40 g) using 30% MeOH–CHCl3 as a solvent fol-lowed by repeated CC over silica gel using CHCl3 as sol-vent to give 3 (28 mg).

Fraction 4 (1.1 g) was subjected to CPTLC (4 mm silicagel disc) using 25% MeOH–CHCl3 to give two fractions Aand B. Fraction B (0.5 g) was separated by CPTLC (2 mmsilica gel disc) using 20% MeOH–CHCl3–NH3 to yieldedtwo sub-fractions a and b. The sub-fraction a (200 mg)was purified by LH-20 (30 g) using 50% MeOH–H2O toafford 6 (80 mg). The sub-fraction b (65 mg) was purified

by RP-column (30 g) using 40% H2O–MeOH as a solventto give 5 (17 mg).

3.4. 6-Acetylantirrinoside (1)

Gum, [a]D �100� (c; 0.04 in MeOH); UV kmax (MeOH)nm (log e): 202 (3.61), 275 (2.24); IR (film) mmax cm�1: 3411,2923, 1734, 1375, 1240, 1101, 1076, 1047, 1016 and 960; 1Hand 13C NMR: see Table 1; EIMS m/z (rel. int. %) 241[M � 163]+ (0.25), 225 (0.58), 207 (1.8), 165 (3.3), 145(3.5), 129 (12.4), 114 (6.5), 97 (19.9), 87 (21.9), 85 (12.1),73 (15.8), 71 (11.7), 69 (10.1), 57 (17.3), 45 (44.7) and 43(100); HRFABMS: 405.1393 ([M + H]+); (calc. for[C17H24O11 + H] 405.1397).

3.5. 6 0-O-p-Hydroxybenzoylantirrinoside (2)

Amorphous powder, mp. 132–134 �C; [a]D �51.3� (c;0.07 in MeOH); UV kmax (MeOH) nm (log e): 202 (4.82),257 (4.58), 320 (3.62); IR (film) mmax cm�1: 3420, 3411,2920, 1701, 1608, 1313, 1279, 1236, 1167, 1101, 1074,1045, 1012, 771 and 617; 1H and 13C NMR: see Table 1;EIMS m/z (rel. int. %) 483 [M + 1]+ (0.31), 198 (1.2), 177(4.9), 173 (8.8), 163 (4.7), 138 (14.8), 121 (26), 93 (9.0), 73(22.3), 69 (16), 60 (20.9), 57 (29.3), 55 (23.9), 45 (43.5), 44(100) and 41 (37.8); HRFABMS: 483.1500 ([M + H]+);(calc. for [C22H26O12 + H] 483.1503).

Acknowledgements

The authors sincerely thank Dr. Amr Mansour, MassSpectroscopy Unit, National Research Center, Cairo,Egypt, for HRFABMS. Also we thank Mr. MohammedMukhair for technical assistances.

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Five labdane diterpenoids from the seeds of Aframomum zambesiacum

Marguerite Kenmogne b, Elise Prost a, Dominique Harakat d, Marie-Jose Jacquier a,Michel Frederich c, Lucas B. Sondengam e, Monique Zeches a, Pierre Waffo-Teguo a,*

a FRE 2715 CNRS, Laboratoire de Pharmacognosie, IFR 53 Biomolecules, Universite de Reims Champagne-Ardenne, Batiment 18,

CPCBAI, Moulin de la Housse, BP 1039, 51687 Reims Cedex 02, Franceb Department of Chemistry, Faculty of Science, University of Dschang, Box 67, Dschang, Cameroon

c University of Liege, Natural and Synthetic Drugs Research Center, Laboratory of Pharmacognosy, Avenue de l�Hopital 1, B36, B-4000 Liege, Belgiumd Laboratoire ‘‘Reactions Selectives et Applications’’, UMR CNRS 6519, Faculte des Sciences, Universite de Reims Champagne-Ardenne, B.P. 1039, 51687

Reims Cedex 2, Francee Department of Organic Chemistry, Faculty of Science, University of Yaounde I, Box 812, Yaounde, Cameroon

Received 6 September 2005; received in revised form 10 October 2005Available online 29 November 2005

Abstract

Five labdane diterpenoids, (3–5), zambesiacolactone A (7) and zambesiacolactone B (8), were isolated from the seeds of Aframomum

zambesiacum (Baker) K. Schum., along with five known labdanes and a linear sesquiterpene, nerolidol. Their structures were elucidatedby spectroscopic analysis. Their antiplasmodial activity was evaluated in vitro against Plasmodium falciparum. Compound 3 was the mostactive with an IC50 value of 4.97 lM.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Aframomum zambesiacum; Zingiberaceae; Labdane diterpenoids; Antiplasmodial activity; Plasmodium falciparum

1. Introduction

The genus Aframomum of the Zingiberaceae familyincludes 40 species and is most common in tropical andsubtropical regions (Thomas et al., 1989). Twenty speciesare found in Cameroon, where they are widely used in tra-ditional medicine, for spiritual purposes and as spices(Thomas et al., 1989). The compounds isolated from plantsof this genus include flavonoids (De Bernardi et al., 1976;Ayafor and Connolly, 1981), diaryl heptanoids (Kamnainget al., 2003), sesquiterpenes (Ayafor and Connolly, 1981)and labdane diterpenoids, specially in Aframomum albovio-

laceum (Abreu and Noronha, 1997), Aframomum aulaco-

carpos (Ayafor et al., 1994a), Aframomum daniellii

(Kimbu et al., 1979, 1987), Aframomum escapum (Ayimeleet al., 2004), and Aframomum sceptrum (Tomla et al.,2002). A great deal of interest has been focused on thelabdanes from Aframomum species, some of which exhibitantifungal, cytotoxic, and other biological activity (Ayaforet al., 1994a,b). In general, many labdanes from terrestrialplants and marine sources show antibacterial, antifungal,anti-inflammatory, antileishmanial, cardiotonic, cytotoxic,enzyme inhibitory (Singh et al., 1999), and trypanocidal(Scio et al., 2003) activities. Several Aframomum species(i.e., Aframomum angustifolium, A. danielli, Aframomumsanguineum, andAframomum sulcatum) were traditionallyused to treat fevers in Africa (Iwu, 1993), and recently,the antiplasmodial activity of some labdanes from A. scep-

trum and Aframomum latifolium has been investigated(Duker-Eshun et al., 2002).

This paper describes the first phytochemical investiga-tion of the seeds of Aframomum zambesiacum (Baker) K.Schum. This species was selected in the framework of a


doi:10.1016/j.phytochem.2005.10.015

* Corresponding author. Tel.: +33 (0) 3 26 91 82 08; fax: +33 (0) 3 26 9135 96.

E-mail addresses: [email protected], [email protected](P. Waffo-Teguo).



PHYTOCHEMISTRY



screening program to discover novel active compoundsfrom Cameroonian medicinal plants. The structural eluci-dation of the isolated compounds was followed by evalua-tion of their in vitro antiplasmodial activity againstPlasmodium falciparum.


The dry, powdered seeds of A. zambeciacum were suc-cessively extracted with petroleum ether, chloroform, andmethanol. Chromatographic purification of the two lesspolar extracts afforded 10 labdanes (1–10), five of whichare new, and nerolidol (11) which was previously isolatedfrom Aframomum pruinosum (Ayafor and Connolly,1981) and A. escapum (Ayimele et al., 2004). The structureswere assigned by analysis of spectroscopic data and bycomparison with literature values. The five known labd-anes were identified as aulacocarpin A (1) and aulacocarpinB (2), previously isolated from Aframomum aulacocarpus

(Ayafor et al., 1994a) and A. escapum (Ayimele et al.,2004), and galanolactone (6), aframodial (9) and galanalA (10), isolated from Alpinia galanga (Morita and Itokawa,1988).

COOCH3

O

HO

4

CH2OH

5

CH2OH

OHO

O

O

O

COOCH3

O

R1

O

R2

1

36

8

17

1214

R1 R2

3

2

OH H

OH OH

H H

6

8

7

H H

OH H

OH OH

R2

R1

R3

R1 R2

H

OH

OH

R3

14

14 15

14

15

8

17

15

16

16

1316

Compound 3 was obtained as a white powder. Its molec-ular formula, C21H32O4, was established by positiveHRESI-MS (m/z [M + Na]+ 371.2196). The strong IRabsorptions at tmax 1724 and 1645 cm�1 suggested the pres-ence of an a,b-unsaturated ester. This is in agreement withthe three 13C NMR resonances for sp2 carbons correspond-ing to an ester carbonyl (d 166.8) and a trisubstituted olefin(d 127.5 (CH) and d 150.7 (C)). Thus, the compound is tet-racyclic. The 1H NMR spectrum revealed three tertiarymethyls (d 0.87, 0.9, 0.93) and a methyl ester (d 3.75). Otherproton signals included a deshielded vinyl proton [d 6.81 (t,J = 6.5 Hz, H-12)], a monosubstituted epoxide [d 3.60

(brm, H-14), 2.81 (dd, J = 5.5, 2.8 Hz, H-15a) and 2.99(dd, J = 5.5, 4.3 Hz, H-15b)], a disubstituted epoxide [d2.30 (d, J = 3.9 Hz, H-17a) and 2.51 (d, J = 3.9 Hz, H-17b)]. These data suggested that compound 3 was a lab-dane diterpenoid related to aulacocarpin A (1) and B (2)(Ayafor et al., 1994a). All 1H and 13C NMR signals for 3were assigned by analysis of COSY, HSQC, and HMBCspectra. The 1H and 13C NMR data (Table 1) of 3 werealmost identical to those of aulacocarpin A (1). In the13C NMR spectrum, the main difference was the replace-ment of the C-3 methine (d 78.8) of 1 by a methylene (d42.1) in 3. This difference was also evident in the 1HNMR spectrum, where H-3 (d 3.24, dd) of 1 was replacedby two methylene protons at d 1.17 and 1.41 (both m).The relative configuration at C-8 was deduced from aNOESY correlation between H-17 and H-9 and by chemi-cal shift comparison with aulacocarpin A (1) and B (2)(Ayafor et al., 1994; Morita and Itokawa, 1988). As inthe case of 1, the E configuration of the 12,13 double bondwas deduced from NMR data (Ayafor et al., 1994a). Thus,compound 3 is 3-deoxyaulacocarpin A, or methyl-8b,17:14n,15-diepoxy-12E-labden-16-oate.

Compound 4, C21H32O4 (m/z [M + Na]+ 371.2211HRESI-MS in positive mode), a colorless oil, had bandsin its IR spectrum at tmax 3417 cm�1, 1714, and1644 cm�1 in agreement with the presence of a hydroxyland an a,b-unsaturated ester. The 1H and 13C NMR spec-tral data of 4 (Table 1) were very similar to those of 1. Theonly significant differences were the absence of the signalsof the H2-17 epoxide protons of 1 and the presence of an8,17-exomethylene group [d 4.86 (d, J = 0.9 Hz) and 4.49(d, J = 0.9 Hz)] in 4. The b-orientation of the hydroxylgroup at C-3 was deduced from the coupling constants ofH-3 [d 3.27 (dd, J = 11.7 and 4.3 Hz)] and from theNOESY spectrum, while the E configuration of the 12,13double bond follows from the deshielded nature of H-12.Therefore, 4 is methyl-14n,15-epoxy-3b-hydroxy-8(17),12E-labdadien-16-oate. The corresponding 3-deoxy-derivative has been reported from the seeds of A. danielli

(Kimbu et al., 1987).Compound 5, C20H34O4 (m/z [M + Na]+ 361.2349

HRESI-MS in positive mode), was obtained as white pow-der, m.p. 154–155 �C, and showed an hydroxyl absorption(tmax 3402 cm�1) in its IR spectrum. The 1H and 13C NMRdata (Table 1) of 5 indicated that it was also a labdane dit-erpenoid. The 1H NMR spectrum showed three methylsinglets at d 0.88, 0.92, and 0.92, the protons of three oxym-ethylene groups [dH 3.50, (dd, J = 11.3, 7.6 Hz, H-15a) and3.60 (dd, J = 11.3, 4.6 Hz, H-15b), dH 4.04 (d, J = 12.9 Hz,H-16a) and 4.15 (d, J = 12.9 Hz, H-16b), and 2.27(d,J = 4.1 Hz, H-17a) and dH 2.69 (d, J = 4.1 Hz, H-17b)]and a proton of an oxymethine (dH 4.6 (dd, J = 7.8,4.3 Hz, H-14)). Comparison of the 13C NMR data of 5

with those of 3 showed that the monosubstituted epoxidehad been replaced by an oxymethylene (dC 66.1, C-15)and an oxymethine (dC 71.9, C-14). Further analyses ofthe NMR spectra led to the assignments of all protons

434 M. Kenmogne et al. / Phytochemistry 67 (2006) 433–438

Table 11H and 13C NMR data of 3, 4, 5, 7 and 8 in CDCl3

3 4 5a 7 8b

dH dc dH dc dH dC dH dC dH dC

1ax 0.96 dd (13.0, 3.4) 39.3 1.27 td (13.1, 3.5) 37.2 1.03 td (12.9, 3.1) 40.3 1.15 td (12.9, 3.9) 37.6 1.12 dd (12.9, 3.7) 39.91eq 1.78 brd (13.0) 39.3 1.79 dd (13.1, 3.5) 37.2 1.83 dd (12.7, 3.6) 1.79 dt (12.9, 3.4) 1.73 dt (13.1, 3.6)2ax 1.43 m 18.7 1.60 qd (13.3, 3.4) 28.0 1.48 dq (13.6, 3.4) 19.7 1.62 qd (13.3, 3.6) 27.3 1.60 m 26.52eq 1.58 m 1.72 dq (13.3, 3.7) 1.65 m 1.69 m 1.65 m

3ax/3 1.17 td (13.6, 4) 42.1 3.27 dd (11.7, 4.3) 78.8 1.24 td (13.5, 3.7) 43.1 3.27 dd (11.5, 4.4) 78.8 3.12 dd (11.2, 4.6) 78.63eq 1.41 m 1.43 dtd (13.1, 3.2, 1.5)4 33.7 – 39.4 – 34.4 – 39.3 – 40.05 1.02 dd (12, 2.8) 55.2 1.12 dd (12.5, 2.6) 54.7 1.10 dd (12.3, 2.3) 56.2 1.02 dd (12.5, 3.5) 54.2 0.94 d (1.9) 55.86ax 1.58 m 20.2 1.41 qd (13.1, 4.2) 23.9 1.61 m 21.2 1.72 m 19.9 4.46 td (3, 1.8) 67.96eq na 1.75 m 1.72 m 1.72 m

7ax 1.36 dq (13.8, 2.5) 36.0 2.00 td (13, 4.9) 37.9 1.31 dq (1.39, 2.6) 37.1 1.40 ddd (13.9, 3.7, 2.6) 35.9 1.15 dd (14.8, 2.5) 43.87eq 1.93 td (13.8, 5.3) 2.41 dq (13, 2.3) 1.97 td (13.7, 5) 1.94 td (13.9, 5.8) 2.18 dd (14.8, 3.6)8 – 57.7 – 147.8 – 59.3 – 58.1 – 57.29 1.57 m 53.0 1.77 m 56.6 1.60 d (10.6) 54.6 1.66 m 52.3 1.66 m 52.510 – 40.0 – 39.5 – 40.9 – 39.6 – 39.811a 2.02 m 21.0 2.50 ddd (16, 11.6, 7.6) 23.8 1.78 brt (8.3) 20.7 2.08 ddd (16.5, 9.1, 7.8) 22.6 2.10 m 22.411b 2.34 dd (18.4, 6.1) 2.61 ddd (16.6, 6.3, 2.8) 1.98 m 2.27 brdd (16.5, 6.8) 2.31 d (6.6)12 6.81 t (6.5) 150.7 6.87 t (7.00) 149.6 5.44 ddd (8.1, 4.7, 1) 133.3 6.85 td (7.1, 1.6) 149.2 6.77 td (6.9, 1.8) 148.713 – 127.4 – 127.5 – 138.8 – 128.4 – 128.814 3.60 brs 48.6 3.65 t (3.4) 49 4.6 dd (7.8, 4.3) 71.9 5.03 t (5.6) 66.1 4.92 d (6.1) 65.415a/15 2.81 dd (5.5, 2.8) 47.9 3.00 dd (5.5, 4.4) 47.8 3.5 dd (11.3, 7.6) 66.1 4.27 dd (10.4, 2) 74.5 4.18 dd (10.2, 4.6) 75.015b 2.99 dd (5.5, 4.3) 2.78 dd (5.5, 2.8) 3.6 dd (11.3, 4.6) 4.47 dd (10.4, 6.1) 4.41 dd (10.2, 6.2)16a/16 – 166.8 – 166.8 4.04 d (12.9) 63.9 – 170.2 – 171.316b 4.15 d (12.9)17a 2.30 d (3.9) 49.2 4.49 d (0.9) 108.2 2.27 d (4.1) 50.3 2.34 d (3.6) 49.4 2.27 d (3.5) 47.417b 2.51 d (3.9) 4.86 d (0.9) 2.69 d (4.1) 2.73 d (3.6) 2.75 d (3.5)18 0.90 s 33.7 0.79 s 28.5 0.92 s 34.0 1.04 s 28.5 1.06 s 28.019 0.87 s 21.9 1.00 s 15.6 0.88 s 22.2 0.85 s 15.7 1.22 s 16.820 0.93 s 14.8 0.74 s 14.6 0.92 s 15.2 0.95 s 14.9 1.17 s 16.7O-CH3 3.75 s 52.1 3.73 s 51.9

na: not assigned.a Measured in CD3OD.b Measured in CDCl3 with some drops of CD3OD.

M.

Ken

mo

gn

eet

al.

/P

hy

toch

emistry

67

(2

00

6)

43

3–

43

8435

and carbons, the E configurations of the double bond, andthe b configuration of the 8,17-epoxide. Thus, structure 5 is8b,17-epoxy-12E-labdene-14n,15,16-triol.

Compound 7, C20H30O5 (m/z [M + Na]+ 373.1974HRESI-MS in positive mode) a white powder, showedIR absorption bands at tmax 3415 and 1743 cm�1. Its 1Hand 13C NMR spectra data were similar to those of 6 (Mor-ita and Itokawa, 1988) except for the absence of two meth-ylene signals (C-3 and C-14) and the appearance of twooxymethine carbon resonances at d 66.1 (C-14) and 78.8(C-3) in the 13C NMR spectrum of 7. H-3 appeared at d3.27 (dd, J = 11.5,4.4 Hz) indicating that the hydroxylattached to C-3 is b. Thus compound 7, zambesiacolactoneA, is 8b,17-epoxy-3b,14n-dihydroxy-12(E)-labden-16,15-olide.

Analysis of the 1H and 13C NMR spectral data (Table 1)of compound 8, C20H30O6 (m/z [M + Na]+ 389.1956HRESI-MS in positive mode; tmax 3417 and 1739 cm�1),indicated that it was closely related to compound 7. Themain difference was the presence of signals for an oxyme-thine [dH 4.46 (td, J = 3.0,1.8 Hz, H-6); dC 67.9 (C-6)] in8 replacing the C-6 resonances in 7. The b axial orientationof the C-6 hydroxyl of 8 was deduced from the small cou-pling constant (J = 1.9 Hz) between H-5 and H-6. Further-more, the upfield resonance of H-5 (d 0.94) is reminiscentof some 6b-hydroxylated Scapania labdane derivatives(Huneck et al., 1986). Thus, compound 8, zambesiacolac-tone B, was assigned the structure 8b,17-epoxy-3b,6b,14n-trihydroxy-12(E)-labden-16,15-olide.

The relative configuration at C-14 remains undeter-mined in all the new compounds, as in aulacocarpin A(1) and aulacocarpin B (2) (Ayafor et al., 1994a).

The in vitro antiplasmodial activity of the labdanes 1–9

was determined against an FCB1 chloroquine-resistantstrain of P. falciparum relative to artemisinine, chloro-quine, and quinine (Table 2). The amount of compound10 was insufficient to allow evaluation of its biologicalactivity. Compounds 1, 3, 7, and 8 showed moderate activ-ity with IC50 values between 4 and 20 lM. Amongst theactive compounds, compound 3, the least polar compound,was the most active with an IC50 of 4.97 lM (1.73 lg/ml).

3. Experimental

3.1. General experimental procedures

Melting points were determined on a Reichert apparatusand are uncorrected. 1H and 13C NMR spectra wererecorded on a Bruker Avance DRX 500 (1H at 500 MHzand 13C at 125 MHz). 2D experiments were performedusing standard Bruker microprograms. ESI-MS andHRESI-MS were obtained on a Micromass Q-TOF microspectrometer. IR spectra were obtained with a NicoletAVATAR 320 FT-IR spectrophotometer. Optical rota-tions were determined in MeOH or CHCl3 with a Per-kin–Elmer 241 polarimeter. Centrifugal TLC was carriedout on a chromatotron, Model 7924T (Harrison Research).The plate was coated with silica gel 60 F254 Merck. TLCwas performed on pre-coated silica gel 60 F254 Merckand detection was achieved by spraying with a vanillin sul-phuric reagent containing (3 g Vanillin, 100 ml EtOH, and3 ml H2SO4). CC was carried out on Kieselgel 60 (63–200mesh) Merck.

3.2. Plant material

Seeds of A. zambesiacum (Baker) K. Schum. were col-lected in Nyassosso, a small locality of the district of Tom-bel in south-west of Cameroon in November 2003. Avoucher specimen (accession number 37737HNY) has beendeposited at the National Herbarium Yaounde Cameroon.The identification was confirmed by Dr. Tchiengu and P.Mezili, botanists of the Cameroon National Herbarium.


Dried and finely powdered seeds (97 g) were extractedsuccessively with petroleum ether (2.5 l), chloroform(2.5 l), and methanol (2.5 l) at room temperature by perco-lation in an open column after a maceration step for 36 h.The extracts were concentrated to yield respective residues3.55 g (petroleum ether I), 6.78 g (chloroform II) and 3.54 g(methanol III).

The petroleum ether extract I (3.55 g) was subjected tocolumn chromatography (CC) on silica gel with a mixtureof hexane/EtOAc of increasing polarity to give 15 fractions(Fr. 1–15). Fr. 3 (50 mg) eluted with hexane/EtOAc (98/2)was purified by preparative TLC in hexane/EtOAc (8/2) togive nerolidol (11) (10 mg). Fr. 6 (60 mg) eluted with hex-ane/EtOAc (92.5/7.5) was purified by preparative TLCusing the same conditions as above to give compound 3

(30 mg). Aframodial (9) (50 mg) was obtained from Fr. 8(70 mg) eluted with hexane/EtOAc (9/1) after preparativeTLC in hexane/EtOAc (8/2).

The chloroform extract II (6.78 g) was fractionated onsilica gel CC eluting with a mixture of hexane/EtOAc/MeOH of increasing polarity to give 22 fractions (Fr. 1–22). Fr. 8 eluted with hexane/EtOAc (8/2) was further sub-jected to a Sephadex LH20 column, eluting with MeOH/

Table 2In vitro antiplasmodial activity of compounds 1–9, quinine, chloroquine,and artemisinine on FCB1 line of Plasmodium falciparum

compound IC50 (lM) na

Aulacocarpin A (1) 13.68 ± 6.89 3Aulacocarpin B (2) 21.10 ± 4.55 23 4.97 ± 2.27 24 39.94 ± 12.58 25 >133.13 2Galanolactone (6) 92.79 ± 12.83 2Zambesiacolactone A (7) 17.20 ± 3.05 3Zambesiacolactone B (8) 15.51 ± 4.20 3Aframodial (9) >94.33 3Quinine 0.55 ± 0.10 3Chloroquine 0.30 ± 0.03 5Artemisinine 0.01 ± 0.00 3

a n = number of experiments.


CH2Cl2 (1/1) and purified by preparative TLC in hexane/EtOAc (7/3) to give galanolactone (6) (14 mg). Galanal A(10) (15 mg) and compound 4 (3.8 mg) were obtained fromFr. 9 (20 mg) eluted with hexane/EtOAc (8/2), and Fr. 11(70 mg) eluted with hexane/EtOAc (75/25), respectively,after purification with CC over Sephadex LH20 elutingwith MeOH/CH2Cl2 (1/1) and preparative TLC in hex-ane/EtOAc (6/4). Fr. 13 (73 mg) eluted with hexane/EtOAc(75/25) was subjected to silica CC eluting with hexane/EtOAc (6/4) to give aulacocarpin A (1) (42 mg). Aulaco-carpin B (2) (70 mg) was obtained from Fr. 17 (100 mg)eluted with hexane/EtOAc (6/4) by recrystallization in amixture hexane/EtOAc (1/1). Fr. 19 (80 mg) eluted withhexane/EtOAc (3/7) was fractioned on Sephadex LH20CC eluting with CH2Cl2/MeOH (1/1) and compound 7

(5.8 mg) was purified by centrifugal TLC using CHCl3/MeOH (99/1) and preparative TLC in CH2Cl2/acetone(8/2). Fr. 20 (224 mg) eluted with EtOAc/MeOH (95/5)was fractioned on Sephadex LH20 as above to give Fr. a(37 mg) and Fr. b (34 mg) which were respectively purifiedby centrifugal TLC using a mixture CHCl3/MeOH (98/2)to yield compound 5 (20 mg) by recrystallisation in MeOHand compound 8 (10 mg) after a preparative TLC inCH2Cl2/acetone (6/4).

3.4. Aulacocarpin A (1)

ESI-MS (positive ion mode) m/z 365 [M + Na]+; spec-troscopic data as in Ayafor et al. (1994a).

3.5. Aulacocarpin B (2)

ESI-MS (positive ion mode) m/z 381 [M + Na]+; spec-troscopic data as in Ayafor et al. (1994a).

3.6. 3-Deoxyaulacocarpin A (3)

White powder: m.p. 88–89 �C; ½a�20D +40.3� (CHCl3, c

1.14); IR (KBr) tmax 2952, 2923, 1724, 1645, 1433, 1389,1262, 1245, 1214 cm�1; 1H and 13C NMR (CDCl3): seeTable 1; HRESI-MS: [M + Na]+ Calc. 371.2198; found371.2196; ESI-MS (positive ion mode) m/z 371 [M + Na]+.

3.7. Methyl-14n,15-epoxy-3b-hydroxy-8(17),12E-

labdadien-16-oate (4)

Colorless oil: ½a�20D + 28.2� (CHCl3, c 1.13); IR (film) tmax

3496, 2940, 2848, 1714, 1644, 1439, 1386, 1260 cm�1 1Hand 13C NMR (CDCl3): see Table 1; HRESI-MS:[M + Na]+ Calc. 371.2198; found 371.2211; ESI-MS (posi-tive ion mode) m/z 371 [M + Na]+.

3.8. 8b,17-Epoxy-12E-labdene-14n,15,16-triol (5)

White powder: m.p. 154–155 �C; ½a�20D + 13.2� (MeOH, c

1.19); IR (KBr) tmax 3402, 2929, 1647, 1434, 1387,1218 cm�1; 1H and 13C NMR (CD3OD): see Table 1;

HRESI-MS: [M + Na]+ Calc. 361.2355; found 361.2349;ESI-MS (positive ion mode) m/z 361 [M + Na]+.

3.9. Galanolactone (6)

ESI-MS (positive ion mode) m/z 341 [M + Na]+; spec-troscopic data as in Morita and Itokawa (1988).

3.10. Zambesiacolactone A (7)

White powder: m.p. 164–166 �C; ½a�20D +73.6� (CHCl3, c

0.8); IR (KBr) tmax 3415, 2938, 2868, 1743, 1673, 1460,1214 cm�1; 1H and 13C NMR (CDCl3): see Table 1; HRE-SIMS: [M + Na]+ Calc. 373.1991; found 373.1974 ESI-MS(positive ion mode) m/z 373 [M + Na]+.

3.11. Zambesiacolactone B (8)

White powder: m.p. 141–142 �C; ½a�20D +73.6� (MeOH, c

0.8); IR (KBr) tmax 3417, 2929, 2868, 1739, 1673, 1463,1421, 1364, 1215 cm�1; 1H and 13C NMR (CDCl3/CD3OD):see Table 1; HRESI-MS: [M + Na]+ Calc. 389.1940; found389.1956 ESI-MS (positive ion mode) m/z 389 [M + Na]+.

3.12. Aframodial (9)


3.13. Galanal (10)


3.14. Antiplasmodial assays

Continuous cultures of asexual erythrocytic stages of anFCB1 chloroquine-resistant strain of P. falciparum weremaintained following the procedure of Trager and Jensen(1976) and as described previously (Frederich et al.,2002). Artemisinin (Sigma, Bornem, Belgium), chloroquinediphosphate (Sigma C6628), and quinine base (Aldrich14590-4) were used as antimalarial references. Each testsample was applied in a series of eight fourfold dilutions(final concentrations ranging from 20 to 0.0012 lg/ml)and was tested in duplicate and triplicate. Parasite growthwas estimated by determination of lactate dehydrogenaseactivity as described by Delhaes et al. (1999) and Makleret al. (1993) and slightly modified. Briefly, in a new micro-titer plate, a 20 ll subsample of the contents of each wellwas mixed with 100 ll of a substrate solution containing1 mg lithium L-lactate (Sigma), 0.2 mg 3-acetyl pyridineadenine dinucleotide (APAD, Sigma), 0.2 ll Triton X-100(Sigma), 10 lg saponine (Merck) in TRIS buffer (pH 8,Sigma). After incubation for 20 min, 20 ll of a mix ofnitroblue tetrazolium (NBT, 2 mg/ml in TRIS pH 8 buffer,SIGMA) and phenazine ethosulfate (PES, 0.1 mg/ml in

M. Kenmogne et al. / Phytochemistry 67 (2006) 433–438 437

TRIS pH 8 buffer, Sigma) was added to each well. Afteranother 30 min of incubation, the formation of the reducedform of APAD was measured at 595 nm.

Acknowledgements

We are grateful to ISF (International Foundation forScience) for partial financial support of this work, and toAUF (Agence Universitaire de la Francophonie) for a trav-elling fellowship (Marguerite Kenmogne).

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species. Pure & Appl. Chem. 66, 2327–2330.Ayimele, G.A., Tane, P., Connolly, J.D., 2004. Aulacocarpin A and B,

nerolidol and b-sitosterol glucoside from Aframomum escapum.Biochem. Syst. Ecol. 32, 1205–1207.

De Bernardi, M., Vidari, G., Vita-Finzi, P., 1976. Dehydrozyngerone fromAframomum giganteum. Phytochemistry 15, 1785–1786.

Delhaes, L., Lazaro, J.-E., Gay, F., Thellier, M., Danis, M., 1999. Themicroculture tetrazolium assay: another colorimetric method of testingPlasmodium falciparum chemosensitivity. Ann. Trop. Med. Parasitol.93, 31–40.

Duker-Eshun, G., Jaroszewski, J.W., Asomaning, W.A., Oppong-Boa-chie, F., Olsen, C.E., Christensen, S.B., 2002. Antiplasmodial activity

of labdanes from Aframomum latifolium and Aframomum sceptrum.Planta Med. 68, 642–644.

Frederich, M., Jacquier, M.J., Thepnier, P., De Mol, P., Tits, M., Philippe,G., Delaude, C., Angenot, L., Zeches-Hanrot, M., 2002. Antiplasmo-dial activity of alkaloids from various Strychnos species. J. Nat. Prod.65, 1381–1386.

Huneck, S., Connolly, J.D., Harrison, L.J., Joseph, R., Phillips, W.R.,Rycroft, D.S., Ferguson, G., Parvez, M., 1986. New labdane diterp-enoids from the liverwort Scapania undulata. J. Chem. Res., Synopses,162–163.

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Kamnaing, P., Tsopmo, A., Tanifum, E.A., Tchuendem, M.H.K., Tane,P., Ayafor, J.F., Sterner, O., Rattendi, D., Iwu, M.M., Schuster, B.,Bacchi, C., 2003. Trypanocidal diarylheptanoids from Aframomum

letestuianum. J. Nat. Prod. 66, 364–367.Kimbu, S.F., Njimi, T.K., Sondengam, B.L., Akinniyi, J.A., Connolly,

J.D., 1979. The structure of a labdane dialdehyde from Aframomum

daniellii (Zingiberaceae). J. Chem. Soc., Perkin Trans. 1, 1303–1304.Kimbu, S.F., Ngadjui, B., Sondengam, B.L., 1987. A new labdane

diterpenoid from the seeds of Aframomum daniellii. J. Nat. Prod. 50,230–231.

Makler, M., Ries, J., Williams, J., Bancroft, J., Piper, R., Gibbins, B.,Hinrichs, D., 1993. Parasite lactate dehydrogenase as an assay forPlasmodium falciparum drug sensitivity. Am. J. Trop. Med. Hyg. 48,739–741.

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Hydroxylation of the sesterterpene leucosceptrine by the fungusRhizopus stolonifer

Muhammad Iqbal Choudhary a,*, Rosa Ranjit a, Atta-ur-Rahman a,Krishna Prasad Devkota a, Syed Ghulam Musharraf a, Tirtha Maiya Shrestha b

a H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistanb Department of Plant Resources, Ministry of Forest and Soil Conservation, Thapathali, Kathmandu, Nepal

Received 26 July 2005; received in revised form 19 November 2005Available online 19 January 2006

Abstract

The microbial transformation of leucosceptrine (1), the first member of class leucosesterterpenes, by Rhizopus stolonifer afforded twometabolites, 1a-hydroxyleucosceptrine (2), and 8a-hydroxyleucosceptrine (3).� 2005 Elsevier Ltd. All rights reserved.

Keywords: Leucosceptrine; Sesterterpene; Fungal transformation; Rhizopus stolonifer; 1a-Hydroxyleucosceptrine; 8a-Hydroxyleucosceptrine

1. Introduction

Selective functionalization by chemical methods at unac-tivated carbon atoms has been a major challenge in organicsynthesis, and thus microbiological methods have frequentlybeen used for this purpose (Fraga et al., 1996). Biotransfor-mations involve the use of enzymes or microorganisms toperform chemical reactions in which the starting substancesand products are of comparable chemical complexity.

Leucosceptrine (1), C25H36O7, the first member of a newclass of sesterterpene named as leucosesterterpenes, wasisolated from a medicinal plant Leucosceptrum canum

Smith, belonging to the family Lamiaceae, by our researchgroup (Choudhary et al., 2004a,b). L. canum, locallyknown as Bhusure (Hooker, 1983) is traditionally used asan insecticidal agent in remote areas of Nepal. Compound1 has exhibited prolylendopeptidase (PEP) inhibitory activ-ity (IC50 = 80 lM) in a mechanism-based assay (Choudh-ary et al., 2004a,b). The novel structure of leucosceptrine(1) stimulated us to carry out microbiological transforma-tion on this compound by employing Rhizopus stolonifer.


Screening scale experiments have shown that the R.

stolonifer was capable of converting compound 1 into polarmetabolites 2–3. Large scale fermentation was thus carriedout to produce sufficient quantities of metabolites 2–3 forstructure elucidation (Scheme 1). Two sets of controls wereused to ensure the authenticity of metabolites. Metaboliteswere isolated from the culture medium by chloroformextraction. The residues obtained were fractionated by col-umn chromatography. The PEP inhibitory activity of themetabolites could not be screened due to insufficient quan-tities after structure determination.

1a-Hydroxyleucosceptrine (2) was isolated as a colorlesssolid from the chloroform extract of culture broth. Thecompound 2 showed the strong IR absorptions at 3320(OH), 1735 (C@O), and 1664 (CH@CH) cm�1.

The FAB MS of compound 2 showed the (M+ � H)peak at m/z 463 (C25H36O8), 16 amu higher than the sub-strate 1. The HREI-MS spectrum showed an ion at m/z446.3312 (calcd 446.3246) supporting the formula C25H34-O7 and representing a loss of H2O from the M+.

The 1H NMR spectrum (CDCl3) of compound 2 showedclose resemblance with the substrate 1. Five methyl signals,


doi:10.1016/j.phytochem.2005.11.021

* Corresponding author. Tel.: +92 21 4824924 5/4819010; fax: +92 214819018 9.

E-mail address: [email protected] (M.I. Choudhary).



PHYTOCHEMISTRY


including three secondary methyl groups were resonated atd 1.15 (d, J22,6 = 6.4 Hz), 1.06 (d, J23,10 = 6.8 Hz), and 1.23(d, J24,14 = 7.1 Hz), and assigned to the C-22, C-23, and C-24 methyl protons, while the tertiary methyl singlets at dH

1.75 and 2.04 were assigned to the C-21 and C-25 methylprotons, respectively.

The 13C NMR spectrum showed the resonances for fivemethyl carbons, resonated at d 17.1, 12.5, 21.2, 13.8, and12.1, which were assigned to C-21, C-22, C-23, C-24, andC-25, respectively. Two trisubstituted double bond carbonswere appeared at d 142.0, 123.6, 168.3, and 117.2 andassigned to C-2, C-3, C-18, and C-19, respectively. Fourmethylene carbons were resonated at d 35.3, 32.4, 28.1,

and 26.3 due to C-8, C-9, C-15, and C-16, respectively.Six methine carbons were appeared at d 104.2 (C-1), 46.7(C-6), 47.7 (C-7), 35.3 (C-10), 57.4 (C-11), and 41.7 (C-14). Similarly six quaternary carbons, including two car-bonyl carbons, were resonated at d 75.2, 101.2, 86.5,218.3, and 174.1, and were assigned to C-4, C-5, C-12, C-13, and C-20, respectively.

The main difference between compound 2 and the sub-strate 1 was the absence of oxymethylene protons and pres-ence of a downfield proton signal at d 5.82 in compound 2which indicated that the C-2 oxymethylene was oxidizedinto an OH-containing methine. The presence of a down-field signal at d 104.2 further supported the presence of a

O

O

H3C

CH3

CH3O

H3C

O

OH

HO

H

H

H

H

H

H3C

HO2

3

4

5

68

9

1011

12

13

14

15

16

17

18

1920

21

22

24

25

7

23

1

O

O

H3C

CH3

CH3O

H3C

O

OH

HO

H

H

H

H

H

H3C

HO2

3

4

5

68

9

1011

12

13

14

15

16

17

18

1920

21

24

25

7

23

1

O

O

H3C

CH3

CH3O

H3C

O

OH

HO

H

H

H

H

H

H3C

HO2

3

4

5

68

9

1011

12

13

14

15

16

17

18

1920

21

22

24

25

7

23

1

HOOH

1

32

Rhizopus stolonifer14 days

Scheme 1. Metabolism of compound 1 by Rhizopus stolonifer.

440 M.I. Choudhary et al. / Phytochemistry 67 (2006) 439–443

hydroxyl group at C-1 in compound 2. The mass fragmentsat m/z 265, 247, 219, 108, and 110 further supported thepresence of a hydroxyl group at C-1 in hemiacetal ring.In the HMBC spectrum (Fig. 1), H-1 was found to be cou-pled with C-2 (d 142.0), C-3 (d 123.6) and C-5 (d 101.2).The Horeau’s method (Horeau and Kagan, 1964) wasemployed to deduce the stereochemistry of the newly intro-duced hydroxyl group at C-1 in compound 2. The sign ofrotation of residual acid was negative (R), which indicated

the stereochemistry of newly formed hydroxyl group as S

(a). These observations supported the structure of com-pound 2 as 1a-hydroxyleucosceptrine.

The compound 3 was isolated as a colorless solid fromthe chloroform extract of broth of R. stolonifer. Compound3 showed the strong IR absorptions at 3300 (OH), 1725(C@O), and 1665 (C@C) cm�1.

The FAB MS of compound 3 showed the (M+ � H)peak at m/z 463, 16 amu higher than the substrate 1. TheHREI-MS showed (M+ � H2O) peak at m/z 446.3012(C25H34O7, calcd 446.2946).

The 1H NMR spectrum (CDCl3) of 3 showed closeresemblance with the substrate 1. The only difference beingthe appearance of a downfield methine proton at d 4.29,geminal to an OH group.

The broad-band (BB) decoupled 13C NMR spectrum(CDCl3) of compound 3 indicated the presence of 25 car-bons including 5 methyls, 4 methylene, 10 methine and 6quaternary carbons. The 13C NMR spectrum of 3 was dis-tinctly similar to substrate 1, with a downfield methine car-bon at d 72.1, indicating the introduction of a OH group.

The mass fragment ions at m/z 265, 247 and 127 alsosupported the presence of an additional hydroxyl groupin metabolite 3. The methine proton resonated at dH 4.29showed COSY 45� correlations with H-7 (d 3.01) and H2-9 (d 1.19, 1.52), indicated the position of hydroxyl groupat C-8 in ring C. The HMBC interactions between C-11methine proton (2.19) and C-8 (dC 72.1), and between H-7 (d 3.01) and C-8, further supported the assigned positionof hydroxyl group at C-8 (Fig. 2). The C-9 methylene pro-tons (d 1.19, 1.52) also showed HMBC interactions withthe C-8.

The relative stereochemistry in compound 3 were inferredfrom the cross peaks in NOESY spectrum (Fig. 2). TheNOE correlations between H-6/H-8, H-8/H-11, and

O

O

H3C

CH3

CH3O

H3C

O

OH

HO

HO

H

H

H

H

HH3C

1

2

34

5

68

9

1011

12

13

14

15

16

17

18

1920

21

22

24

25

7

23

HO

H

H

Fig. 1. Key HMBC correlations in compound 2.

O

O

H3C

CH3

CH3O

H3C

O

OH

HO

HO

H

H

H

H

HH3C

1

2

34

5

6 8

9

1011

12

13

14

15

16

17

18

1920

21

22

24

25

7

23

H

H

H

OHH

H

H

O

H3C

CH3O

H3C

O

OH

HO

H

H

H

H

HH3C

OOHH

H

H3C

1

2 34

5

68

9

10

11

12

13

14

15

16

1718

1920

21

22

24

25

7

23

(a) (b)

Fig. 2. (a) Key HMBC, and (b) NOESY correlations in compound 3.

M.I. Choudhary et al. / Phytochemistry 67 (2006) 439–443 441

H3-23/H-11 indicated that H-6, H-8, and H-11 were in b-configuration (see Fig. 2).

3. Experimental

3.1. General methods

The melting points were recorded on a micro meltingpoint apparatus and are uncorrected. Optical rotations weremeasured on a digital polarimeter in methanol on a Jascodigital polarimeter (model DIP-3600). Ultraviolet spectrawere recorded in methanol on a Hitachi UV 3200 spectro-photometer. Infrared spectra were recorded on a Jasco A-302 IR spectrophotometer. The mass spectra were recordedon a double focusing mass spectrometer. Accurate massmeasurements were performed with FAB source using glyc-erol as matrix. The HREI-MS were recorded on a Jeol HX110 mass spectrometer. The 1H NMR spectra were recordedon 300 MHz, while 13C NMR spectra were recorded on Bru-ker AMX-500 operating at 125 MHz using CDCl3 as sol-vent. Methyl, methylene and methine carbons weredistinguished by DEPT experiments. Homonuclear 1H con-nectivities were determined by using the COSY experiment.One-bond 1H–13C connectivities were determined withHMQC gradient pulse factor selection. Two- and three-bond1H–13C connectivities were determined by HMBC experi-ment. Chemical shifts were reported in d (ppm) and couplingconstants (J) were measured in Hz. Precoated TLC plates(silica gel) were used to check the purity of compounds,and ceric sulphate spraying reagent was used for the stainingof compounds on TLC. All reagents used were of analyticalgrades.

3.2. Organism

Cultures of R. stolonifer were obtained from the Amer-ican Type Culture Collection (ATCC). All cultures weremaintained on Sabouraud dextrose agar (SDA) slantsand stored in a refrigerator at 4 �C prior to use.

3.3. Incubation of leucosceptrine (1)

R. stolonifer was grown in shake cultures at 25 �C in fiveconical flasks (250), each containing 100 mL of a sterilemedium comprising (per dm3) glucose (10 g), peptone(5 g), KH2PO4 (5 g), Yeast extract (5 g), Glycerol(10 mL), and NaCl (5 g). The media solution was adjustedto pH 7.0 before sterilization by autoclaving at 121�C for15 min. Incubations were initiated by suspending the sur-face growth from slants in sterile medium and using thesuspensions to inoculate stage I cultures. Cultures wereincubated with shaking on a shaker. After two days ofincubation in the above-described medium, stage I culturewas used as inoculum for stage II culture.

Leucosceptrine (1) (76 mg), dissolved in 5 mL ofDMSO, was uniformly distributed among five flasks

(250), each containing 100 mL of a sterile medium compris-ing (per dm3) glucose (10 g), peptone (5 g), KH2PO4 (5 g),Yeast extract (5 g), Glycerol (10 mL), and NaCl (5 g). Cul-ture controls consisted of fermentation blanks in whichmicroorganisms were grown under identical conditionsbut without the substrate. Substrate controls consisted ofsterile medium containing the same amount of substrateand were incubated under the same conditions. After 14days, the fermentation products were filtered, extractedwith chloroform, and concentrated. The organic layer werescreened for biotransformation by TLC methods.

3.4. Isolation of metabolites

The mycelium was filtered and the culture filtrate wasextracted with CHCl3. The extract was dried over Na2SO4

and the solvent evaporated to obtain a residue (122 mg). Thismaterial was chromatographed on a silica gel column with apet. ether-CHCl3 gradient, to afford 1a-hydroxyleucoscep-trine (2) (5 mg), and 8a-hydroxyleucosceptrine (3) (2 mg).

3.4.1. 1a-Hydroxyleucosceptrine (2)Colorless crystals, m.p. 154–157 �C, Rf = 0.41 (5%

MeOH/CHCl3), ½a�25D 143� (CHCl3, c 0.03), UV (MeOH)

nm (log e) kmax 380 (2.78), 351 (2.76); kmin nm 391 (1.06),361 (2.16), 182 (2.56); IR (CHCl3) mmax 3456 (OH), 2935and 2868 (CH), 1705 (C@O) cm�1; 1H NMR (CDCl3,300 MHz) and 13C NMR (CDCl3, 100 MHz) data, seeTable 1; FAB MS (M+ � 1) m/z 463, HREI-MS m/z446.3312 (calcd for C25H36O7, m/z 446.3246), EI-MS m/z(rel. int. %): 446 (30), 265 (16), 247 (100), 219 (18), 181(18), 153 (17), 139 (19), 125 (24), 110 (46), 97 (66), 83 (62).

3.4.2. 8a-Hydroxyleucosceptrine (3)

Colorless crystals, m.p. 145–147 �C, Rf = 0.35 (5%MeOH/CHCl3), ½a�25

D 240� (CHCl3, c 0.06); UV (MeOH)nm (log e): kmax 365 (2.78), 301 (2.76), kmin 382 (1.79),346 (2.02), 261 (2.16), IR (CHCl3); mmax 3456 (OH), 2935and 2868 (CH), 1705 (C@O) cm�1; 1H NMR (CDCl3,300 MHz) and 13C NMR (CDCl3, 100 MHz) data, seeTable 1; FAB MS (M+ � 1) m/z 463, HREI-MS m/z446.3012 (calcd for C25H36O7, m/z 446.2946), EI-MS m/z(rel. int. %): 446 (30), 265 (11), 247 (19), 181 (18), 155(10), 153 (18), 127 (22), 125 (48), 110 (42), 97 (68), 92(17), 83 (100).

3.5. Horeau’s procedure

Compound 2 (5 mg, ca. 0.01 mmol) was added to a solu-tion of 2-phenyl butyric anhydride (0.1 mL) in 0.5 mLC5H5N. The resulting mixture was stirred overnight atroom temperature. Distilled water (3.0 mL) was addedand the reaction mixture allowed to stand for 30 min,0.1 M NaOH was then added dropwise until the pHbecame 9 and the solution was then extracted with CHCl3.The aqueous layer was acidified to pH 3 using 1.0 M HCland the acidic layer was extracted with C6H6 (10 mL).

442 M.I. Choudhary et al. / Phytochemistry 67 (2006) 439–443

The benzene extract was evaporated to adjust the volumeto 1.0 mL. The optical rotation of the resulting 2-phenylbutyric acid was found to be negative (R), indicating the‘‘S’’ configuration at C-20 in compound 2.

Acknowledgments

One of us, Dr. Rosa Ranjit, acknowledges the supportof the Third World Organization for Women in Science(TWOWS), Trieste, Italy, through a Ph.D. scholarship tostudy at the H.E.J. Research Institute of Chemistry, Kar-achi-75270, Pakistan. We are also grateful to the ThirdWorld Academy of Sciences (TWAS), Trieste, Italy, andLG Corporation Limited, Pakistan, for financial supportto Dr. Krishna P. Devkota.

References

Choudhary, M.I., Ranjit, R., Atta-ur-Rahman, Shrestha, T.M., Yasin,A., Parvez, M., 2004a. Leucosceptrine – a novel sesterterpene withpropylendopeptidase inhibitory activity from Leucosceptrum canum. J.Org. Chem. 69, 2906–2909.

Choudhary, M.I., Ranjit, R., Atta-ur-Rahman, Hussain, S.,Devkota, K.P., Shrestha, T.M., Parvez, M., 2004b. Novel sesterter-pens from Leucosceptrum canum of Nepalese origin. Org. Lett. 6,4139–4142.

Fraga, B.M., Guillermo, R., Hanson, J.R., Trunch, A., 1996.Biotransformation of cerdrol and related compounds by Mucor

plumbeus. Phytochemistry 42, 1583–1586.Hooker, J.D., 1983. Flora of British India, vol. IV. Bishen Singh

Mahendra Pal Singh Publishers, Dehradun, pp. 699–700.Horeau, A., Kagan, H.B., 1964. Determination des configurations par

‘‘dedoublement partiel’’ – III alcohols steroides. Tetrahedron 20,2431–2441.

Table 11H and 13C NMR data of 1a-hydroxyleucosceptrine (2) and 8a-hydroxyleucosceptrine (3) in CDCl3

Position 2 3

dH (m, J in Hz)a dCb Multiplicity dH (m, J in Hz)a dC

b Multiplicity

1 5.82 (s) 104.2 CH 3.99, 4.21 (d, 17.1) 64.7 CH2

2 – 142.0 C – 137.2 C3 5.61 (brs) 123.6 CH 4.96 (brs) 121.3 CH4 – 75.2 C – 73.4 C5 – 101.2 C – 96.8 C6 1.95 (m) 46.7 CH 2.10 (m) 41.6 CH7 1.55 (m) 47.7 CH 3.01 (m) 42.6 CH8 1.45, 1.91 (m) 35.3 CH2 4.29 (m) 72.1 CH9 1.23, 1.44 (m) 32.4 CH2 1.19, 1.52 (m) 38.5 CH2

10 1.67 (m) 35.3 CH 1.85 (m) 36.6 CH11 1.55 (m) 57.4 CH 2.19 (m) 52.2 CH12 – 86.5 C – 82.2 C13 – 218.3 C@O – 221.2 C@O14 3.01 (m) 41.7 CH 3.37 (m) 38.6 CH15 1.25, 1.86 (m) 28.1 CH2 1.21, 2.01 (m) 28.9 CH2

16 1.15, 1.75 (m) 26.3 CH2 1.15, 1.85 (m) 26.8 CH2

17 4.81 (brd, 6.2) 84.5 CH 4.81 (brd, 6.7) 84.2 CH18 – 168.3 C – 167.7 C19 5.94 (m) 117.2 CH 5.81 (m) 117.3 CH20 – 174.1 C – 181.6 C21 1.80 (brd, 1.3) 17.1 CH3 1.65 (brs) 14.7 CH3

22 1.15 (d, 6.4) 12.5 CH3 0.99 (d, 6.7) 10.8 CH3

23 1.06 (d, 6.8) 21.2 CH3 0.71 (d, 6.9) 17.7 CH3

24 1.23 (d, 7.1) 13.8 CH3 1.06 (d, 6.7) 13.7 CH3

25 2.05 (s) 12.1 CH3 2.05 (s) 13.4 CH3

a 1H NMR data, 300 MHz.b 13C NMR data, 125 MHz.

M.I. Choudhary et al. / Phytochemistry 67 (2006) 439–443 443

Clerodane and labdane diterpenoids from Nuxia sphaerocephala

Lengo Mambu a,*, Philippe Grellier b, Loic Florent a, Roger Joyeau a,David Ramanitrahasimbola c, Philippe Rasoanaivo c, Francois Frappier a,z

a USM 0502-UMR5154 CNRS Chimie et Biochimie des substances naturelles, Departement Regulations, Developpement, Diversite Moleculaire,

Museum National d’Histoire Naturelle, 63 rue Buffon, 75231 Paris Cedex 05, Franceb USM 0504 Biologie fonctionnelle des protozoaires, Departement Regulations, Developpement, Diversite Moleculaire, Museum National d’Histoire

Naturelle, 61 rue Buffon, 75231 Paris Cedex 05, Francec Laboratoire de Pharmacognosie Appliquee aux Maladies Infectieuses, Institut Malgache de Recherches Appliquees, B.P. 3833,

101-Antananarivo, Madagascar

Received 30 March 2005; received in revised form 17 November 2005Available online 19 January 2006

Abstract

Seven diterpenoids including four clerodane and three labdane derivatives, (13S)-ent-7b-hydroxy-3-cleroden-15-oic acid (1), ent-7b-hydroxy-2-oxo-3-cleroden-15-oic acid (2), ent-2,7-dioxo-3-clero-den-15-oic acid (3), ent-18-(E)-caffeoyloxy-7b-hydroxy-3-cleroden-15-oic acid (4) (13S)-ent-18-(E)-coumaroyloxy-8(17)-labden-15-oic acid (5), ent-18-(E)-caffeoyloxy-8(17)-labden-15-oic acid (6), ent-15-(E)-caffeoyloxy-8(17)-labden-18-oic acid (7), have been isolated from an ethyl acetate extract of the leaves of Nuxia sphaerocephala,together with 17 known compounds. 3-Oxolup-20(29)-en-30-al (3-oxolupenal) (8) and 3b-hydroxylup-20(29)-en-30-al (3b-hydroxy-lupe-nal) (9) showed the best inhibitory activity against Plasmodium falciparum with the IC50 values between 1.55 and 4.67 lg/ml in vitro,respectively. The structure and the relative stereochemistry of the compounds were established on the basis of their spectroscopic prop-erties. The absolute configuration at C-13 of 1 and 5 was determined by the PGME amide procedure.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Nuxia sphaerocephala; Loganiaceae; Clerodanes; Labdanes; PGME amide method; Antiplasmodial activity

1. Introduction

Nuxia sphaerocephala Baker (Loganiaceae) is a shrubgrowing in the Eastern rainforests of Madagascar. Leavesare used in traditional medicine to treat splenomegaly asso-ciated with malaria, and also infantile hederosyphilis (Boi-teau, 1999). Previous phytochemical studies on the genusNuxia led to the isolation of the eight-carbon iridoid gluco-side unedoside and its derivatives (Jensen et al., 1998; Fred-eriksen et al., 1999). To the best of our knowledge, nostudy has been reported on N. sphaerocephala. As part ofour search for novel antimalarial agents from plants, we

investigated the leaves of N. sphaerocephala, the ethyl ace-tate extract of which showed antiplasmodial activity withan IC50 value of 4.2 lg/ml (Rasoanaivo et al., 2004). Sevennew compounds, including four clerodane diterpenoids (1,2, 3, 4) and three labdane diterpenoids (5, 6, 7) along with17 known compounds were isolated, of which ten wereidentified as triterpenes namely 3-oxolupenal (3-oxolup-20(29)-en-30-al) (8), 3b-hydroxylupenal (3b-hydroxylup-20(29)-en-30-al) (9), lup-20(29)-ene-3b,30-diol (Wijeratneet al., 1981), 3-oxolupenol (30-hydroxylup-20(29)-en-3-one) (10) (Bohlmann and Jakupovic, 1979), lupeol, uvaol(Dehmlow et al., 1998), ursolic acid, 3b-acetylursolic acid(Houghton and Lian, 1986), 3b-acetyloleanolic acid (11)(Chavez and Delgado, 1994) and oleanolic acid (12); threewere found to be diterpenes, ent-15-hydroxy-8(17)-labden-19-oic acid (13) (Zdero et al., 1991a), ent-18-hydroxy-8(17)-labden-15-oic acid (14) (Zdero et al., 1991b) and ent-18-


doi:10.1016/j.phytochem.2005.11.024

* Corresponding author. Tel.: +33 1 40 79 56 07; fax: +33 1 40 79 31 35.E-mail address: [email protected] (L. Mambu).

z In memoriam.



PHYTOCHEMISTRY


hydroxy-3-cleroden-15-oic acid (15) (Tsichritzis andJakupovic, 1990), three were identified as flavones, 5-hydroxy-7-methoxyflavone (Mongkolsuk and Dean,1964), 5-hydroxy-4 0,7-dimethoxyflavone (Biftu and Steven-son, 1978) and 5,7-dihydroxy-4 0-methoxyflavone (Gaydouand Bianchini, 1978) and one as a sterol, 3-O-b-D glyco-pyranosylsitosterol (Ahmad et al., 1992). All clerodaneand labdane diterpenoids isolated from this plant were rec-ognized as members of ent-series through their optical rota-tion values. In this paper, we describe the isolation andstructural elucidation of 1–7 as well as the inhibitory activ-ity of all isolated compounds against Plasmodium

falciparum.


A crude EtOH extract of leaves of N. sphaerocephala

was partitioned successively with cyclohexane and ethylacetate. The ethyl acetate soluble extract showed significantinhibitory activity against P. falciparum with an IC50 of4.2 lg/ml and was subjected to a series of bioassay-guidedcolumn chromatographic purification steps over silica gelto afford seven new compounds 1–7 along with 17 knowncompounds.

Compound 1 was obtained as colourless oil. Its molecu-lar formula C20H34O3 was established from its HRCIMS atm/z 340.2845 [M + NH4]+ and confirmed by 13C NMRdata which also revealed the presence of a trisubstituteddouble bond and a carboxyl group. The molecule is there-

fore bicyclic. The 1H NMR spectrum (Table 2) displayedcharacteristic signals for an olefinic proton at d 5.11 (1H,m), an oxymethine proton at d 3.98 (1H, q, J = 3.3 Hz),two tertiary methyl singlets at d 0.96 and 1.26 and threedoublet methyl signals at d 0.96 (d, J = 6.9 Hz), 0.98 (3H,d, J = 7.2 Hz) and 1.59 (d, J = 1.4 Hz) and suggested aclerodane skeleton. The 13C NMR spectral data indicatedthat compound 1 contained 20 carbons, including fivemethyls, five methines, six methylenes and four quaternarycarbons (Table 1). The COSY spectrum revealed three spinsystems associated with ring A, ring B and the side chain asin 1. HMBC correlations confirmed the clerodane nucleusand the position of the functionality. Thus H-3 showed cor-relations with C-2, C-5 and C-18 while H-7 correlated withC-5, C-6, C-8, C-9 and C-17. The structure of the side chainwas established inter alia by the HMBC correlations fromH-13 to C-11, C-12, C-14, C-15 and C-16. Other useful cor-relations of C-5 included those from Me-18 and Me-19.The configuration of the hydroxyl group at C-7 wasdeduced to be a (axial) on the basis of the small couplingconstants of H-7. NOEs from Me-19 to Me-20 and fromH-7 to Me-17 completed the relative stereochemistry of 1apart from the A/B ring junction which was assumedto be trans on the basis of the lack of a NOE between

Me-19 and H-10. The configuration at C-13 could not beresolved by spectroscopic means.

The absolute configuration at C-13 was determined byapplying the phenylglycine methyl ester method (PGME)developed for carboxylic acids having a chiral center at

R

COOHH1

410

1113 15

17

18 19

20R

R27

1 . R = H, H; R = OH; R = H 4

2 . R = O; R1 = OH; R2 = H

3 . R = O; R , R2 = O

COOH

O

R1

OH

R2

HO

OH

OHO

OOH

H

H1

518

17

20

1315

8'

7'1'

4'

19

5 R1 =OH; R2 = H 7

6 R1 = OH; R2 = OH

Diterpenoids 1-7 from N.sphaerocephala

21

1

2

OH

COOH

O

OOH

OH

H

7

18

7'

8'

1'

4'

3'

L. Mambu et al. / Phytochemistry 67 (2006) 444–451 445

the b-position (Yabuuchi and Kusumi, 2000). Two por-tions of 1 (each 4 mg) were condensed in DMF with (S)-and (R)-PGME in the presence of PyBOP, HOBT, andN-methylmorpholine which afforded the (S)- and (R)-PGME amide derivatives (1s and 1r). Assignments of theproton signals of PGME amides were based on the corre-lations of the 1H–1H COSY and NOESY spectra includingHSQC and HMBC. The chemical shift difference valuesDdRS = dR � dS of the individual protons of 1s and 1r areshown in Fig. 1. The systematic arrangement of positiveand negative DdRS values was observed and unambiguouslyestablished the absolute configuration of C-13 to be S asindicated by the structure 1 which belongs to the enantio

series. Accordingly, compound 1 was assigned as (13S)-ent-7b-hydroxy-3-cleroden-15-oic acid.

The FABMS spectrum of 2 exhibited a pseudomolecularion peak at m/z 337 [M + H]+ consistent with a molecularformula C20H32O4 and five double bond equivalents. Its13C spectrum, UV and the IR spectra showed the presenceof an a,b-unsaturated ketone (d 200.6 (C-2), 125.0 (C-3)and 173.1 (C-4); mmax 1657 cm�1; kmax 238 nm). The 1Hand 13C NMR data for 2 (Tables 1 and 2) were similarto those of 1 apart from the presence of a ketone at C-2,confirmed by HMBC correlations from H-10 and 2H-1to C-2. The relative stereochemistry of 2 was establishedin the same way as that of 1 with additional support com-ing from NOEs between H-6, H-8 and H-10 indicating thatall these protons were axial (b). Thus, compound 2 wasestablished as ent-7b-hydroxy-2-oxo-3-cleroden-15-oicacid.

The molecular formula of compound 3 was determinedas C20H30O4 from combined analysis of HRCIMS and 13Cdata. Its 1H and 13C NMR data were similar to those of 1

except for the lack of an oxymethine proton (H-7) and thepresence of two ketone carbonyl carbons at d 198.8 and211.3. Long-range correlations of H-6, H-8 and H3-17 tothe carbonyl at d 211.3 allowed us to place the carbonylgroup at C-7 and the influence of this group was observedby an upfield shift of C-17 (d 7.7 instead of 12.4) in the 13Cspectrum. The second carbonyl group was located at C-2 asshown by the connectivities of H2-1 and H-10 with this car-bon in HMBC spectrum. The relative stereochemistry of 3

was assigned by analysis of NOESY correlations as for 1

and 2. Me-20 showed strong correlations with 2H-11,Me-17 and Me-19 suggesting that they were all on the sameface of the molecule. Compound 3 was therefore identifiedas ent-2,7-dioxo-3-cleroden-15-oic acid.

For compound 4, the HRFABMS showed a pseudomo-lecular ion peak at m/z 523.2687 [M + Na]+ (calcd.507.2672 for C29H40O7Na) in agreement with the molecu-lar formula of C29H40O7. Its 1H NMR spectrum was mark-edly similar to that of 1 except for the presence of a caffeoylmoiety, a primary oxygenated carbon (dH 4.67 (br s, 2H-18), dC 66.0 (C-18)) and the lack of a vinyl methyl group.The spectroscopic data for the caffeoyl residue are inTables 1 and 2 and in Section 3. An HMBC correlationfrom 2H-18 to the ester carbonyl at dC 169.1 establishedthe connection of the caffeoyl group to the clerodane skel-eton. The NOESY spectrum revealed the same relative ste-reochemistry as 1. Hence, compound 4 was elucidated asent-18-E-caffeoyloxy-7b-hydroxy-3-cleroden-15-oic acid.

Compound 5 (mmax 3352, 1663, 1587 and 1416 cm�1,kmax 312, 296 and 226 nm), a colorless oil, contained ester,carboxylic acid and conjugated aromatic functionality. Itsmolecular formula C29H40O5 was established from the[M + NH4]+ ion at m/z 486.3227 (calcd. 486.3219 forC29H44O5N) in its HRCIMS spectrum. Its 1H NMR spec-trum (Table 3) revealed the presence of two tertiary methylsdH 0.84 and 0.7, a secondary methyl signal dH 0.96 (d,J = 6.6 Hz), an exomethylene group dH 4.47 and 4.80 (boths), an oxygenated methylene group dH 3.73, 3.96 (both d,J = 10.9 Hz) and a trans p-hydroxycinnamoyl moiety (cou-

Table 113C NMR data for compounds 1–7

Position 1a 2a 3a 4b 5a 6b 7b

1 17.8 34.7 35.0 18.8 38.5 39.8 39.52 26.7 200.6 198.9 27.5 18.6 19.7 19.53 119.9 125.0 126.0 126.4 36.0 37.2 38.44 144.9 173.1 169.5 145.1 37.0 38.3 48.05 37.5 39.4 44.3 38.6 49.5 50.8 51.26 42.8 41.4 51.2 43.6 24.3 25.6 28.07 73.9 73.3 211.3 74.0 38.0 39.2 39.18 39.2 38.8 50.1 40.6 148.1 149.6 149.09 38.0 38.1 44.3 39.2 56.8 58.4 58.710 46.5 45.9 45.7 47.8 39.5 40.6 40.011 36.6 35.9 35.3 37.8 20.9 22.0 22.212 29.5 29.2 29.2 30.7 35.6 36.9 36.913 30.8 30.7 30.5 32.2 30.7 32.1 31.714 41.3 41.1 40.8 42.7 41.8 43.3 37.015 178.6 177.0 176.0 177.1 178.6 177.6 63.916 19.9 19.9 19.9 20.3 19.5 20.0 20.317 12.4 12.3 7.7 13.0 106.6 107.2 107.418 18.0 19.0 18.8 66.0 72.8 73.7 182.219 21.8 20.2 19.4 23.7 17.6 18.0 17.120 20.2 19.7 19.1 20.6 14.9 15.4 15.210 127.5 127.0 127.7 127.820 115.2 130.0 115.2 115.230 146.8 115.9 147.0 146.840 149.6 157.9 149.5 149.550 116.5 115.9 116.5 116.560 122.9 130.0 123.0 122.970 146.8 144.6 146.9 146.880 115.4 115.5 115.0 115.3CO 169.1 167.8 169.3 169.4

a Spectra were recorded in CDCl3.b Spectra were recorded in CD3OD.

CH3

CH3

CH3

O

N

O

OCH3

H PhH

CH3

CH3-0.089

-0.001

+0.018

+0.015

-0.059

+0.021+0.015

+0.021

+0.001

OH

H

Ph H

(R )

(S)

Fig. 1. Dd(R � S) values (in ppm) for PGME amide derivatives of 1r and1s in CDCl3.

446 L. Mambu et al. / Phytochemistry 67 (2006) 444–451

maroyl, see Tables 1 and 3). The COSY spectrum revealedspin systems associated with rings A and B and the sidechain of a labdane skeleton which was confirmed by severalcorrelations in the HMBC spectrum, in particular from H-7 to C-8, C-9 and C-17, H-5 to C-4, C-9, C-10 and C-20, H-1 to C-5, C-10 and C-20, H-3 to C-4 and C-19. Support forthe side chain came from correlations of H-14 to C-15 andC-16 and from H-12 to C-9 and C-11. The oxymethyleneprotons clearly belonged to C-18 in view of HMBC corre-lations to C-3, C-4, C-5 and C-19. A further correlation ofthese to the coumarate ester carbonyl group at d 167.8 con-firmed the position of attachment of the ester. NOE inter-actions between Me-19 and Me-20, 2H-18 and H-5, andH-5 and H-9 indicated a typical A/B trans labdane system.

The absolute configuration at C-13 was also determinedby applying the phenylglycine methyl ester method(PGME). The chemical shift difference valuesDdRS = dR � dS of (R)-(5) and (S)-(5) PGME amides areshown in Fig. 2. From these data the absolute configura-tion of the chiral carbon at C-13 was identified to be S

and compound 5 belongs to the enantio series. Thus, thestructure of 5 was established as (13S)-ent-18-E-couma-royloxy-8(17)-labden-15-oic acid (see Fig. 2).

The HRFABMS of compound 6 gave a pseudomolecu-lar ion peak at m/z 507.2725 [M + Na]+ (calcd. 507.2723for C29H40O6Na) corresponding to a molecular formulaC29H40O6. Its UV spectrum was characteristic of a caffeoyl

moiety with absorption maxima at 327, 295, 245, 220 and203 nm. The 1H NMR spectrum of 6 resembled that of 5

apart differences in the aromatic region (Table 3). Exami-nation of the proton and carbon data readily revealed thatthe coumaroyl moiety of 5 had been replaced by a caffeoylmoiety. The structure of 6 was determined as ent-18-E-caf-feoyloxy-8(17)-labden-15-oic acid.

Compound 7 was obtained as colourless oil with amolecular formula of C29H40O6 (m/z 507.2739) based onHRFABMS and 13C data. The spectroscopic data, includ-ing the UV (kmax 328, 293, 245, 220, 203 nm), indicated thepresence of a caffeoyl moiety. The lack of 2H-14 reso-nances, present in 1–6 between dH 2.08 and 2.33, suggestedthat C-15 was no longer a carboxyl group but had beenreduced to an alcohol. The spectroscopic properties indi-cated that 7 was a derivative of the C-4 epimer of ent-15-hydroxy-8(17)-labden-19-oic acid (Zdero et al., 1991a).The downfield shift 2H-15 and an HMBC correlation from2H-15 to the caffeoyl carbonyl group revealed the positionof attachment of the ester. NOEs between H-9 and H-5 andMe-19 and Me-20 established the trans nature of the ringjunction and the presence of a C-18 carboxyl group. Thuscompound 7 was assigned as 15-E-caffeoyloxy-8(17)-lab-den-18-oic acid.

All the isolated compounds were tested for their abilityto inhibit in vitro the growth of the chloroquine-resistantstrain FcB1 of P. falciparum. The antiplasmodial activity

Table 21H NMR data of clerodane diterpenoids

Position 1a 2a 3a 4b

1 1.53, m 2.34, dd (3.9, 17.5) 2.34, m 1.59, m

2.44, dd (13.7, 17.5)2 1.96, m 2.13, m

2.01, m

3 5.11, m 5.67, d (1.3) 5.77, d (1.2) 5.58, br s

6 1.37, dd (3.3, 14.1) 1.50, dd (3.5, 14.0) 2.34, m 1.53, dd (3.1, 13.5)2.07, dd (2.6, 14.1) 2.17, dd (2.8, 14.0) 2.51, d (11.9) 2.10, dd (2.8, 13.5)

7 3.98, q (3.3) 4.06, ddd (2.8, 3.2, 3.5) 3.97, ddd (2.8, 3.1, 3.4)8 1.51, m 1.53, dd (3.2, 7.2) 2.56, q (6.3, 12.8) 1.56, m

10 1.37, m 1.89, dd (3.9, 13.7) 2.46, dd (4.1, 11.2) 1.47, dd (2.1, 11.4)11 1.28, m 1.25, m 1.26, m 1.41, dd (9.8, 12.6) 1.31, m

12 1.00, m 1.00, m 1.09, m 1.21, m

1.16, ddd (5.5, 12.3) 1.09, m 1.26, m

13 1.84, m 1.83, m 1.88, m 1.80, m

14 2.14, dd (7.9, 15.1) 2.15, dd (8.7, 15) 2.19, dd (7.3, 15.1) 2.15, dd (14.8, 7.2)2.33, dd (6.2, 15.1) 2.29, dd (6.4, 15) 2.28, dd (6.4, 15.1) 2.23, dd (14.8, 7.0)

16 0.96, d (6.9) 0.95, d (6.7) 0.98, d (6.7) 0.97, d (6.7)17 0.98, d (7.2) 1.00, d (7.2) 0.92, d (6.3) 1.00, d (6.1)18 1.59, d (1.4) 1.89, d (1.3) 1.85, d (1.2) 4.67, br s

19 1.26, s 1.37, s 1.08, s 1.40, s

20 0.97, s 1.05, s 0.77, s 1.02, s

2 0 7.03, d (2.0)5 0 6.77, d (8.2)6 0 6.93, dd (2.0, 8.2)7 0 7.52, d (15.9)8 0 6.25, d (15.9)



of new compounds 1–7 and the most active known com-pounds (8–12) are summarized in Table 4. The other com-pounds displayed IC50 value over 10 lg/ml. Compounds 8

and 9 displayed significant antimalarial activity with IC50

value between 1.55 and 4.67 lg/ml depending upon theP. falciparum strain tested (Fig. 3). In comparison tochloroquine which was used as a positive control, thesecompounds have to be considered as moderately active(Table 4).

For 8 and 9, the C-30 aldehyde and the C-3 ketone prob-ably have a major influence on the activity. The activity of10, with a C-30 hydroxyl group, was lower and withhydroxyls at both C-3 and C-30 the activity diminishedconsiderably.

Ursolic acid exhibited antiplasmodial activity with IC50

value of 16 lg/ml which is consistent with that published(Azas et al., 2002). In vitro antiplasmodial activity of 12has been previously reported with IC50 value similar tothose found in the present work (Sairafianpour et al.,2003). The lupane triterpenoids betulinic acid and lupeol,as well as labdane diterpenoids have been reported to exhi-bit moderate antiplasmodial activity in vitro (Bringmannet al., 1997; Ziegler et al., 2002; Asili et al., 2004).

It has been demonstrated that terpenoids can be incor-porated into the lipid bilayer of erythrocyte membranesirreversibly and induce shape transformation of this mem-brane (Ziegler et al., 2002; Asili et al., 2004). In this case,

Table 31H NMR data of labdane diterpenoids

Position 5a 6b 7b

1 0.95, m 1.09, m 1.16, dd (4.7, 7.8)1.71, m 1.83, m 1.79, m

2 1.55, m 1.59, m 1.51, m

1.70, m

3 1.35, m 1.47, m 1.51, m

1.71, m

5 1.40, m 1.51, m 1.96, dd (2.2, 12.5)6 1.35, m 1.40, m 1.32, m

1.40, m 1.69, m 1.44, m

7 1.96, dd (4.9, 13.2) 1.99, m 2.02, dd (4.7, 13.1)2.35, m 2.38, m 2.36, m

9 1.59, d (9.5) 1.65, m 1.65, m

11 1.35, m 1.42, m 1.44, m

1.44, m 1.52, m 1.51, m

12 1.14, m 1.16, m 1.24, m

1.31, m 1.37, m 1.32, m

13 1.90, m 1.89, m 1.51, m

14 2.12, dd (7.8, 15.1) 2.08, dd (7.7; 14.6) 1.51, m

2.27, dd (6.2, 15.1) 2.24, dd (6.6, 14.6) 1.71, m

15 4.17, m

16 0.96, d (6.6) 0.96, d (6.6) 0.96, d (6.3)17 4.47, br s 4.53, s 4.55, s

4.80, br s 4.83, s 4.84, s

18 3.73, d (10.9) 3.75, d (10.8)3.96, d (10.9) 3.99, d (10.8)

19 0.84, s 0.89, s 1.12, s

20 0.70, s 0.77, s 0.74, s

2 0 7.42, d (8.7) 7.06, d (2.0) 7.04, d (2.0)3 0 6.82, d (8.6)5 0 6.82, d (8.6) 6.78, d (8.2) 6.77, d (8.3)6 0 7.42, d (8.7) 6.97, dd (2.0, 8.2) 6.94, dd (2.0, 8.3)7 0 7.59, d (15.9) 7.55, d (15.9) 7.52, d (15.9)8 0 6.30, d (15.9) 6.29, d (15.9) 6.24, d (15.9)


H

O

N

O

OCH3

H Ph

O

O

OH

H

+0.025

-0.023

+0.014

+0.035+0.021

+0.121 -0.054

+0.053

+0.023

H

Ph

(R)

(S)H

Fig. 2. Dd(R � S) values (in ppm) for PGME amide derivatives of 5r and5s in CDCl3.

Table 4In vitro antiplasmodial activity of compounds 1–12

Compound IC50 ± SD (lg/ml) IC50 ± SD (lg/ml)FcB1 FcM29

1 14.6 ± 1.42 4.3 ± 0.93 8.0 ± 0.24 7.3 ± 0.85 11.4 ± 1.16 21.0 ± 1.857 16.0 ± 0.878 1.55 ± 0.06 4.67 ± 0.099 3.15 ± 0.07 4.06 ± 0.5310 9.05 ± 1.06 15.56 ± 2.1111 7.65 ± 0.4912 9.83 ± 3.1Chloroquine 0.05 ± 0.002

Results are expressed as IC50 and IC90 values (lg/ml) ± standard devia-tions. All experiments were realised in triplicate.

R2

H

R

R11

3

13

15

19

28

24 23

25 26

27

30 20

29

22H

H

H

8 R, R1= O , R2 = CHO

9 R = OH, R1 = H, R2 = CHO

10 R, R1 = O, R2 = CH2OH

Fig. 3. Structures of lupane derivatives 8–10.


the inhibition of parasite growth observed in vitro could beattributed to indirect effects due to stomatocytic or echino-cytic modifications of the host cell membrane.

Non-parasitized erythrocytes were then incubated withincreasing concentrations of compounds 8, 9 and 10 underthe same conditions as previously described (Ziegler et al.,2002). No lysis of cells and no change of erythrocyte mem-brane shape in echnocytic forms were observed at the con-centrations up to 50 lg/ml by phase-contrast microscopy.There was also no evidence of stomatocytic forms. How-ever, such modification might not be clearly detectable byphotonic microscopy, further investigation using transmis-sion electron microscopy will be required to confirm ourobservations.

3. Experimental


Optical rotations were measured with a Perkin–Elmermodel 341 polarimeter at 20 �C. IR spectra were takenon a Nicolet Impact 400D spectrophotometer. The UVspectra were recorded on a Kontron spectrometer. 1Hand 13C NMR spectra were recorded at 400.13 and100.61 MHz, respectively, on a Bruker AVANCE-400spectrometer at 298 K, equipped with 1H-broad-bandreverse gradient probehead. Temperature was controlledby a Bruker BCU-05 refrigeration unit and a BVT 3000control unit. The 1H and 13C NMR chemical shifts wereexpressed in ppm relative to TMS, with coupling constants(J) given in Hz. High-resolution mass spectra and FAB-MSwere recorded on a JEOL MS700 apparatus. Mass spectradata were recorded using an electrospray time of flightmass spectrometer (ESI-TOF-MS) operating in the positivemode (QSTAR Pulsar I of Applied Biosystems). TLC wascarried out on precoated Si gel 60 F254 plates (Merck).Spots were detected under UV (254 and 366 nm) beforespraying with phosphomolybdic acid solution in EtOH orLiebermann–Burchard reagent or vanillin-sulfuric solutionfollowed by heating the plate at 110 �C. Column chroma-tography was performed on 200–400 mesh silica gel 60(Merck). Preparative medium-pressure liquid chromatog-raphy (MPLC) was performed with a pump K-120(Knauer) and Flashsmart cartridges (Si gel 20–40 lm,AIT, France).

3.2. Plant material

The plant material was collected in March 2000 inAnkazobe, middle West located at 100 km North fromAntananarivo (Madagascar) and was identified by ArmandRakotozafy by comparison with authentic specimens heldin the Department of Botany, Parc Botanique et Zoologi-que de Tsimbazaza, Antananarivo. A voucher specimen(ANKA 15/AR/2000) was deposited at the InstitutMalgache de Recherches Appliquees.

3.3. Bioassay

The in vitro antiplasmodial tests, based on the inhibitionof [3H]-hypoxanthine uptake by P. falciparum cultured inhuman blood, were conducted as previously described(Frappier et al., 1996).


Air-dried powdered leaves of N. sphaerocephala (600 g)was exhaustively extracted with ethanol (3 · 500 ml) atroom temperature to give 31 g of crude extract. The ethan-olic extract exhibited an IC50 value of 7.1 lg/ml against thegrowth of P. falciparum. A portion of the crude extract(14 g) was partitioned between ethyl acetate (3 · 500 ml)and water (300 ml). The organic fraction (10.8 g) showedan IC50 value of 4.2 lg/ml whereas the aqueous fractionwas inactive (1.27 g; IC50 > 25 lg/ml). A portion (5 g) ofthe ethyl acetate extract was chromatographed on a silicagel column using a mixture of cyclohexane–EtOAc ofincreasing polarity as eluant to give 22 fractions. The puri-fication of the most potent fractions F1 and F2 on silica gelcolumn chromatography (cyclohexane–EtOAc 90:10)yielded 27 mg of compound 8. Chromatography of F6 withcyclohexane–EtOAc 85:15 followed by crystallization (ace-tone) afforded 42 mg of compound 9. Repeated chroma-tography of fraction F11 on MPLC column (AIT,France) with CH2Cl2–MeOH 98:2 furnished 8 mg of com-pound 1. Chromatography of fraction F12 yielded addi-tional compound 1. From fraction F15, compound 5(8 mg) was obtained after repeated purification on silicagel with cyclohexane–EtOAc 75:25 followed by CH2Cl2–MeOH 97:3. F16 was purified by silica gel column chroma-tography (CH2Cl2–MeOH 98:2) to afford 13 mg ofcompound 7. Compound 6 (9 mg) was isolated from fractionF17 by successive chromatography on silica gel with cyclo-hexane–EtOAc 60:40 and on a Sephadex column usingMeOH as eluting solvent. F18 was purified by silica gel col-umn chromatography (cyclohexane–EtOAc 60:40 andCH2Cl2–MeOH 95:5) to afford 11 mg of compound 4.Fraction F22 afforded compound 3 (2 mg) and compound2 (5 mg) by further purification by column chromatogra-phy on silica gel eluting with CH2Cl2–MeOH 98:2.

3.4.1. (13S)-ent-7b-Hydroxy-3-cleroden-15-oic acid (1)

Colourless oil; ½a�20D � 32:3 (c 0.4, CHCl3); IR (CHCl3)

mmax 2925, 1712, 1470, 1066 cm�1; UV (MeOH) kmax (loge)237 (3.06), 203 (3.48) nm; 1H NMR and 13C NMR data,see Tables 1 and 2; HRCIMS, m/z: 340.2845([M + NH4]+) (calcd. for C20H38O3N, 340.2852).

3.4.2. ent-7b-Hydroxy-2-oxo-3-cleroden-15-oic acid (2)

Colourless oil; ½a�20D � 30 (c 0.195, CHCl3); IR (CHCl3)

mmax 2925, 1657, 1470, 1029 cm�1; UV (MeOH) kmax (loge)281 (2.90), 238 (3.56), 203 (3.52) nm; 1H NMR and 13CNMR data, see Tables 1 and 2; HRFABMS, m/z:337.2373 ([M + H]+) (calcd. for C20H33O4, 337.2379).


3.4.3. ent-2,7-Dioxo-3-cleroden-15-oic acid (3)

Colourless oil; ½a�20D � 10 (c 0.215, CHCl3); IR (CHCl3)

mmax 2931, 2372; 1663, 1458, 1079 cm�1; UV (MeOH) kmax

(loge) 276 (2.70), 229 (3.29), 203 (3.47) nm; 1H NMR and13C NMR data, see Tables 1 and 2; HRCIMS, m/z:335.2224 ([M + H]+) (calcd. for C20H31O4, 335.2222).

3.4.4. ent-18-E-Caffeoyloxy-7b-hydroxy-3-cleroden-15-oic

acid (4)

Colourless oil; ½a�20D � 29 (c 0.165, MeOH); IR (CHCl3)

mmax 2926, 1660, 1610, 1418, 1279, 1165, 1032, 797 cm�1;UV (MeOH) kmax (loge) 329 (4.02), 295 (3.91), 245 (3.81),218 (3.97), 204 (4.02) nm; 1H NMR and 13C NMR data,see Tables 1 and 2; HRFABMS, m/z: 523.2687([M + Na]+) (calcd. for C29H40O7Na, 523.2672).

3.4.5. (13S)-ent-18-E-Coumaroyloxy-8(17)-labden-15-oic

acid (5)

Colourless oil: ½a�20D � 2:4 (c 0.365, MeOH); IR (CHCl3)

mmax 2928, 1663, 1587, 1416, 1158, 1024 cm�1; UV (MeOH)kmax (loge) 312 (3.70), 296 (3.69), 226 (3.58), 203 (3.83) nm;1H NMR and 13C NMR data, see Tables 1 and 3; HRC-IMS, m/z: 486.3227 ([M + NH4]+) (calcd. forC29H44O5N, 486.3219).

3.4.6. ent-18-E-Caffeoyloxy-8(17)-labden-15-oic acid (6)

Colourless oil; ½a�20D � 8:9 (c 0.373, MeOH); IR (CHCl3)

mmax 2371, 1640, 1270, 1165, 1038 cm�1; UV (MeOH) kmax

(loge) 327 (3.93), 295 (3.84), 245 (3.76), 220 (3.96), 203(4.15) nm; 1H NMR and 13C NMR data, see Tables 1and 3; HRFABMS, m/z: 507.2725 ([M + Na]+) (calcd.for C29H40O6Na, 507.2723).

3.4.7. ent-15-E-Caffeoyloxy-8(17)-labden-18-oic acid (7)

Colourless oil; ½a�20D � 21 (c 0.21, MeOH); IR (CHCl3)

mmax 2371, 1659, 1273, 1170, 1026 cm�1; UV (MeOH) kmax

(loge) 328 (3.78), 294 (3.84), 245 (3.64), 220 (3.83), 203(4.00) nm; 1H NMR and 13C NMR data, see Tables 1and 3; HRFABMS, m/z: 507.2739 ([M + Na]+) (calcd.for C29H40O6Na, 507.2723).

3.4.8. ent-15-Hydroxy-8(17)-labden-19-oic acid (13)

Colourless oil; ½a�20D � 23 (c 0.165, CHCl3), no lit. value

available. 1H NMR and 13C NMR spectral data consistentwith the literature values (Zdero et al., 1991a).

3.4.9. ent-18-Hydroxy-8(17)-labden-15-oic acid (14)

Colourless oil; ½a�20D � 34 (c 0.40, CHCl3); lit. ½a�20

D � 37 (c2.3, CHCl3) as methyl ester (Hugel et al., 1966). 1H NMRand 13C NMR spectral data consistent with the literaturevalues (Zdero et al., 1991b).

3.4.10. ent-18-Hydroxy-3-cleroden-15-oic acid (15)

Colourless oil; ½a�20D � 28:4 (c 0.25, CHCl3), no lit. value

available. 1H NMR and 13C NMR spectral data consistentwith the literature values (Tsichritzis and Jakupovic, 1990).

3.4.11. Preparation of the (R)- and (S)-PGME amide

derivatives of 1 and 5(R)- and (S)-phenylglycine methyl ester were obtained

by esterification of phenylglycine (Nagai and Kusumi,1995).

Two portions of compound 1 (each 4 mg; 0.0124 mmol)were separately stirred with (R)-PGME (3.1 mg;0.0155 mmol) and (S)-PGME (3.1 mg, 0.0155 mmol) indry DMF (0.5 ml). To these solutions were added succes-sively 1H-benzotriazol-1-yloxytripyrrolidinophosphoniumhexafluorophosphate (6.5 mg, 0.0155 mmol), 1-hy-droxybenzotriazole (2.4 mg, 0.0155 mmol) and N-meth-ylmorpholine (5 ll) at 0 �C. After stirring at roomtemperature for 5 h, ethyl acetate was added to the reactionmixture which was successively washed with HCl 0.1 N,saturated NaHCO3 solution and brine. The organic layerswere dried over Na2SO4 and concentrated under vacuum.Purification on silica gel column chromatography (cyclo-hexane–EtOAc 75:25) afforded 3 mg of compound 1r and1s in 52% yield. 1H NMR data of the (R)-PGME amidederivative (1r): (400 MHz, CDCl3) d 7.341–7.312 (5H, m,Ar-H), 6.358 (1H, d, J = 7.2 Hz, NH), 5.570 (1H, d,J = 7.2 Hz, H-a), 5.113 (1H, d, J = 1.4 Hz, H-3), 3.971(1H, q, J = 3.3 Hz, H-7), 3.708 (3H, s, CO2CH3), 2.249(1H, dd, J = 5.7, 13.9 Hz, H-14a), 2.043 (1H, dd, J = 2.7,14.2 Hz, H-6a), 1.952 (1H, m, H-2a), 1.930 (1H, dd,J = 5.3, 13.9 Hz, H-14b), 1.872 (1H, m, H-2b), 1.806 (1H,m, H-13), 1.590 (3H, d, J = 1.3 Hz, CH3-18), 1.476 (3H,m, 2H-1, H-8), 1.312 (2H, m, H-6b, H-10), 1.255 (3H, s,CH3-19), 1.233 (2H, m, 2H-11), 1.072 (1H, m, H-12a),0.953 (3H, d, J = 7.3 Hz, CH3-17), 0.949 (4H, br s, H-12b, CH3-20), 0.859 (3H, d, J = 6.5 Hz, CH3-16); 1HNMR data of the (S)-PGME amide derivative (1s):(400 MHz, CDCl3) d 7.344–7.320 (5H, m, Ar-H), 6.327(1H, d, J = 7.1 Hz, NH), 5.570 (1H, d, J = 7.1 Hz, H-a),5.099 (1H, d, J = 1.4 Hz, H-3), 3.957 (1H, q, J = 3.3 Hz,H-7), 3.713 (3H, s, CO2CH3), 2.213 (1H, dd, J = 5.7,14.1 Hz, H-14a), 2.037 (1H, dd, J = 2.7, 14.1 Hz, H-6a),1.952 (3H, m, H-2a, H-14b), 1.864 (1H, m, H-2b), 1.824(1H, m, H-13), 1.572 (3H, d, J = 1.2 Hz, CH3-18), 1.485(2H, m, 2H-1), 1.476 (1H, m, H-8), 1.313 (2H, m, H-6b,H-10), 1.248 (3H, s, CH3-19), 1.212 (2H, m, 2H-11),1.051 (1H, m, H-12a), 0.934 (7H, m, H-12b, CH3-17,CH3-20), 0.917 (3H, d, J = 6.6 Hz, CH3-16).

3.4.12. Preparation of the (R)- and (S)-PGME amidederivatives of 5

Compound 5 (each 6 mg) was condensed with (R)- and(S)-PGME under the same conditions above to yield afterpurification on silica gel column chromatography (cyclo-hexane–EtOAc 80:20) 3 mg of 5r and 5s. 1H NMR dataof the (R)-PGME amide derivative (5r): (400 MHz, CDCl3)d 7.355–7.326 (5H, m, Ar-H), 7.273 (2H, d, J = 8.4 Hz, H-2 0, H-6 0), 6.939 (1H, d, J = 12.4 Hz, H-7 0), 6.774 (2H, d,J = 8.6 Hz, H-3 0, H-5 0), 6.517 (1H, d, J = 6.8 Hz, NH),5.768 (1H, d, J = 12.4 Hz, H-8 0), 5.567 (1H, d,J = 7.0 Hz, H-a), 4.713 (1H, br s, H-17a), 4.363 (1H, br s,


H-17b), 3.795 (1H, d, J = 11.4 Hz, H-18a), 3.716 (3H, s,CO2CH3), 3.553 (1H, d, J = 11.4 Hz, H-18b), 2.199 (1H,m, H-7a), 2.152 (2H, m, 2H-14), 1.855 (1H, m, H-13),1.630 (1H, m, H-7b), 1.579 (1H, m, H-1a), 1.480 (1H, m,H-3a), 1.420 (2H, m, 2H-2), 1.350 (1H, m, H-6a), 1.257(2H, m, 2H-11), 1.176 (1H, m, H-6b), 1.154 (3H, m, H-9,2H-12), 1.132 (1H, m, H-3b), 0.896 (1H, m, H-1b), 0.876(1H, m, H-5), 0.860 (3H, d, J = 6.5 Hz, CH3-16), 0.686(3H, s, CH3-19), 0.563 (3H, s, CH3-20); 1H NMR data ofthe (S)-PGME amide derivative (5s): (400 MHz, CDCl3)d 7.360–7.321 (5H, m, Ar-H), 7.113 (2H, d, J = 8.3 Hz,H-2 0, H-6 0), 6.922 (1H, d, J = 12.5 Hz, H-7 0), 6.534 (2H,d, J = 8.6 Hz, H-3 0, H-5 0), 6.647 (1H, d, J = 6.7 Hz, NH),5.754 (1H, d, J = 12.5 Hz, H-8 0), 5.580 (1H, d,J = 6.8 Hz, H-a), 4.691 (1H, br s, H-17a), 4.328 (1H, br

s, H-17b), 3.754 (1H, d, J = 11.4 Hz, H-18a), 3.722 (3H,s, CO2CH3), 3.539 (1H, d, J = 11.4 Hz, H-18b), 2.314(1H, m, H-14a), 2.277 (1H, m, H-14b), 2.125 (2H, m, 2H-7), 1.873 (1H, m, H-13), 1.554 (2H, m, 2H-1), 1.482 (3H,m, 2H-2, H-3a), 1.267 (2H, m, 2H-6), 1.233 (2H, m, 2H-11), 1.089 (2H, m, 2H-12), 1.033 (1H, m, H-9), 0.913 (3H,d, J = 6.5 Hz, CH3-16), 0.809 (1H, m, H-5), 0.669 (3H, s,CH3-19), 0.548 (3H, s, CH3-20).

Acknowledgment

This work was supported by a grant from the pro-gramme VIH-Pal, Ministere Francais de la Recherche.

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Rings B,D-seco limonoids from the leaves of Swietenia mahogani

Samir A.M. Abdelgaleil a, Matsumi Doe b, Yoshiki Morimoto b, Munehiro Nakatani c,*

a Department of Pesticide Chemistry, Faculty of Agriculture, Alexandria University, Alexandria, Egyptb Analytical Division, Graduate School of Science, Osaka City University, 3-3-7 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan

c Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065, Japan

Received 17 March 2005; received in revised form 6 October 2005Available online 26 January 2006

Abstract

Seven phragmalin limonoids of swietephragmins A-G, and two other different types of 2-hydroxy-3-O-tigloylswietenolide and deace-tylsecomahoganin, were isolated along with three known limonoids from the leaves of Swietenia mahogani (Meliaceae). Their structureswere determined by spectroscopic methods.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Swietenia mahogani; Meliaceae; Phragmalins; Swietephragmins A-G; Mexicanolide; 2-Hydroxy-3-O-tigloylswietenolide; Rings B,D-secolimonoid; Deacetylsecomahoganin

1. Introduction

Swietenia mohogani JACQ. is a valuable meliaceous tim-ber tree closely related to the African genus Khaya and oneof the most popular traditional medicines in Africa. Thedecoction of the bark of these mahoganies is extensivelyused as febrifuge, which could be associated with its useas an antimalarial drug (Nagalakshmi et al., 2001). Thisgenus is one of the main sources of rings B,D-seco limo-noids of mexicanolides and phragmalins. Ever since amexicanolide was isolated (Adeoye and Bekoe, 1965),many limonoids having bicyclo[3,3,1] and tricyclo[3.3.1.1]ring systems have been reported (Taylor, 1984).

In our continuing search for limonoid antifeedantsfrom the family Meliaceae, we have reported the isolationof ring D opened phragmalin-type limonoids from thestem bark of Swietenia mahogani, collected at Alexandria,Egypt (Saad et al., 2003). A subsequent study of the leavesisolated seven new phragmalins possessing an orthoestergroup at 8,9,30-position, named swietephragmins A (1)–

F (6) and G (9), together with two new different type ringsB,D-seco limonoids, 2-hydroxy-3-O-tigloylswietenolide (7)and deacetylsecomahoganin (8), along with three knownlimonoids of methyl 6-hydroxyangolensate (10) (Adesoganand Taylor, 1968), swietemahonin G (11) (Kadota et al.,1990b) and 7-deacetoxy-7-oxogedunin (12) (Kadotaet al., 1990a). We describe herein the isolation, and struc-tural elucidation of these new limonoids. The antifeedantactivity of the isolated compounds was also describedbriefly.


After partition with hexane and methylene chloride ofthe ether extract of the leaves dissolved in H2O–MeOH(1:1), the methylene chloride layer was subjected to chro-matographic separation using SiO2 with MeOH–CH2Cl2as an eluant system, with the resulting limonoid fractiondivided into four fractions by SiO2 rechromatography withhexane–AcOEt (1:1) for elution. The first limonoid fractionwas purified by a combination of TLC and reversed phaseHPLC to give nine new compounds, 1–9, and three knowncompounds, 10–12.


doi:10.1016/j.phytochem.2005.11.020

* Corresponding author. Tel.: +81 99 285 8114; fax: +81 99 285 8117.E-mail address: [email protected] (M. Nakatani).



PHYTOCHEMISTRY


O

OOH

MeO2C

OR

OR

OTig

O

O

O

O

O

MeO2C

ROTig

H OH

H OO

O

O

O

HO

CO2MeCH2ORO

H

3

7

2

2

6

3

1: 2

1

2

2: R = Ac, R = H, R = CH(CH )CH CH3 R = Ac, R = H, R = CH(CH )

18

2

3

2

20

3

4

1

1

12

23

1911

23

4: R = H, R = H, R = CH(CH )

1 2

13

21

3: R = H, R = H, R = CH(CH )CH CH1

3

13

28

22

29

31

3

2

2

5: R = H, R = OH, R = CH(CH )CH CH3

2

26: R = H, R = H, R = CH CH

Tig =

9: R = H, R = H, R = CH

16

1 2

2

98

1 30

1415

3 2

17

1

15

5

6

6

9

9

8

8

7

7

5

30

30

RH

3

3

3

3

3

3

3

3

O

3

15

7: R = OH13: R = H

8: R = H14: R = Ac15: R= β-D-glu

Swietephragmin A (1) was found to possess a molecularformula of C38H46O13 (16 unsaturations) as determinedfrom the HRFAB-MS (m/z: 711.3009[M + 1]+, D�0.8 mmu) and 13C NMR spectrum. The IR spectrumrevealed absorption bands for hydroxyl (3600–3200 cm�1)saturated (1740 cm�1) and unsaturated ester carbonyl(1724 and 1715 sh cm�1) groups. The UV spectrum alsoindicated the presence of an a,b-unsaturated ester groupat 215 nm. From the 1H and 13C NMR spectra, it was clearthat eight of the elements of unsaturation were present asdouble bonds: four carbon–carbon double bonds (onefuran ring) and four CO (as esters). Therefore, the moleculeis octacyclic. The presence of a b-furyl group was recog-nized together with each one of acetyl, tigloyl, methoxyand hydroxyl groups.

All protons directly bonded with carbon atoms wereassigned by analysis of the HMQC spectrum. From thesubsequent 2D NMR spectroscopic studies of the 1H–1H

COSY, HMBC and NOESY spectra, it was strongly sug-gested that 1 was a phragmalin limonoid (Tables 1 and2). Thus, a characteristic low-field singlet at d 5.71 due toH-17 was observed and the H2-6 protons at d 2.34 (dd,J = 16.4 and 11.7 Hz) and 2.40 (br d, J = 16.4 Hz),attached to a methylene carbon adjacent to an ester car-bonyl, were coupled with the H-5 broad doublet protonat d 2.48 (br d, J = 11.7 Hz). These observations stronglysuggested that 1 was a rings B,D-seco limonoid. In additionto this knowledge, the absence of two tertiary methyl sig-nals due to 4~b-Me (Me-29) and 8b-Me (Me-30) groups inthe basic limonoid skeleton, and the presence of two pro-ton resonances at d 1.73 and 1.94 (each d, J = 11.5 Hz)assigned to the 29-methylene group, strongly supportedthat 1 had a tricyclo [3.3.12,10.11,4] decane ring system.The presence of an orthoester group in 1 was also pre-sumed from the characteristic orthocarbon resonance of d122.9. Almost all of the phragmalins isolated so far havebeen reported to be 1,8,9- or 8,9,14-orthoacetates exceptfor some exceptions (Olmo et al., 1997; Nakatani et al.,2001; Saad et al., 2004), and their orthocarbon signals havebeen observed around d 119 (Taylor, 1984).

The 1H and 13C NMR spectra due to the tricyclodecanering of 1 were similar to those of swietenialide A (Saadet al., 2003) isolated from the stem bark of the same plant.All of the carbons due to the ring system, including Me-19and 28 were, respectively, assigned by long-range C–H cor-relations (Fig. 1) of a broad doublet (J = 11.7 Hz, H-5) andtwo methine singlets at d 5.34 (H-3) and 5.44 (H-30) withthe corresponding carbon signals. A W-type long rangecoupling between the H-5 signal and one (Pro-S) of29-methylene signals observed at d 1.73 and the NOE cor-relation of the other H-29 signal at d 1.94 (Pro-R) withthe 10a-Me (Me-19), confirmed the ring structure and theirrelative stereochemistry. The presence of 3-tigloyl and 1-OH groups was elucidated by HMBC correlations of theH-3 and OH signals with the tigloyl carbonyl carbon andC-1 signals, respectively. On the other hand, compound 1showed the presence of one trisubstituted double bond atdC151.8 (s, C-14) and 123.4 (d, C-15) conjugated with a lac-tone carbonyl. An olefinic proton at d 6.32 (s, H-15) corre-lating to the carbon at 163.6 (C-16) in the HMBC spectrum,showed additional correlations with two quaternary car-bons of C-8 and C-13. In the HMBC correlations(Fig. 1), the H2-11, H2-12 and H-17 signals correlated withC-8, C-9 and C-11–C-17 resonances, and characterized thesecond fragment of C and D rings including 13-Me (Me-18)and a furan ring. Finally, the orthoester moiety, identifiedas an isobutylate group, was located at the positions8,9,30 by the HMBC correlation of the H-30 resonancewith the orthocarbon signal at d 122.9 and a considerationof the molecular model. This was supported further by theNOEs of the acetyl signal of 2-OAc with the H-3 and H-30signals.

Stereochemistry of 1 was elucidated by the considerationof NOE correlations (Fig. 2) using a molecular model.Strong cross-peaks of H-5 with H-12b and H-17, and

S.A.M. Abdelgaleil et al. / Phytochemistry 67 (2006) 452–458 453

Table 11H NMR spectroscopic data for swietephragmins A–F (1–6) and G (9)

No. 1 2 3 4 5 6 9

3 5.34 s 5.30 s 4.83 s 4.83 s 4.73 s 4.83 s 4.83 s

5 2.48 br d (11.7) 2.48 br d (11.5) 2.55 br d (11.5) 2.55 br d (11.6) 2.63 br s 2.55 br d (11.5) 2.54 br d (11.4)6 2.34 dd (16.4, 11.7) 2.34 dd (16.5, 11.7) 2.38 dd (16.6, 11.5) 2.38 dd (16.6, 11.6) 4.57 br s 2.38 dd (16.5, 11.5) 2.38 dd (16.6, 11.4)

2.40 br d (16.4) 2.40 br d (16.3) 2.43 dd (16.6, 2.0) 2.44 dd (16.6, 1.8) 2.43 br d (16.5) 2.43 br d (16.6)11a 1.87 ddd (15.8, 15.0, 4.0) 1.87 dt (4.2, 15.4) 1.86 m 1.85 dt (3.9, 14.9) 1.89 dt (3.9, 15.5) 1.89 dt (3.7, 14.7) 1.91 dt (3.6, 14.7)11b 2.17 dt (15.0, 3.5) 2.11 dt (15.0, 3.0) 2.08 dt (14.9, 3.2) 2.08 dt (14.9, 3.4) 2.21 dt (14.9, 3.2) 2.09 dt (14.9, 1.9) 2.10 dt (14.9, 2.0)12a 1.16 dt (14.1, 3.5) 1.16 dt (14.1, 3.7) 1.22 dt (14.3, 3.6) 1.13 dt (14.5, 3.7) 1.21 dt (13.8, 3.3) 1.13 ddd (14.3, 3.7, 1.9) 1.13 dt (14.3, 3.0)12b 1.57 ddd (15.8, 14.1, 3.2) 1.56 dt (3.7, 14.1) 1.51 br dt (2.7, 14.7) 1.51 br dt (3.6, 14.8) 1.58 br dt (2.9, 14.5) 1.50 br dt (2.0, 14.5) 1.50 br t (14.5)15a 6.32 s 6.39 s 5.97 s 5.97 s 5.94 s 5.97 s 5.97 s

17 5.71 s 5.71 s 5.68 s 5.68 s 5.52 s 5.69 s 5.69 s

18 1.36 s 1.35 s 1.33 s 1.33 s 1.29 s 1.34 s 1.35 s

19 1.30 s 1.29 s 1.28 s 1.29 s 1.54 s 1.28 s 1.28 s

21 7.48 br s 7.48 br s 7.47 br s 7.47 br s 7.47 br s 7.47 br s 7.47 br s

22 6.46 br d (1.1) 6.46 br d (1.1) 6.44 br d (1.4) 6.44 br d (1.1) 6.40 br s 6.44 br s 6.44 br s

23 7.42 br t (1.5) 7.42 br t (1.3) 7.42 t (1.5) 7.41 t (1.5) 7.43 br s 7.41 br s 7.41 br s

28 0.68 s 0.73 s 0.82 s 0.82 s 0.95 s 0.82 s 0.82 s

29pro-R 1.94 d (11.5) 1.94 d (11.6) 1.82 d (11.4) 1.83 d (11.1) 2.27 d (10.7) 1.83 d (11.3) 1.83 d (11.5)29pro-S 1.73 br d (11.5) 1.72 br d (11.6) 1.78 br d (11.4) 1.78 br d (11.1) 1.73 d (10.7) 1.78 br d (11.3) 1.78 br d (11.5)30 5.44 s 5.43 s 4.49 s 4.50 s 4.47 s 4.51 s 4.51 s

OMe 3.68 s 3.68 s 3.72 s 3.72 s 3.85 s 3.72 s 3.72 s

2-OAc 2.19 s 2.19 s 2.17 s

1-OH 3.44 s 3.45 s 3.49 s 3.49 s 3.53 s 3.50 s 3.50 s

2-OH 3.55 s 3.56 s 3.57 s 3.56 s 3.56 s

6-OH 2.80 s

Tigloyl

30 6.62 qq (6.9, 1.4) 6.62 qq (6.9, 1.4) 6.91 qq (7.1, 1.2) 6.91 qq (7.0, 1.3) 6.75 br q (7.0) 6.91 br q (7.0) 6.91 br q (6.8)40 1.71 br d (6.9) 1.71 br d (7.0) 1.75 br d (7.1) 1.75 dq (7.0, 0.9) 1.74 br d (6.9) 1.75 br d (7.0) 1.75 br d (6.8)20-Me 1.87 br s 1.87 br s 1.85 br s 1.85 br s 1.85 br s 1.85 br s 1.85 br s

Orthoesters

20 0 2.19 quint (6.9) 1.94 m 1.92 m 2.17 quint (7.0) 1.93 m 1.96 dq (14.5, 7.6) 1.67 s

1.93 dq (14.5, 7.6)300-a 1.04 d (6.9) 1.24 m 1.22 dq (9.6, 7.5) 1.04 d (7.0) 1.23 m 1.03 t (7.6)300-b 1.68 m 1.71 m 1.71 m

400 0.93 t (7.5) 0.93 t (7.5) 0.93 t (7.5)200-Me 1.04 d (6.9) 1.02 d (6.9) 1.02 d (6.9) 1.04 d (7.0) 1.02 d (6.9)

All spectra were measured in CDCl3 at 600 MHz. Chemical shifts are expressed in ppm. J values in parentheses are in Hz.

454S

.A.M

.A

bd

elga

leilet

al.

/P

hy

toch

emistry

67

(2

00

6)

45

2–

45

8

H-12b with H-17 in the NOESY spectrum indicated a borientation for these three protons. In addition to across-peak between H-30 and H-15, a NOE correlationof H-11b with H3-19 (10a-Me) indicated that 1 was presentin a folded conformation containing a quasi chair C ring asshown in Fig. 2. The NOE observation of 2-OAc with H-3and H-30 also supported this structure assignment. Thus,swietephragmin A was identified as the 8,9,30-ortho-isobu-tylate (1) of methyl 2-acetoxy-3b-tigloyloxy-1,8,9,30-tetra-hydroxy-[3.3. 12,10. 11,4]-tricyclomeliac-16,17-lactonic-14(15)-en-7-oate.

Swietephragmin B (2) was obtained as a white amor-phous powder. The molecular formula (C39H48O13) wasdetermined by the HRFAB-MS (m/z: 725.3154 [M + 1]+ ,D �1.9 mmu) and NMR spectra, which suggested the pres-ence of one additional –CH2– unit in 2 compared to 1. TheIR and NMR (Tables 1 and 2) spectroscopic data of 2 wereextremely similar to those of 1, with the only differencebeing observed in the change of the orthoester group to a2-methylbutanoate moiety in 2. The 8,9,30-orthoesterbonding in 2 was also confirmed by both the similarHMBC correlation between the H-30 signal and the ortho-ester carbon resonance observed at d 123.0, as well as theNOEs of the 2-OAc signal with the 1-OH and H-3resonances.

Swietephragmin C (3) exhibited the molecular formulaof C37H46O12 by HRFAB-MS (m/z: 683.3059 [M + 1]+, D�0.8 mmu) and NMR spectroscopic data. The NMRspectra were similar to those of 2 including the presenceof tigloyl and 2-methylbutanoyl orthoester groups, exceptfor the change of an acetoxy group to a hydroxyl group.In the HMBC spectrum, two OH groups were correlatedto C-1, C-2 and C-29 and to C-1, C-2 and C-3, respec-tively, to elucidate clearly the structure having the8,9,30-orthoester group. Although the NOE correlationsin 3 resembled those in 1 and 2 to suggest not so largeconformation change in 3, the H-30 signal was observed

Table 213C NMR spectroscopic data for swietephragmis A–F (1–6) and G (9)

No. 1 2 3 4 5 6 9

1 84.7 84.6 84.6 84.6 84.6 84.7 84.62 84.0 84.0 75.7 75.7 75.6 75.7 75.73 84.8 84.7 86.6 86.6 87.5 86.7 86.74 44.7 44.7 43.7 43.7 43.4 43.7 43.85 39.6 39.6 40.1 40.1 45.5 40.1 40.26 33.7 33.7 33.7 33.7 71.6 33.7 33.87 173.9 173.9 173.9 173.9 174.5 173.9 173.68 83.9 83.7 83.6 83.8 83.6 83.8 84.09 85.7 85.7 86.6 86.7 87.0 86.9 87.210 48.1 48.0 47.2 47.2 48.4 47.3 47.311 26.9 26.0 25.7 25.7 25.6 25.9 26.212 29.3 29.3 29.0 29.0 29.4 29.0 29.213 37.7 37.7 37.7 37.7 37.8 37.7 37.914 151.8 151.8 153.1 153.0 153.1 153.0 152.715 123.4 123.4 122.5 122.5 122.1 122.6 122.516 163.6 163.6 163.2 163.2 162.7 163.1 162.817 79.8 79.8 79.7 79.7 80.0 79.7 79.718 19.2 19.2 19.8 19.8 19.8 19.8 20.119 15.4 15.4 15.5 15.4 17.4 15.4 15.620 119.9 119.9 119.8 119.8 119.6 119.8 119.421 142.0 142.0 142.0 142.0 141.4 142.1 141.922 110.2 110.2 110.1 110.1 109.8 110.1 110.023 143.3 143.2 143.2 143.2 143.2 143.2 143.128 13.8 13.8 14.4 14.4 15.6 14.4 14.629 39.0 39.0 38.5 38.5 39.8 38.6 38.730 73.9 73.7 77.9 78.0 77.7 78.1 78.1OMe 52.1 52.1 52.1 52.1 52.3 52.1 52.2OAc 170.3 170.2

21.8 21.8

Tiglate

1 0 167.9 167.9 168.1 168.2 167.7 168.2 167.92 0 130.8 130.8 130.0 130.0 130.2 130.1 129.93 0 136.1 136.1 139.9 139.9 139.0 139.9 139.84 0 13.5 13.5 14.2 14.2 14.4 14.2 14.42 0-Me 12.9 12.8 12.3 12.3 12.5 12.3 12.4

Orthoester

100 122.9 123.0 122.9 122.8 122.5 121.3 119.7200 28.9 35.4 35.5 28.9 35.5 23.2 16.6300 16.6 23.6 23.6 16.6 23.7 7.6400 13.5 11.4 11.6200-Me 16.6 12.8 13.1 16.6 13.3

21

14

11

3

CO2Me

6

Me

O

OAc

Tig

Me

H

Me

H

H

5

H

HH

H

H

H H

R

H

CH2

HO

H

H

H

1

2

4

7

8

910

12

13

15

16

17

18

19

20

22

23

28

29

O30

O

O

1'

O

O

O

Fig. 2. Significant NOE correlations in 1.

O

Me

OOH

MeMeO2C

O

OCOCH3

OTig

Me

O

O

O

H

H

CH(CH3)2

H

HH

HH

H

H

H

HH

H

H

H

H

1 13 CH

Fig. 1. Selected HMBC correlations in 1.


at a high-field of d 4.49 compared to d 5.44 and 5.43 in 1

and 2. This high-field shift was attributed to an aniso-tropic effect of the carbon–oxygen double bond of the3-tigloyl group fixed by a seven membered hydrogenbonding with the 2-OH group.

The molecular formula of swietephragmin D (4) wasdetermined as C36H44O12 by HRFAB-MS (m/z: 669.2914[M + 1]+, D +0.3 mmu) and NMR data. The 1H and 13CNMR spectroscopic data (Tables 1 and 2) were extremelysimilar to those of 3 except for the replacement of 2-meth-ylbutanoyl orthoester in 3 by 2-methylpropanoate in 4.

Swietephragmin E (5) was shown to have the molecularformula C37H46O13 by HRFAB-MS (m/z: 699.3021[M + 1]+, D +0.6 mmu). Although the NMR spectra of5 were also similar to those of 3 and 4, it showed the pres-ence of an additional hydroxyl group. The structure of 5

as a 6-hydroxyl derivative of 3 was readily elucidatedfrom that of a hydroxymethine signal at d 4.57 (H-6)weakly coupled to an OH resonance at d 2.80 and showeda HMBC correlation with an ester carbonyl carbon at d174.5 (C-7). The R configuration at C-6 was inferred fromthat of some mexicanolides, swietenins and swietemaho-nins (Connolly et al., 1965; Kadota et al., 1990a,b; Saadet al., 2004) isolated from the same plant S. mahogani.This was supported by the NOE correlations observedbetween the H-6 signal and the H-5, H-12b and 10-Me(Me-19) resonances, the 6-OH signal and the H-28Pro-S

and 4a-Me (Me-28) resonances, and the 7-carboxymethylsignal and the H-17 and tigloyl 3 0-H and 3 0-Me reso-nances. The latter implied that the 7-CO2Me group wasoriented to the same b-side as H-17 and 3-tigloyl of themolecule. The H-28Pro-S signal showed a W-type longrange coupling with the H-5 signal and a strong NOEwith the H-3a resonance, which also accounted well forthe stereochemistry of 5.

The structure of swietephragmin F (6), C35H42O12;HRFAB-MS (m/z: 655.2736[M + 1]+, D �1.9 mmu) wasreadily deduced from the spectroscopic data. The 1H and13C NMR spectra were very similar to those of compounds3 and 4 except for the change of the orthoester groups to anorthopropyonate moiety in 6.

The molecular formula of 7 (2-hydroxy-3-O-tigloylswi-etenolide) was determined to be C32H40O10 by HRFAB-MS (m/z: 585.2673 [M + 1]+, D �2.6 mmu). The UV andIR spectra showed similar absorption bands to those ofthe phragmalins, (1)–(6), and the NMR spectra (Table 3)suggested a mexicanolide structure for 7. Thus the presenceof four tertiary methyls, due to the basic limonoid skeleton,and one methyl ester moiety was observed along with eachketo and lactonic carbonyl and tigloyl groups and twohydroxyl groups. The 1H NMR spectrum resembled 3-O-tigloylswietenolide (13) isolated from the same species(Kadota et al., 1990) except for the presence of an addi-tional hydroxyl group in 7. The presence of C-8/C-14 dou-ble bond was elucidated by HMBC correlations of theH-15b and H-30 signals with the C-8 and C-14 resonances.An additional OH group at d 4.14 was located in C-2 by

HMBC correlations of the OH signal with the C-1, C-2and C-3 resonances at d 217.2, 77.9 (s) and 87.5 (d). Signif-icant NOE correlations of the H-5 signal with the 4b-Me(28) and the two tigloyl methyl signals and the 10a-Me(19) signal with the H-6 and H-9 resonances clarified therelative stereochemistry of these protons in the dicy-clo[3.3.1]nonane ring system. Finally, the configuration atC-6 was assumed to be the same R as that of known mex-icanolides (Taylor, 1969; Kadota et al., 1990a,b) from thesame specimen.

The molecular formula of compound 8, 6-O-deacetox-ysecomahoganin, was determined to be C27H34O8 byHRFAB-MS (m/z: 487.2327 [M + 1]+, D �0.5 mmu).Compound 8 showed UV and IR absorptions due to a con-jugated enone system at kmax 230 nm and mmax 1680 cm�1

Table 31H and 13C NMR spectroscopic data for compounds 7 and 8

No. 7 8

dH dC dH dC

1 217.2 6.67 d (10.6) 152.62 77.9 5.93 d (10.6) 126.53 4.78 s 87.5 203.64 39.7 44.95 3.30 br s 45.0 1.94 br t (4.7) 51.56 4.54 br s 73.1 3.94 m, 4.11 m 61.17 175.3 176.78 126.4 43.19 2.10 m 53.0 3.32 d (11.1) 43.110 52.5 50.811a 1.80 m 18.8 1.57 m 21.511b 1.90 m 1.42 m

12a 1.19 ddd (15.0, 10.3, 3.1) 29.6 1.45 m 32.512b 1.74 dd (15.0, 3.1) 1.77 m

13 38.2 37.814 132.8 68.215a 3.26 br d (21.3) 33.2 3.67 s 51.415b 3.60 d (21.3)16 169.0 166.917 5.41 s 80.9 5.42 s 78.418 0.99 s 17.7 1.22 s 19.319 1.53 s 17.7 1.19 s 18.220 120.7 120.021 6.40 br s 141.1 7.38 s 141.222 7.41 br s 109.7 6.33 br s 109.923 7.43 br t (1.4) 143.2 7.38 s 143.328 0.84 s 23.3 1.26 s 24.229 1.05 s 22.2 1.07 s 23.230a 1.76 br d (14.8) 44.7 1.25 s 15.130b 3.04 d (14.8)OMe 3.86 s 53.2 3.80 s 53.32-OH 4.14 br s

6-OH 2.81 s 2.16 br s

Tig

10 167.020 129.230 6.91 br q (7.0) 138.740 1.82 br d (7.0) 14.520-Me 1.90 br s 12.4

All spectra were measured in CDCl3 at 600 MHz. Chemical shifts areexpressed in ppm. J value in parentheses are in Hz.

456 S.A.M. Abdelgaleil et al. / Phytochemistry 67 (2006) 452–458

different from the above compounds, and the NMR spec-trum (Tables 3) also revealed several differences. The 1Hand 13C NMR spectroscopic data showed the presenceof six methyls (five tertiary and one methoxy), three meth-ylenes, nine methines (five olefinic), and nine quaternarycarbons (one olefinic and one keto and two ester carbo-nyls). Thus 9 is tetracyclic. The 1H NMR spectrumshowed a characteristic H-15 epoxide proton as a singletat 3.67 and five methyls due to the basic limonoid skeletonat d 1.07, 1.19, 1.22, 1.25 and 1.26. The presence of thelactonic D ring was also confirmed by the characteristicH-17 singlet at d 5.42. A conjugated enone system at dH

6.67 and 5.93 (each d, J = 10.6 Hz); dC152.6 (d), 126.5(d) and 203.6, was assigned to ring A by analysis of theHMBC correlations. The H-1 signal at d 6.67 showed cor-relations with the C-3, C-5, C-10 and C-19 signals. On theother hand, the HMBC and NOESY spectra clarified thatring B was opened at C6–C7. Thus, the HMBC correla-tions of the H-9 signal with the C-1, C-5, C-7, C-19 andC-30 resonances, and the NOE correlations of the H-9 sig-nal with the H-5 and H-19 resonances, the H-1 signal withthe H-11b and Me-30 resonances, and the H-5 signal withthe H-11a resonance, suggested that rings A and C in themolecule were twisted about 90� through the C9–C10

bonding in a preferential conformer. These data clarified9 to have the same aglycone moiety as secomahoganin(12) (Kadota et al., 1990b) and khayanoside (13) (Naka-tani et al., 2002).

The structure of the last compound, swietephragmin G(9), C34H40O12; HRFAB-MS (m/z: 641.2604 [M + 1]+, D0.6 mmu) was readily elucidated from analysis of the spec-troscopic data. The 1H and 13C NMR spectra were verysimilar to those of compounds 4, 5 and 7 except for theorthoester group being orthoacetate in 9.

The antifeedant activity of the isolated compounds (1–12) was tested by a conventional leaf disk method (Wadaand Munakata, 1968) against the third-instar larvae ofSpodoptera littoralis (Boisd.). All of the compounds exceptfor 7-deacetoxy-7-oxogedunin (12) were active at1000 ppm, corresponding to a concentration of ca. 20 lg/leaf-cm2, in which swiemahonin G (11) was most activeand swietephragmins 1–6 and 9, showed moderate activi-ties. Details will be reported together with another biolog-ical activities of cytotoxity and antiviral activity againstHIV-1 replication in the near future.

3. Experimental

3.1. General

1H and 13C NMR spectra were measured at 600 and150 MHz in CDCl3 on JEOL FX-600 spectrophotometer.IR (KBr) and UV (MeOH) spectra were recorded onJASCO FT/IR 5300 and Shimadzu UV-210A spectropho-tometers. HPLC were performed on Waters lBondapakC18 column by using 35–65% H2O/MeOH as solvent.

3.2. Plant material

The leaves of S. mahogani were collected in April 2001 atAlexandria in Egypt. The plant material was identified byDr. Khaleil Darweish of Alexandria University and a vou-cher specimen is deposited in the Faculty of Agriculture,Alexandria University.

3.3. Extraction and isolation of compounds 1–6

Air-dried leaves of S. mahogani (1.9 kg) were extractedwith Et2O (15 l) at room temperature for four weeks to givea crude extract (102.2 g). The Et2O extract was suspendedin 1 l of H2O–MeOH (1:2), fractionated successfully withhexane (3 · 500 ml) and CH2Cl2(3 · 500 ml) to give hexane(67.7 g) and CH2Cl2 (32.3 g) extract. The CH2Cl2 extract(10 g) was subjected to SiO2 (500 g) cc with a 0–10%MeOH/CH2Cl2 gradient eluent to give 50 fractions. Thelimonoid fractions of 18–41 eluted with 2.5% MeOH/CH2Cl2 were further applied to SiO2 (150 g) with hexane/AcOEt (1:1) to give 50 fractions, which were combined asneeded to give three limonoid and two non-limonoid frac-tions. The first fraction (1.4 g) was roughly separated into13 fractions through HPLC with 25% H2O/MeOH as sol-vent, followed by TLC separation with 3% MeOH/CH2Cl2and HPLC purification with 20–30% H2O/MeOH to give 1

(31.5 mg), 2 (40 mg), 3 (16 mg), 4 (12.5 mg), 5 (14.5 mg), 6

(9.5 mg), 7 (5 mg), 8 (5.5 mg), 9 (6 mg), 10 (15 mg), 11

(12 mg) and 12 (0.5 mg).

3.3.1. Swietephragmin A (1)

White amorphous powder; C38H46O13; HRFAB-MS m/z:

711.3009 [M + 1]+ (calc. 711.3017); UV kmax nm (e): 215(16,000); IR mmax cm�1: 3600–3200, 1740, 1724, 1715 sh,1635; for 1H and 13C NMR spectroscopic data, see Tables1 and 2.

3.3.2. Swietephragmin B (2)White amorphous powder; C39H48O13;HRFAB-MS

m/z: 725.3154 [M + 1]+ (calc. 725.3173); UV kmax nm(e): 215 (14,000); IR mmax cm�1: 3600–3300, 1740–1710;for 1H and 13C NMR spectroscopic data, see Tables 1and 2.

3.3.3. Swietephragmin C (3)

White amorphous powder; C37H46O12;HRFAB-MSm/z: 683.3059 [M + 1]+ (calc. 683.3067); UV kmax nm(e): 215 (14,000); IR mmax cm�1: 3550–3200, 1735–1710;for 1H and 13C NMR spectroscopic data, see Tables 1and 2.

3.3.4. Swietephragmin D (4)



3.3.5. Swietephragmin E (5)


3.3.6. Swietephragmin F (6)

White amorphous powder; C35H42O12; HRFAB-MSm/z: 655.2736 [M + 1]+ (calc. 655.2755); UV kmax nm (e):215 (16,000); IR mmax cm�1: 3600–3300, 1740–1710; for1H and 13C NMR spectroscopic data, see Tables 1 and 2.

3.3.7. 3-O-Tigloylswietenolide (7)White amorphous powder; C32H40O10;HRFAB-MS

m/z: 585.2673 [M + 1]+ (calc. 585.2699); UV kmax nm (e):215 (12,000); IR mmax cm�1: 3500–3200, 1745–1710; for1H and 13C NMR spectroscopic data, see Tables 3.

3.3.8. 6-O-Deacetylsecomahoganin (8)

White amorphous powder; C27H34O8;HRFAB-MSm/z: 487.2327 [M + 1]+ (calc. 487.2332); UV kmax nm(e): 230 (12,000); IR mmax cm�1: 3500–3300, 1740, 1720,1680; for 1H and 13C NMR spectroscopic data, seeTables 3.

3.3.9. Swietephragmin G (9)


3.4. Antifeedant test

The antifeeding potential of the isolated compounds wastested three times by a conventional leaf disk methodagainst thrid-instar larvae of S. littoralis. Five leaf disks(diameter 1.2 cm) of Chinese cabbage (Brassica campestris

var. chinensis) were immersed in an acetone solution of thesample for 2 s. The treated disks were arranged alterna-tively with another five control disks (immersed only inacetone) close to the wall of a Petri dish. Ten larvae wereplaced in the centre of each Petri dish. The eaten areas oftreated and untreated leaf disks were evaluated at appro-priate intervals for 3–10 h. The experiment was terminated

after the larvae had eaten approximately 50% of one of thedisks.

Acknowledgements

We are grateful to Mr. K. Takezaki, Kagoshima Prefec-tural Agricultural station, for the supply of the insects.

References

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N.S., 1965. Tetranortriterpenoids Part I. The constitution of swiete-nine. J. Chem. Soc., 6935–6939.

Kadota, S., Marpaung, L., Kikuchi, T., Ekimoto, H., 1990a. Constituentsof the seeds of Swietenia mahogani JACQ.I. Isolation, structures, and1H- and 13C-nuclear magnetic resonance signal assignments of newtetranortriterpenoids related to swietenine and swietenolides. Chem.Pharm. Bull. 38, 639–651.

Kadota, S., Marpaung, L., Kikuchi, T., Ekimoto, H., 1990b. Constituentsof the seeds of Swietenia mahogani JACQ. II. Structures of swietema-hoganin A, B, C, D, E, F, and G and swietemahonolide. Chem.Pharm. Bull. 38, 894–901.

Nagalakshmi, M.A.H., Thangadurai, D., Muralidara, D., Pullaiah, R.T.,2001. Phytochemical and antimicrobial study of Chukrasia tabularis

leaves. Fitoterapia 72, 62–64.Nakatani, M., Abdelgaleil, S.A.M., Kurawaki, J., Okamura, H., Iwagawa,

T., Doe, M., 2001. Antifeedant rings B and D opened limonoids fromKhaya senegalensis. J. Nat. Prod. 64, 1261–1265.

Nakatani, M., Abdelgaleil, S.A.M., Kassem, Sh.M.I., Takezaki, K.,Okamura, H., Iwagawa, T., Doe, M., 2002. Three new modifiedlimonoids from Khaya senegalensis. J. Nat. Prod. 65, 1219–1221.

Olmo, L.R.V., da Silva, M.F.G.F., Fo, E.R., Vieira, P.C., Fernandes, J.B.,Pinheiro, A.L., Vilela, E.F., 1997. Limonoids from the leaves of Khaya

senegalensis. Phytochemistry 44, 1157–1165.Saad, M.M.G., Iwagawa, T., Doe, M., Nakatani, M., 2003. Swietenia-

lides, novel ring D opened phragmalin limonoid orthoesters fromSwietenia mahogani JACQ. Tetrahedron 59, 8027–8033.

Saad, M.M.G., Iwagawa, T., Okamura, H., Doe, M., Nakatani, M., 2004.Three new mexicanolides from the stem bark of Swietenia mahogani

JACQ. Heterocycles 63, 389–399.Taylor, D.A.H., 1969. Extractives from Swietenia mahogani (L) Jacq. J.

Chem. Soc. Chem. Commun., 58–59.Taylor, D.A.H., 1984. In: Herz, W., Grisebach, H., Kirby, G.W. (Eds.),

Progress in the Chemistry of Organic Natural Products. Springer, NewYork, pp. 1–102.

Wada, K., Munakata, K., 1968. Naturally occurring insect controlchemicals. J. Agr. Food Chem. 16, 471–474.

458 S.A.M. Abdelgaleil et al. / Phytochemistry 67 (2006) 452–458

Flavones and isoflavones from the west AfricanFabaceae Erythrina vogelii

Alain F. Kamdem Waffo a,b,c, Philip H. Coombes a, Dulcie A. Mulholland a,d,*

Augustin E. Nkengfack c, Zacharias T. Fomum c

a Natural Products Research Group, School of Chemistry, University of KwaZulu-Natal, Howard College Campus, 4041, Durban, South Africab Department of Chemistry, Faculty of Science, University of Douala, P.O. Box 24157 Douala, Cameroon

c Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812 Yaounde, Cameroond School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK

Received 18 August 2005; received in revised form 20 September 2005Available online 17 November 2005

Abstract

The CH2Cl2/MeOH extract of the stem bark of Erythrina vogelii (Fabaceae) from Nigeria has yielded two novel isoflavones, 7,4 0-dihy-droxy-8-(c,c-dimethylallyl)-200n-(400-hydroxyisopropyl)dihydrofurano[100,300:5,6]isoflavone (vogelin H) (1) and 7,4 0-dihydroxy-8-[(2000n,3000-dihydroxy-3000-methyl)butyl]-200,200-dimethyl-300,400-dehydropyrano[100,400:5,6]isoflavone (vogelin I) (2), a novel flavone, 7,4 0-dihy-droxy-200,200-dimethyl-300,400-dehydropyrano[100,400:5,6]flavone (vogelin J) (3), and eight known flavonoids.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Erythrina vogelii; Fabaceae; Stem bark; Isolation; Isoflavones; Flavones; 7,4 0-Dihydroxy-8-(c,c-dimethylallyl)-200n-(400-hydroxyisopropyl)-dihydrofurano[100,300:5,6]isoflavone (Vogelin H); 7,4 0-Dihydroxy-8-[(2000n,3000-dihydroxy-3000-methyl)butyl]-200,200-dimethyl-300,400-dehydropyrano[100,400:5,6]-isoflavone (Vogelin I); 7,40-Dihydroxy-200,200-dimethyl-300,400-dehydropyrano[100,400:5,6]flavone (Vogelin J); 6,8-Diprenylgenistein; 8-Prenylluteone;Warangalone; Scandenone; Auriculatin; 2,3-Dihydroauriculatin; Carpachromene

1. Introduction

The genus Erythrina L. (Fabaceae), comprising approx-imately 130 species of ‘‘coral trees’’ distributed throughoutthe tropical and subtropical regions of the world, has beenwidely studied. More than 340 extractives have been iso-lated to date, with some 30 of these secondary metabolitesvariously reported to display antimicrobial (Ingham andMarkham, 1980; Kamat et al., 1981; Mitscher et al.,1988a,b; Chacha et al., 2005), antibacterial (Fomumet al., 1986), antifungal (Tahara et al., 1984; Bojase et al.,2001), anti-inflammatory (Wandji et al., 1994; Chachaet al., 2005), antiemetic and antitussive (Abbasoglu et al.,1991), and cytotoxic (Hou et al., 2001) properties, and also

to act as phytoalexins (Dagne et al., 1993) and phospholi-pase A2 inhibitors (Hegde et al., 1997).

Previous studies (Atindehou et al., 2002; Queiroz et al.,2002) on the root bark of Erythrina vogelii Hook. fromIvory Coast have yielded vogelins A–G, seven new ring Bprenylated isoflavonoids, and five known isoflavonoids.


In continuation (Fomum and Ayafor, 1983; KamdemWaffo, 2000; Njamen et al., 2004) of our studies on speciesof this genus, we now report on the isolation of two novelisoflavones and a novel flavone, together with eight knownflavonoids, from the CH2Cl2/MeOH (1:1) extract of thestem bark of E. vogelii collected in Nigeria.

Compound 1 was assigned a molecular formula ofC25H26O6 on the basis of HREIMS data, and showed a


doi:10.1016/j.phytochem.2005.09.022

* Corresponding author. Tel.: +27 31 260 1395; fax: +27 31 260 3091.E-mail addresses: [email protected], [email protected]

(D.A. Mulholland).



PHYTOCHEMISTRY



kmax at 271 nm in the UV spectrum. A 1H singlet resonanceat dH 7.75 and corresponding olefinic oxymethine signal atdC 152.5 are characteristic of H-2 and C-2, respectively, inan isoflavone skeleton (Mabry et al., 1970; Agrawal, 1989).From a pair of coupled doublets at dH 7.28 and 6.83 (each2H, J = 8.5 Hz, H-2 0/H-6 0, H-3 0/H-5 0); dC 130.2 (each CH,C-2 0/6 0), 115.4 (each CH, C-3 0/5 0) was deduced the pres-ence of a para disubstituted ring B, while that of a hydroxylgroup at C-4 0 was established from a peak at m/z 118 in theEIMS. Further inspection of the NMR and mass spectrashowed compound 1 to possess a c,c-dimethylallyl (prenyl)substituent (m/z 379 [M � 43]+ and 367 [M � 55]+; dH 3.29(2H, d, J = 7.3 Hz, 2H-1000), 5.23 (1H, m, H-2000), 1.64 (3H, s,3H-4000), 1.73 (3H, s, 3H-5000); dC 21.8 (CH2, C-1000), 121.4(CH, C-2000), 132.2 (C, C-3000), 17.7 (CH3, C-4000), 25.5(CH3, C-5000)). The presence of a hydroxyisopropyl-dihydrofuran ring, suggested by characteristic peaks inthe EIMS at m/z 363 [M � 59]+ and 59 (Tahara et al.,1984, 1989), was supported by the observation of reso-nances attributable to two non-equivalent geminal methylgroups (dH 1.20/1.30 (each 3H, s, 3H-500/600); dC 25.5/24.0(each CH3, C-500/600)), an oxymethine proton (dH 4.74(1H, d, J = 8.3 Hz, H-200); (dC 91.0 (CH, C-200)), two diaste-reotopic protons (dH 3.20 (2H, br d, J = 8.1 Hz, 2H-300; dC

27.1 (CH2, C-300)) and a fully substituted oxygenated car-bon (dC 72.2 (C, C-400)) (Tahara et al., 1989).

The absence of signals in the region dH 5.7 – 6.1, nor-mally attributed to H-6 and H-8, placed both the prenylgroup and dihydrofuran ring on ring A, while the lack ofboth the downfield singlet at dH 12.0–13.2 and theabsorption band at 3280 cm�1 in the IR spectrum, char-acteristic of a chelated OH group at C-5, suggested thelatter substituent to be fused at C-5/C-6, and thus, giventhe normal biosynthetic requirement of a hydroxy groupat C-7, that the prenyl group be located at C-8. Thisplacement was supported by correlations in the HMBCspectrum (Fig. 1) between both the H-2 and 2H-1000 res-onances and a downfield fully substituted carbon signalat dC 151.8, which can only be C-8a, and by further cor-relations between both 2H-1000and 2H-300 to a seconddownfield fully substituted carbon signal at dC 164.5,which must then be C-7. Correlations between the 2H-300 resonance and fully substituted carbon signals at dC

101.8 and 159.7, assigned to C-6 and C-5, respectively,and between H-200 and both of these, confirmed theseassignments. Compound 1 is thus the novel 7,4 0-dihy-droxy-8-(c,c-dimethylallyl)-200n-(400-hydroxyisopropyl)dihy-drofurano[100,300:5,6]isoflavone, which we name vogelin H,and is regioisomeric with senegalensin, from E. senegal-

ensis DC. (Wandji et al., 1990), euchrenone b10, fromEuchresta horsfieldii (Lesch.) Bennet (Mizuno et al.,1990), and lupinisoflavone G, from Lupinis albus L.(Tahara et al., 1989) and Derris scandens Benth. (Sekineet al., 1999); the structures of the former two com-pounds, transposed when originally reported, wererevised on recent isolation from E. suberosa var glabre-

sence Haines (Tanaka et al., 2001).

In similar fashion, compound 2, assigned the molecularformula C25H26O7 by HREIMS, possesses an isoflavoneskeleton (dH 8.01 (1H, s, H-2); dC 154.2 (CH, C-2)) and apara disubstituted C-4 0-hydroxy ring B (dH 7.32 and 6.84(each 2H, J = 8.6 Hz, H-2 0/6 0, H-3 0/5 0); dC 130.9 (eachCH, C-2 0/6 0), 116.0 (each CH, C-3 0/5 0)). However, the sig-nals of both the prenyl group and dihydrofuran ring invogelin H (1) were absent, having been replaced by thoseattributable to a 200,200-dimethyl-300,400-dehydropyran ring(dH 5.64 (1H, d, J = 10.0 Hz, H-300), 6.67 (1H, d,J = 10.0 Hz, H-400), 1.46 (6H, s, 3H-500/3H-600); dC 78.9(C, C-200), 128.7 (CH, C-300), 116.1 (CH, C-400), 28.5/28.5(each CH3, C-500/C-600)), and a 2n, 3-dihydroxy-3-methylbu-tyl group (dH 3.60 (1H, m, H-2000), 2.88 (1H, m, H-1a000), 2.85(1H, m, H-1b 000), 1.28 (6H, s, 3H-4000/3H-5000); dC 25.5 (CH2,C-1000), 78.6 (CH, C-2000), 73.6 (C, C-3 000), 24.4/26.0 (eachCH3, C-4000/C-5000)) (Takashima and Ohsaki, 2002). As invogelin H (1), the absence of the resonances normallyattributable to H-6 and H-8 placed both of these groupson the A ring. Correlations in the HMBC spectrum(Fig. 1) between both H-2 and 2H-1000and a downfield fullysubstituted carbon resonance at dC 156.4, which must be C-8a, and between 2H-1000and a second downfield fully substi-tuted carbon resonance at dC 158.1, which must then be C-7, place the 2n,3-dihydroxy-3-dimethylbutyl group at C-8.

vogelin H (1)

1

2

4

5''

6

7

4a

8a O

O

HO

OOH

HO

1''

1'''

3''

4''6''

2'''

3''' 5'''4'''

1'

4'

2'

6'2''

3'

5'

vogelin I (2)5''

1''3''

4''

6''

2''

OHO

OOH

OH

OH

O

vogelin J (3)

OHO

OO

OH

(2)

H

OHO

OOH

OH

OH

O

HH

H

(1)

O

O

HO

OOH

HO

H

H H

H

H

H

H

Fig. 1. Selected HMBC correlations in vogelins H (1) and I (2).

460 A.F. Kamdem Waffo et al. / Phytochemistry 67 (2006) 459–463

Further correlations between H-400 and C-7, and betweenH-400 and fully substituted carbon signals at dC 105.5 and155.5, assigned to C-6 and C-5, respectively, locate thedehydropyran ring at C-5/C-6. Compound 2 is thus thenovel 7,4 0-dihydroxy-8-[(2000n,3000-dihydroxy-3000-methyl)bu-tyl]-200,200-dimethyl-300,400-dehydro-pyrano[100,400:-5,6]isoflav-one, which we name vogelin I.

The UV and NMR spectra of vogelin J (3), assigned themolecular formula C20H16O5 from HREIMS data, dis-played kmax peaks at 272, 313 and 330 nm, a 1H singlet res-onance at dH 6.46, and corresponding upfield olefinic signalat dC 103.1 (CH) characteristic of H-3 and C-3, respec-tively, in a flavone skeleton (Mabry et al., 1970; Agrawal,1989). In common with vogelins H (1) and I (2), vogelinJ (3) possesses a para disubstituted 4 0-hydroxy ring B (dH

7.60 and 6.85 (each 2H, J = 8.9 Hz, H-2 0/H-6 0, H-3 0/H-5 0); dC 127.7 (each CH, C-2 0/6 0), 116.2 (each CH, C-3 0/5 0)) and a 200,200-dimethyl-300,400-dehydropyran ring (dH

5.54 (1H, d, J = 10.0 Hz, H-300), 6.62 (1H, d, J = 10.0 Hz,H-400), 1.40 (6H, s, 3H-500/3H-600); dC 78.0 (C, C-200), 127.3(CH, C-300), 115.7 (CH, C-400), 27.9/28.0 (each CH3, C-500/C-600)). The latter was placed at C-5/C-6 when a bathochro-mic shift with NaOAc, but not AlCl3, indicated the pres-ence of a hydroxyl group at C-7 only. Vogelin J (3) isthus 7,4 0-dihydroxy-200,200-dimethyl-300,400-dehydropyr-ano[100,400:5,6]flavone, a novel regioisomer of limonianinfrom Citrus limon [L.] Burn. (Chang, 1990), carpachromenefrom Flindersia laevicarpa C.T.White (Picker et al., 1976;Jain et al., 1978) and yinyanghuo C from Vancouveria hex-

andra (Hook.) C. Morren & Decne (Linuma et al., 1993)and Epimedium sagittatum (Siebold & Zucc.) Maxim.(Chen et al., 1996).

The known compounds were identified as 6-prenylapige-nin (Abegaz et al., 1998), 6,8-diprenylgenistein (Shiratakiet al., 1982), 8-prenylluteone (Nkengfack et al., 1989),warangalone (scandenone) (Nkengfack et al., 1989), auric-ulatin (Shabbir et al., 1968; Subba Raju et al., 1981), 2,3-dihydroauriculatin (Shabbir et al., 1968; Taylor et al.,1986), limonianin (Chang, 1990) and carpachromene (Sar-aswathy et al., 1998) by comparison of their physical prop-erties and spectral data with the literature values.

3. Experimental

3.1. General

Melting points were determined on a Kofler micro-hotstage melting point apparatus and are uncorrected. NMRspectra were recorded at room temperature on a400 MHz Varian UNITY-INOVA spectrometer. Chemicalshifts are expressed in d (ppm) units relative to tetrameth-ylsilane (TMS) as internal standard and coupling constantsare given in Hz. 1H NMR, 13C, HMBC, HSQC andNOESY spectra were recorded in CDCl3 and CD3OD.UV spectra were obtained on a Varian DMS 300 UV–vis-ible spectrometer with MeOH as solvent. IR spectra were

recorded on a Nicolet Impact 400D Fourier-TransformInfrared (FT-IR) spectrometer, using NaCl windows withCHCl3 as solvent against an air background. LREIMSand HREIMS were taken on Perkin–Elmer 6890-Agilent5975 GC–MS and Micromass VG 70 SEQ instruments,respectively. Optical rotations were measured at room tem-perature in CHCl3 on a Perkin–Elmer 341 Polarimeter,using a 100 mm quartz microcell flow tube.

3.2. Plant material

Erythrina vogelii Hook. was collected at Ogbomoso,Nigeria, in May 2003 and identified at the University ofIbadan Herbarium and the Cameroon National Herbar-ium, Yaounde, where a voucher specimen (20693/SRFCam.) is retained for verification purposes.

3.3. Extraction and isolation of compounds

The air-dried, ground stem bark material of E. vogelii

(3 kg) was extracted for 72 h at room temperature withCH2Cl2:MeOH (1:1) and concentrated under reduced pres-sure to give 201.3 g of extract. Repeated combinations ofvacuum liquid and gravity column chromatography onMerck 7729 and 9385 silica gels, and PTLC on aluminiumbacked analytical TLC (Merck 5554) plates, using variousmixtures of hexane:EtOAc:MeOH, afforded vogelins H (1)(4.0 mg), I (2) (4.9 mg) and J (3) (15.2 mg), together with 6-prenylapigenin (6.8 mg), 6,8-diprenylgenistein (9.7 mg), 8-prenylluteone (10.2 mg), warangalone (scandenone)(9.0 mg), auriculatin (10.0 mg), 2,3-dihydroauriculatin(10.1 mg), limonianin (6.7 mg) and carpachromene (6.8 mg).

3.3.1. 7,4 0-Dihydroxy-8-(c,c-dimethylallyl)-200n-(400-

hydroxyisopropyl)dihydrofurano-[100,300:5,6]isoflavone,

vogelin H (1)

Pale yellow powder; m.p. 195–197 �C; ½a�20D ¼ �38� (c,

0.0015 in CHCl3); mmax(NaCl) cm�1 3545, 1642, 1610,1512, 1425, 1382, 1270, 1215, 1172, 1075, 836; HREIMS(70 eV) m/z 422.1720 (calc. for C25H26O6 422.1729); EIMS(70 eV) m/z (rel. int.) 422 (98), 407 (20), 379 (70), 367(100),363 (25), 352 (33), 349 (28), 335 (18), 320 (14), 307 (30), 295(40), 118 (35), 59 (10); kmax (MeOH) nm (log e): 203 (4.48),216 (4.37), 271 (4.48), (MeOH + NaOAc) 275; 1H NMRspectral data (400 MHz, CD3OD) dH 7.75 (1H, s, H-2),7.28 (2H, d, J = 8.5 Hz, H-2 0/H-6 0), 6.83 (2H, d,J = 8.5 Hz, H-3 0/H-5 0), 5.23 (1H, m, H-2000), 4.74 (1H, d,J = 8.3 Hz, H-200), 3.29 (2H, d, J = 7.3 Hz, 2H-1000), 3.20(2H, br d, J = 8.1 Hz, 2H-300), 1.73 (3H, s, 3H-5000), 1.64(3H, s, 3H-4000), 1.30/1.20 (each 3H, s, 3H-500/600); 13CNMR spectral data (100 MHz, CD3OD) (Table 1).

3.3.2. 7,4 0-Dihydroxy-8-[(2000n,3000-dihydroxy-3000-methyl)bu-

tyl]-200,200-dimethyl-300,400-de-hydropyrano[100,400:5,6]isoflav-one, vogelin I (2)

Pale yellow powder; m.p. 248–249 �C; ½a�20D ¼ 42� (c,

0.0050 in CHCl3); mmax(NaCl) cm�1 3525, 1640, 1610,

A.F. Kamdem Waffo et al. / Phytochemistry 67 (2006) 459–463 461

1085 and 840; HREIMS (70 eV) m/z 438.1665 (calc. forC25H26O7 438.1678); EIMS (70 eV) m/z (rel. int.) 438(46), 423 (64), 398 (3), 379 (29), 349 (100), 335 (20), 321(60), 295 (13), 236 (5), 203 (8), 166 (9), 152 (7), 118 (4),91 (18), 57 (27), 28 (61); kmax (MeOH) nm (log e): 215(4.41), 268 (4.40), 294 (3.93); 1H NMR spectral data(400 MHz, CDCl3) dH 8.01 (1H, s, H-2), 7.32 (2H, d,J = 8.6 Hz, H-2 0/H-6 0), 6.84 (2H, d, J = 8.5 Hz, H-3 0/H-5 0), 6.67 (1H, d, J = 10.0 Hz, H-400), 5.64 (1H, d,J = 10.0 Hz, H-300), 3.60 (1H, m, H-2000), 2.88 (1H, m, H-1a000), 2.85 (1H, m, H-1b000), 1.46 (6H, s, 3H-500/3H-600),1.28 (6H, s, 3H-4000/3H-5000), 13C NMR spectral data(100 MHz, CDCl3) (Table 1).

3.3.3. 4 0,7-Dihydroxy-200,200-dimethyl-300,400-

dehydropyrano[100,400:5,6]flavone, vogelin J (3)Pale yellow needles; m.p. 238–239 �C; mmax(NaCl) cm�1

3450, 1652, 1589, 1540, 1400, 1272, 1235; HREIMS(70 eV) m/z 336.0932 (calc. for C20H16O5 336.0998); EIMS(70 eV) m/z (rel. int.) 336 (25), 321 (100), 203 (19), 135 (27),118 (43); kmax (MeOH) nm (log e): 236 (4.42), 272 (4.36),313 (4.23), 330 (4.25), 356 (3.78), (MeOH + NaOAc) 277;1H NMR spectral data (400 MHz, CDCl3)dH 7.60 (2H, d,J = 8.9 Hz, H-2 0/6 0), 6.85 (2H, d, J = 8.9 Hz, H-3 0/H-5 0),6.62 (1H, d, J = 10.0 Hz, H-400), 6.46 (1H, s, H-3), 6.34(1H, s, H-8), 5.54 (1H, d, J = 10.0 Hz, H-300), 1.40 (6H, s,3H-500/3H-600); 13C NMR spectral data (100 MHz, CDCl3)(Table 1).

Acknowledgements

We thank Mr. Dilip Jagjivan for the running of NMRspectra, Mr. Bret Parel and Mr. Tommy van der Merwefor GC–MS and HRMS, respectively, Mr. Ernest Makh-aza for his technical assistance, and the University ofKwaZulu-Natal and the National Research Foundation(NRF) for financial aid. Dr. Alain Kamdem gratefullyacknowledges a post-doctoral Fellowship from the NRF.

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Carbon 1 2 3

2 152.5 (CH) 154.2 (CH) 164.5 (C)3 123.0 (C) 124.0 (C) 103.1 (CH)4 180.7 (C) 182.4 (C) 182.5 (C)4a 104.0 (C) 105.8 (C) 102.8 (C)5 159.7 (C) 155.5 (C) 155.7 (C)6 101.8 (C) 105.5 (C) 105.4 (C)7 164.5 (C) 158.1 (C) 159.3 (C)8 107.0 (C) 106.5 (C) 95.2 (CH)8a 151.8 (C) 156.4 (C) 156.9 (C)

10 121.4 (C) 122.7(C) 122.0(C)20 130.2(CH) 130.9(CH) 127.7 (CH)30 115.4 (CH) 116.0 (CH) 116.2 (CH)40 156.0 (C) 158.0 (C) 160.8 (C)50 115.4 (CH) 116.0 (CH) 116.2 (CH)60 130.2(CH) 130.9(CH) 127.7 (CH)

200 91.0 (CH) 78.9 (C) 78.0 (C)300 27.1 (CH2) 128.7 (CH) 127.3 (CH)400 72.2 (C) 116.1 (CH) 115.7 (CH)500 25.5 (CH3)A 28.5 (CH3) 28.0 (CH3)A

600 24.0 (CH3)A 28.5 (CH3) 27.9 (CH3)A

1000 21.8 (CH2) 25.5 (CH2)2000 121.4 (CH) 78.6 (CH)3000 132.2 (C) 73.6 (C)4000 17.7 (CH3) 26.0 (CH3)A

5000 25.5 (CH3) 24.4 (CH3)A

A Values interchangeable within column.

462 A.F. Kamdem Waffo et al. / Phytochemistry 67 (2006) 459–463

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A.F. Kamdem Waffo et al. / Phytochemistry 67 (2006) 459–463 463

Phenolic compounds from the flowers of Garcinia dulcis

S. Deachathai a, W. Mahabusarakam a,*, S. Phongpaichit b, W.C. Taylor c,Y.-J. Zhang d, C.-R. Yang d

a Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailandb Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

c School of Chemistry, University of Sydney, NSW 2006, Australiad Laboratory of Phytochemistry, Kunming Institute of Botany, Chinese Academy of Sciences, China

Received 7 July 2005; received in revised form 14 September 2005Available online 1 December 2005

Abstract

Dulcisxanthones C–F (1–4) and dulcinone (5) together with 22 known compounds were isolated from the flowers of Garcinia dulcis.Their structures were determined by spectroscopic methods. The abilities of some of these compounds to act as radical scavengers andantibacterial agents were investigated.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Garcinia dulcis; Guttiferae; Xanthones; Chromones; Radical scavenging; Antibacterial

1. Introduction

The sub-woody plant Garcinia dulcis Kurz. (Guttiferae),local Thai name Ma-Phut, grows mainly in southeast Asia.Its leaves and seeds have been used in traditional medicineagainst lymphatitis, parotitis, struma and other diseaseconditions (Kasahara and Henmi, 1986). In our previouswork (Deachathai et al., 2005), we have investigated thechemical constituents from its fruit and their biologicalactivities. In our continuing work, we have examined thechemical constituents from the flowers. This investigationhad led to the isolation and structural determination of fivenew and 22 known compounds.


The flowers of G. dulcis were sequentially extracted withacetone. Purification of the extract, produced four new

xanthones named dulcisxanthones C–F (1–4), one newchromone named dulcinone (5), along with 22 known com-pounds: volkensiflavone (6), morelloflavone (7) (Ansariet al., 1976), 1-hydroxy-3,4,5-trimethoxyxanthone (8)(Stout et al., 1973), rhamnazin (9) (Subhadhirasakulet al., 2003), quercetin 3-O-b-galactopyranoside (10) (Kart-nig et al., 1985), podocarpusflavone A (11) (Harrison et al.,1994), xanthochymusside (12), fukugeside (13) (Kono-shima et al., 1970), cowaxanthone (14) (Deachathai et al.,2005), GB-2a (15) (Ansari et al., 1976), xanthochymol(16) (Blount and Williams, 1976), BR-xanthone A (17),1,3,6-trihydroxy-7-methoxy-2,5-bis(3-methyl-2-butenyl)-xanthone (18) (Deachathai et al., 2005), guttiferone E (19)(Gustafson et al., 1992), rheediaxanthone A (20) (DelleMonache et al., 1981), a-mangostin (21), 1,7-dihydroxy-3-methoxy-2-(3-methyl-2-butenyl)xanthone (22) (Deachathaiet al., 2005), 3-isomangostin (23) (Mahabusarakam et al.,1987), kaempferol 3-O-glucopyranosyl-7-O-rhamnopyr-anoside (24), garcinone B (25), morusignin J (26) (Deacha-thai et al., 2005) and b-mangostin (27) (Mahabusarakamet al., 1987).

Dulcisxanthone C (1) is 1-hydroxy-2,3,4,6-tetrameth-oxyxanthone, and a yellow solid, m.p. 125–128 �C. Its


doi:10.1016/j.phytochem.2005.10.016

* Corresponding author. Tel.: +66 7428 8432; fax: +66 7421 2918.E-mail addresses: [email protected], [email protected]

(W. Mahabusarakam).



PHYTOCHEMISTRY



molecular formula of C17H16O7 was established on thebasis of its mass spectrum ([M]+ m/z 332). The UV spec-trum showed absorption maxima at 376, 309, 273,241 nm. The IR spectrum exhibited O–H stretching at3400 cm�1 and C@O stretching at 1646 cm�1. The 1HNMR spectrum showed a singlet signal of a chelatedhydroxy proton (1-OH) at d 12.60. The signals in the aro-matic region, d 7.84 (dd), 7.32 (d) and 7.26 (d) thatappeared as an ABX type were proposed for the signalsof H-7, H-8 and H-5, respectively. These assignment weresupported by the correlations of H-7 to C-5, C-6, C-9; H-8 to C-6, C-8a, C-10a and H-5 to C-6, C-7, C-10a froman HMBC experiment. Four singlet signals at d 4.04,4.16, 3.97 and 4.05 were assigned for the methoxy protonsof 2-OCH3, 3-OCH3, 4-OCH3 and 6-OCH3, respectively,and confirmed by the 3J coupling of the methoxy protonsto C-2, C-3, C-4 and C-6, respectively, in HMBC. Theassigned structure was further confirmed by analysis ofHMBC correlations (Table 1).

Dulcisxanthone D (2) is l,6-dihydroxy-5-(3-methyl-2-butenyl)-2 0,2 0-dimethylchromeno(5 0,6 0:2,3)-2000,2000-dimethyl-chromeno(5000,6000:8,7)xanthone. It is an orange solid, m.p.218–220 �C, with the molecular formula C28H28O6. TheUV spectrum showed absorption maxima at 335, 301,289, 221 nm. Absorption bands of O–H stretching andC@O stretching were shown in the IR spectrum at3424 cm�1 and at 1615 cm�1. The 1H NMR spectrumshowed the signals of a chelated hydroxy proton at d13.70 (1-OH), a non-chelated hydroxy proton at d 6.34(6-OH) and an isolated aromatic proton at d 6.30 (H-4).The characteristic signals of a prenyl group were presentat d 3.57 (H-100), 5.28 (H-200), 1.69 (H-400) and 1.88 (H-500)

and it was located at C-5 according to the HMBC correla-tion of H-100 to C-6, and C-10a. The signals of four methylgroups at d 1.49 (6H, s, 2000-Me) and 1.47 (6H, s, 2 0-Me) andvicinal olefinic protons at d 5.77 (d, H-3000), 7.98 (d, H-4000),5.57 (d, H-3 0) and 6.73 (d, H-4 0) associated with a chromenerings were present. The 3J correlations of H-4000 to C-7 andH-4 0 to C-1 suggested that the chromene rings were con-nected to the parent structure at C-7, C-8 and C-2, C-3.The complete HMBC data (Table 2) confirmed thisstructure.

Dulcisxanthone E (3) is 1,3,6,7-tetrahydroxy-2-(3,7-dimethyl-2,6-octadienyl)-5-(3-methyl-2-butenyl)xanthone.It is a yellow solid with the molecular formula C28H32O6.The UV spectrum showed absorption maxima at 369,322, 258, 242 nm. The IR spectrum showed absorptionbands of O–H stretching at 3450 cm�1 and C@O stretchingat 1690 cm�1. The 1H NMR spectrum exhibited resonancesof a chelated hydroxy proton 1-OH at d 13.19 and aromaticprotons H-4 at d 6.42 and H-8 at d 7.33. The characteristic

O

O OH

HO

O

O

O

O OH

OH

HO

HO O

O OH

OCH3

O

HO

O

O

HO

H3C

OH

CH3

O

O OH

OCH3

OCH3

H3CO

OCH3 1''

2''' 4'''

6

1

2'

4'

5''4'' 2

1'1

1''

4'' 5''

8'

9' 10'

3

1'

4'

5'2''

1

4''

4

2

4

8

5

17

1

10a 10a

10a 10a

4a 4a

4a 4a

8a 8a

8a8a

9a 9a

9a

6

6

9a

6

Table 1HMBC correlation data of compounds 1 and 5

H-position 1 5

3 C-2, C-4, C-4a, 2-Me5 C-6, C-7, C-10a C-4, C-4a, C-6, C-7, C-8a7 C-5, C-6, C-98 C-6, C-8a, C-10a2-OMe C-23-OMe C-34-OMe C-46-OMe C-62-Me C-2, C-37-Me C-6, C-7, C-8

S. Deachathai et al. / Phytochemistry 67 (2006) 464–469 465

signals of a prenyl unit were displayed at d 6.30 (d, H-100),5.28 (br t, H-200), 1.69 (s, H-400) and 1.88 (s, H-500). The pre-nyl unit was linked at C-5 according to the HMBC corre-lation of H-100 to C-5, C-6 and C-10a. It also exhibitedthe typical signal of a geranyl group: three singlets of threemethyl groups at d 1.57 (H-8 0), 1.80 (H-9 0) and 1.52 (H-10 0), a doublet of methylene protons at d 3.37 (H-1 0), twomultiplets of methylene protons at d 1.99 (H-4 0) and 2.03(H-5 0) and two broad triplets of two olefinic methine pro-tons at d 5.28 (H-2 0) and 5.02 (H-6 0). The correlation ofH-1 0 to C-1, C-2 and C-3 in the HMBC indicated thatthe geranyl side chain was at C-2. The complete HMBC(Table 2) supported this structure.

Dulcisxanthone F (4) is 1,6-dihydroxy-2-(3-methyl-2-butenyl)-3-methoxy-200,200-dimethylchromeno(500,600:8,7)-xanthone. It is a yellow solid with the molecular formulaC24H24O6. The UV spectrum showed absorption maximaat 331, 322, 265, 244 nm. The IR spectrum exhibitedabsorption bands of O–H stretching at 3479 cm�1 andC@O stretching at 1675 cm�1. As was found for compound2, the 1H NMR spectrum indicated the presence of a che-lated hydroxy group 1-OH at d 13.35 (s), aromatic protonsH-4 at d 6.36 (s), H-5 at d 6.83 (s), methoxy protons 3-OCH3 at d 3.91 (s) and proton resonances correspondedto a prenyl group at d 1.80 (H-5 0, s), 1.68 (H-4 0, s), 3.36(H-1 0, d) and 5.23 (H-2 0, br t). The characteristic signalsof two methyl groups (H-500, H-600) and vicinal olefinic pro-tons (H-300 and H-400) associated with a chromene ring wereshown at d 1.50, 5.83 and 8.04, respectively. The deshieldedeffect on the resonance of H-400 indicated that the chromenering was attached to the xanthone nucleus close to its car-bonyl group. The HMBC correlation of H-1 0 to C-1, C-2

and C-3 indicated that the prenyl unit was at C-2. Themethoxy group was assigned at C-3 according to theHMBC correlations of H-4 to C-3 and methoxy protonsto C-3. Irradiation of the methoxy protons at d 3.91 in aNOE experiment, enhanced the signal of H-4 and this alsoconfirmed the placement of the methoxy group. The com-plete HMBC (Table 2) supported the assigned structure.

Dulcinone (5) is 6,8-dihydroxy-2,7-dimethyl-4H-chro-men-4-one. It is a yellow solid with molecular formulaC11H10O4. The IR spectrum showed absorption bands ofO–H stretching at 3343 cm�1 and C@O stretching at1657 cm�1. The 13C NMR spectrum also showed the reso-nance of a carbonyl carbon at d 181.5. The 1H NMR spec-trum showed a singlet signal of an olefinic proton H-3 at d5.99, a singlet signal of an aromatic proton H-5 at d 6.33, asinglet signal of methyl protons 2-Me at d 2.39 and a singletsignal of methyl protons 7-Me at d 2.13. These assignmentwere supported by the HMBC correlations: H-3 to C-2, C-4, C-4a; H-5 to C-4a, C-6, C-7, C-8a; 2-Me to C-2, C-3 and7-Me to C-6, C-7, C-8. The assigned structure was furtherconfirmed by HMBC correlations (Table 1).

Compounds 1–5 are new natural products. Compounds6, 7, 11, 14, 15, 17, 18, 21, 22, 24, 25, and 26 were previ-ously isolated from G. dulcis (Ansari et al., 1976; Harrisonet al., 1994; Deachathai et al., 2005). This is the first reportof the known compounds 8, 9, 10, 12, 13, 16, 19, 20, 23, 27

in G. dulcis. Evaluation of the radical scavenging activitiesof some of the compounds at the concentration of 10 lM(Table 3) revealed that none of the newly isolated com-pounds showed any antioxidant activity. However 10, 13,16 and 19 had potent antioxidant properties produced57%, 56%, 60% and 59% scavenging properties, respec-

Table 2HMBC correlation data of compounds 2–4

H-position 2 3 4

4 C-2, C-3, C-4a, C-9, C-4 0 C-2, C-3, C-4a, C-9a C-2, C-3, C-4a, C-9a5 C-6, C-7, C-8a, C-10a8 C-6, C-7, C-8a, C-9, C-10a10 C-1, C-2, C-3, C-2 0, C-30 C-1, C-2, C-3, C-2 0, C-30

20 C-10, C-40 C-2, C-40, C-50

30 C-2, C-20, 20-Me40 C-1, C-20 C-30, C-50, C-60 C-20, C-30, C-50

50 C-30, C-40, C-60 C-20, C-30, C-40

60 C-50, C-80, C-100

80 C-60, C-70, C-100

90 C-20, C-30, C-40, C-50

10 0 C-60, C-70, C-80

100 C-6, C-8a, C-10a, C-200, C-300 C-5, C-6, C-10a, C-200, C-300

200 C-5, C-400, C-500 C-100, C-400, C-500

300 C-8, C-400

400 C-200, C-300, C-500 C-200, C-300, C-500 C-200

500 C-200, C-300, C-400 C-200, C-300, C-400

3000 C-8, C-2000

4000 C-7, C-2000

1-OH C-1, C-2, C-9a C-9a, C-1, C-23-OMe C-320-Me C-20, C-30

200-Me C-300

2000-Me C-2000, C-3000

466 S. Deachathai et al. / Phytochemistry 67 (2006) 464–469

tively. These values corresponded to IC50 values of 10.50,11.40, 8.50 and 10.10 lM, respectively. Compound 9 and16 were tested for the antibacterial activity. Compound16 was found to inhibit the growth of both the penicillin-sensitive strain, ATCC 25923, and the methicillin-resistantstrain, MRSA SK1, of Staphylococcus aureus with MICvalues of 8 lg/mL whereas 9 showed only weak activitywith MIC values greater than 128 lg/mL. The results ofradical scavenging and antibacterial activities of com-pounds 7, 14, 17, 18, 21, 22, 24, 25, 26 have been reportedin the previous work (Deachathai et al., 2005).

3. Experimental

3.1. General methods

Melting points were measured on a digital Electrother-mal 9100 Melting Point Apparatus and are uncorrected.Infrared spectra were recorded on an FTS 165 FT-IR spec-trometer. Ultraviolet absorption spectra were recordedusing a UV-160A spectrometer (SHIMADZU). 1H and13C NMR spectra were performed on a Varian UNITYINOVA 500 spectrometer and on a 300 MHz FTNMR,Bruker spectrometer.

The high resolution mass spectra were recorded on anMS25RFA spectrometer. Pre-coated TLC sheets of silicagel 60 PF254 were used. Quick column chromatography(QCC) was performed with silica gel 60H. Column chro-matography (CC) was performed with silica gel 100 andsephadex LH-20.

3.2. Plant material

The flowers of G. dulcis were collected from Songkhlaprovince in the southern part of Thailand. The voucher

specimen (Coll. No. 02, Herbarium No. 0012652) has beendeposited at Prince of Songkla University Herbarium, Biol-ogy Department, Faculty of Science, Prince of SongklaUniversity, Thailand.


The flowers of G. dulcis (1.2 kg) were extracted at roomtemperature sequentially with acetone (5 and 7 days).Removal of the solvents from the extracts yielded the ace-tone extracts A (148.2 g) and B (38.4 g). An aliquot of theacetone extract A (92.0 g) was further separated by QCCand eluted with CH2Cl2, CH2Cl2–Me2CO, Me2CO,Me2CO–MeOH gradient solvent system. The eluted frac-tions were combined into 17 fractions (A1–A17) on thebasis of their TLC behaviour. Fraction A6 (7.7 g) was sub-jected to CC on sephadex LH-20 and eluted with a gradientof H2O–MeOH to give compounds 1 (3.0 mg), 8 (9.0 mg), 9

(62.0 mg), 10 (27.0 mg) and 11 (10.0 mg). Fractions A5(2.2 g), A7 (4.8 g) and A13 (1.0 g) were further purifiedproducing 6 (29.1 mg), 7 (1.5 g), 12 (148.0 mg) and 13

(51.0 mg). The acetone extract A (55.8 g) was also fraction-ated by dissolving in CH2Cl2 to give a soluble (15.2 g) andinsoluble (40.2 g) fraction. This CH2Cl2 soluble fractionwas subjected to silica gel CC and eluted sequentially withCH2Cl2, and a CH2Cl2–Me2CO gradient to afford 2

(10.0 mg), 14 (21.5 mg), 15 (38.2 mg) and 16 (2.3 g). TheCH2Cl2 insoluble fraction after CC produced 17

(15.2 mg) and 18 (5.4 mg). Extraction of the acetoneextract B (38.4 g) with CH2Cl2, EtOAc and then Me2COgave CH2Cl2 soluble – (8.7 g), EtOAc soluble – (8.5 g)and Me2CO soluble (3.5 g) fractions. After CC of CH2Cl2soluble fraction and eluting with CH2Cl2–Me2CO in apolarity gradient manner, compounds 19 (10.2 mg), 20

(5.5 mg), 3 (14.3 mg), 21 (31.7 mg), 22 (20.2 mg), 23

(3.2 mg) and 24 (12.3 mg) were obtained. The EtOAc solu-ble fraction was subjected to silica gel CC eluted withCH2Cl2–Me2CO in a polarity gradient manner to afford 4(23.8 mg) and 5 (5.2 mg), whereas the Me2CO soluble frac-tion subjected to silica gel CC and eluted with CH2Cl2–MeOH in a polarity gradient manner gave 25 (2.6 mg),26 (2.3 mg) and 27 (15.2 mg).

3.3.1. Dulcisxanthone C (1)

Yellow solid, m.p. 125–128 �C. HRESIMS m/z 332.0892[M]+ (calcd. for C17H16O7, 332.0896). UV (CH3OH) kmax

(nm) (log e): 376 (3.37), 309 (3.78), 273 (3.87), 241 (3.91).IR (neat), m (cm�1): 3400, 1646. 1H NMR (CDCl3) (dppm): 12.60 (1H, s, 1-OH), 7.84 (1H, dd, J = 7.8, 1.5 Hz,H-7), 7.32 (1H, d, J = 7.8 Hz, H-8), 7.26 (1H, d,J = 1.5 Hz, H-5), 4.16 (3H, s, 3-OMe), 4.05 (3H, s, 6-OMe), 4.04 (3H, s, 2-OMe), 3.97 (3H, s, 4-OMe). EIMSm/z (% rel. int): ([M]+ 332, 100), 317 (98), 302 (21), 287(15), 259 (17), 203 (10), 175 (13). 13C NMR (CDCl3) (dppm): 181.7 (C-9), 154.1 (C-3), 150.6 (C-1), 148.7 (C-6),146.4 (C-10a), 145.7 (C-4a), 135.4 (C-4), 132.7 (C-2),123.7 (C-8), 120.9 (C-8a), 116.5 (C-7), 115.9 (C-5), 104.5

Table 3Radical scavenging activity of compounds from the flower of G. dulcis

(10 lM)

Compounds % Scavenging of DPPH IC50 (lM)

1 2 –2 16 –3 15 –4 2 –5 3 –6 5 –8 2 –9 8 –

10 57 10.5011 5 –12 36 –13 56 11.4015 33 –16 61 8.5019 59 10.1020 8 –23 3 –27 2 –BHT 43 19.00


(C-9a), 61.9 (2-OMe), 61.7 (3-OMe), 61.2 (4-OMe), 56.5 (6-OMe).

3.3.2. Dulcisxanthone D (2)

Yellow solid, m.p. 218–220 �C. HRFABMS m/z460.1929 [M]+ (calcd. for C28H28O6 460.1886). UV(CH3OH) kmax (nm) (log e): 335 (4.10), 301 (4.24), 289(4.30), 221 (4.21), 205 (4.25). IR (neat) m (cm�1): 3424,1615, 1440 cm�1. 1H NMR (CDCl3) (d ppm): 13.70 (1H,s, 1-OH), 7.98 (1H, d, J = 10.2 Hz, H-4000), 6.73 (1H, d,J = 10.2 Hz, H-4 0), 6.34 (1H, s, 6-OH), 6.30 (1H, s, H-4),5.77 (1H, d, J = 10.2 Hz, H-3000), 5.57 (1H, d, J = 10.2 Hz,H-3 0), 5.28 (1H, br t, J = 5.7 Hz, H-200), 3.57 (2H, d,J = 7.5 Hz, H-100), 1.88 (3H, s, H-500), 1.69 (3H, s, H-400),1.49 (6H, s, 2000-Me), 1.47 (6H, s, 2 0-Me). FABMS m/z (%rel. int.): ([M]+ 460, 100), 444 (50), 61 (42). 13C NMR(CDCl3) (d ppm): 182.8 (C-9), 159.8 (C-1), 157.7 (C-3),156.5 (C-10a), 150.9 (C-4a), 148.6 (C-6), 136.5 (C-7),132.6 (C-300), 131.3 (C-3000), 127.0 (C-3 0), 120.9 (C-4000),120.9 (C-200), 117.1 (C-8), 115.7 (C-4 0), 115.3 (C-8a),108.3 (C-5), 104.2 (C-9a), 103.8 (C-2), 94.2 (C-4), 77.9(C-2 0), 76.8 (C-2000), 28.3 (2 0-Me), 27.4 (2000-Me), 25.8 (C-400), 22.5 (C-100), 18.0 (C-500).

3.3.3. Dulcisxanthone E (3)


(nm) (log e): 369 (4.06), 322 (4.28), 258 (4.46), 242 (4.50).IR (neat), m (cm�1): 3450, 2923, 1690, 1456. 1H NMR(CDCl3 + CD3OD one drop) (d ppm): 13.19 (1H, s, 1-OH), 7.33 (1H, s, H-8), 6.42 (1H, s, H-4), 6.30 (2H, d,J = 6.0 Hz, H-100), 5.28 (2H, br t, H-2 0, H-200), 5.02 (1H,br t, H-6 0), 3.37 (2H, d, J = 6.0 Hz, H-1 0), 2.03 (2H, m,H-5 0), 1.99 (2H, m, H-4 0), 1.88 (3H, s, H-500), 1.80 (3H, s,H-9 0), 1.69 (3H, s, H-400), 1.57 (3H, s, H-8 0), 1.52 (3H, s,H-10 0). EIMS m/z (% rel. int.): ([M]+ 464, 80), 421 (50),409 (100), 339 (40), 297 (55), 285 (54), 69 (26). 13C NMR(CDCl3 + CD3OD one drop) (d ppm): 180.6 (C-9), 162.5(C-3), 159.8 (C-1), 156.1 (C-4a), 150.7 (C-6), 150.2 (C-10a), 141.9 (C-7), 135.9 (C-300), 132.2 (C-3 0), 131.5 (C-7 0),124.2 (C-6 0), 122.4 (C-2 0), 121.3 (C-200), 115.5 (C-8a),112.7 (C-5), 110.4 (C-2), 105.1 (C-8), 102.5 (C-9a), 93.4(C-4), 39.8 (C-4 0), 25.8 (C-400), 25.5 (C-8 0), 22.4 (C-100),21.7 (C-5 0), 21.4 (C-1 0), 17.8 (C-9 0), 17.6 (C-10 0), 16.2 (C-500).

3.3.4. Dulcisxanthone F (4)


(nm) (log e): 331 (4.44), 322 (4.43), 265 (4.53), 244 (4.54).IR (neat) m (cm�1): 3479, 1675, 1587, 1447. 1H NMR(CDCl3 + DMSO-d6 one drop) (d ppm): 13.35 (1H, s, 1-OH), 8.04 (1H, d, J = 9.7 Hz, H-400), 6.83 (1H, s, H-5),6.36 (1H, s, H-4), 5.83 (1H, d, J = 9.7 Hz, H-300), 5.23(1H, br t, J = 7.0 Hz, H-2 0), 3.91 (3H, s, 3-OMe), 3.36(2H, d, J = 6.7 Hz, H-1 0), 1.80 (3H, s, H-5 0), 1.68 (3H, s,H-4 0), 1.50 (6H, s, 200-Me). EIMS m/z (% rel. int.): ([M]+

408, 60), 393 (55), 365 (70), 353 (100), 169 (15). 13CNMR (CDCl3 + DMSO-d6 one drop) (d ppm): 182.4 (C-9), 163.6 (C-3), 159.6 (C-1), 155.5 (C-4a), 153.0 (C-10a),150.7 (C-6), 136.7 (C-7), 132.2 (C-300), 131.7 (C-3 0), 122.2(C-2 0), 121.0 (C-400), 119.7 (C-8), 111.4 (C-2), 108.7(C-8a), 102.2 (C-9a, C-5), 88.9 (C-4), 71.0 (C-200), 55.8 (3-OCH3), 29.6 (200-Me), 27.3 (200-Me), 25.8 (C-4 0), 21.3 (C-1 0), 17.7 (C-5 0).

3.3.5. Dulcinone (5)


(nm) (log e): 327 (3.51), 300 (3.62), 258 (4.25), 226 (3.96).IR (neat) m (cm�1): 3343, 1657, 1598, 1417. 1H NMR(CDCl3 + DMSO-d6 one drop) (d ppm): 6.33 (1H, s, H-5), 5.99 (1H, s, H-3), 2.39 (3H, s, H-2), 2.13 (3H, s, H-7).EIMS m/z (% rel. int.): ([M]+ 206, 100), 205 (95), 177(11), 165 (8). 13C NMR (CDCl3 + DMSO-d6 one drop)(d ppm): 181.5 (C-4), 165.6 (C-2), 160.9 (C-6), 158.2 (C-8a), 154.6 (C-8), 106.8 (C-3), 102.9 (C-4a), 101.2 (C-7),97.5 (C-5), 19.3 (2-CH3), 6.4 (7-CH3).

3.4. Radical scavenging activity

This was carried out according to the procedure of Dea-chathai et al. (2005).

3.5. Antibacterial activity

This was carried out according to the procedure of Mah-abusarakam et al. (2004).

Acknowledgements

This research was supported by a scholarship to S.D.from the Postgraduate Education and Research Programin Chemistry (PERCH), funded by The Royal Thai Gov-ernment and the Graduate School, Prince of SongklaUniversity.

References

Ansari, W.H., Rahman, W., Barraclough, D., Maynard, M.R., Schein-mann, F., 1976. Biflavonoids and a flavanone–chromone from theleaves of Garcinia dulcis (Roxb.). Kurz. J. Chem. Soc. Perkin Trans I.,1458–1463.

Blount, J.F., Williams, T.H., 1976. Revised structure of xanthochymol.Tetrahedron Lett. 34, 2921–2924.

Deachathai, S., Mahabusarakam, W., Towatana, N., Phongpaichit, S.,Taylor, W.C., 2005. Phenolic compounds from the fruit of Garcinia

dulcis. Phytochemistry 66, 2368–2375.Delle Monache, F., Botta, B., Nicoletti, M., de Barros Coelho, J.S., de

Andrade Lyra, F.D., 1981. Three new xanthones and macluraxanth-one from Rheedia benthamiana PI. Triana (Guttiferae). J. Chem. Soc.Perkin Trans. I., 484–488.

Gustafson, K.R., Blunt, J.W., Munro, M.H.G., Fuller, R.W., McKee,T.C., Cardellina II, J.H., McMahon, J.B., Cragg, G.M., Boyd, M.R.,1992. The guttiferones, HIV-inhibitory benzophenones from Sympho-

468 S. Deachathai et al. / Phytochemistry 67 (2006) 464–469

nia globulifera, Garcinia livingstonei, Garcinia ovalifolia and Clusia

rosea. Tetrahedron 48, 10093–10102.Harrison, L.J., Leong, L.-S., Leong, Y.-W., Sia, G.-L., Sim, K.-Y., Tan,

H.T.-W., 1994. Xanthone and flavonoid constituents of Garcinia dulcis

(Guttiferae). Nat. Prod. Lett. 5, 111–116.Kartnig, Th., Gruber, A., Stachel, J., 1985. Zur kenntnis des flavonoidm-

usters von Asparagus officinalis. Planta Med. 3, 288.Kasahara, S., Henmi, S., 1986. Medicine Herb Index in Indonesia. Eisai

Indonesia, Jakarta, p. 92.Konoshima, M., Ikeshiro, Y., Miyahara, S., Yen, K.-Y., 1970. The

constitution of biflavonoids from Garcinia plants. Tetrahedron Lett.48, 4203–4206.

Mahabusarakam, W., Wiriyachitra, P., Taylor, W.C., 1987.Chemical constituents of Garcinia mangostana. J. Nat. Prod. 50,474–478.

Mahabusarakam, W., Deachathai, S., Phongpaichit, C., Jansakul, C.,Taylor, W.C., 2004. A benzil and isoflavone derivatives from Derris

scandens Benth. Phytochemistry 65, 1185–1191.Stout, G.H., Christensen, E.N., Balkenhol, W.J., Stevens, K.L., 1973.

Xanthones of the Gentianaceae-II Frasera albicaulis Dougl. EXGriesb. Tetrahedron 25, 1961–1973.

Subhadhirasakul, S., Jankeaw, B., Malinee, A., 2003. Chemical constit-uents and antioxidative activity of the extracts from Dyera costulata

leaves. Songklanakarin J. Sci. Technol. 25, 351–353.


Xanthone derivatives from Cratoxylum cochinchinense roots

W. Mahabusarakam a,*, W. Nuangnaowarat a, W.C. Taylor b

a Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailandb School of Chemistry, University of Sydney, New South Wales 2006, Australia

Received 5 April 2005; received in revised form 6 September 2005Available online 28 November 2005

Abstract

Two xanthones and two caged-prenylated xanthones, named cochinchinones A–D, respectively, and a synthetically known caged-prenylated xanthone, together with seven known compounds were isolated from the roots of Cratoxylum cochinchinense (Lour.) Blume.Their structures were assigned on the basis of analyses of spectroscopic data. Some of the compounds exhibited effective antioxidativeproperties.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Cratoxylum cochinchinense; Clusiaceae; Xanthones; Caged-prenylated xanthones; Radical scavenger

1. Introduction

Cratoxylum cochinchinense (Lour.) Blume is called ‘‘tue-gliang’’ locally in Thailand. In traditional medicine, it hasbeen used to treat fevers, coughs, diarrhoea, itches, ulcersand abdominal complaints (Vo, 1997). The bark of thisplant was previously shown to contain triterpenoids, tocot-rienols and xanthones (Bennett et al., 1993; Sia et al., 1995;Nguyen and Harrison, 1998). The use of this plant as a tra-ditional medicine, and the results from a preliminaryscreening of the biological activity of crude extracts fromits roots, led us to examine them further for substances thatact as radical scavengers. Two new xanthones (1–2), twonew caged-prenylated xanthones (3–4), a syntheticallyknown caged-prenylated xanthone (5) and seven knowncompounds (6–12) were isolated. Their structures were elu-cidated from analyses of 1D and 2D NMR spectroscopicdata, including 1H, 13C NMR, NOE, COSY, HMQC andHMBC. Radical scavenging activity of the compoundswas investigated.


Separation of a dichloromethane extract of the roots ofC. cochinchinense produced cochinchinones A–D (1–4), thecaged-prenylated xanthone (5) (Thoison et al., 2000), b-mangostin (6) (Mahabusarakam et al., 1987), l,3,7-trihy-droxy-2,4-bis (3-methyl-2-butenyl)xanthone (7) (Iinumaet al., 1996), mangostin (8) (Mahabusarakam et al.,1987), macluraxanthone (9) (Iinuma et al., 1994), garcinoneB (10) (Sen et al., 1982), celebixanthone (11) (Stout et al.,1962), and garcinone D (12) (Bennett et al., 1993).

Cochinchinone A, 1,3,7-trihydroxy-2-(3-methyl-2-bute-nyl)-4-(3,7-dimethyl-2,6-octadienyl)xanthone (1), was ayellow solid, m.p. 119–120 �C. Its molecular ion of m/z448.2299 was in agreement with the molecular formulaC28H32O5. The IR spectrum showed the presence of O–H(3413 cm�1) and C@O (1641 cm�1) groups. The 1H NMRspectrum (Table 1) exhibited signals of a hydrogen-bondedhydroxy proton at d12.95 (s, 1-OH) and three aromaticprotons which coupled as an ABX system at d7.59 (d,J = 3.0 Hz, H-8), 7.36 (d, J = 9.0 Hz,H-5) and 7.24 (dd,J = 9.0, 3.0 Hz, H-6). The lower field resonance at d7.59was assigned for H-8 according to the anisotropic effectfrom C@O. Since H-8 showed only m-coupling in the 1HNMR spectrum, a hydroxyl group was placed at C-7.


doi:10.1016/j.phytochem.2005.10.008

* Corresponding author. Tel./fax: +66 7421 2918.E-mail address: [email protected] (W. Mahabusarakam).



PHYTOCHEMISTRY


The characteristic signals of protons in a prenyl unit weredisplayed at d3.47 (H-1 0, d), 5.29 (H-2 0, br t), 1.84 (H-4 0,s) and 1.76 (H-5 0, s). In addition, the presence of a geranylside chain was indicated from the resonances at d3.57 (H-100, d), 5.27 (H-200, br t), 2.04 (H-400, m), 2.09 (H-500, m),

5.05 (H-600, br t), 1.57 (H-800, s), 1.88 (H-900, s) and 1.64(H-1000, s). The correlation of H-1 0 to C-1, C-2 and C-3in the HMBC spectrum (Table 1) established that the loca-tion of the prenyl unit was at C-2, whereas the correlationof H-100 to C-3, C-4 and C-4a indicated that the geranyl

O

O OH

OH

HO

O

O OH

OH

HO

HO

O

O R2

R1O

R3

O

1

3

79

1'

4'

5'

1''

4''

8''

9''

10''

1

3

79

1'

4'

5'

1''

4''

8''

9''

10''

1 2

3

8

9

12

1110

15

19

18

1

3 3 R1= H, R2= OH, R3= OCH3

4 4 R1= R2= OH, R3= OCH3

5 5 R1= R2= OH, R3= H

4b

54b 4a

8a 9a

4b

8a 9a

4a

8a 9a

4a

Table 1The 1H NMR and HMBC spectroscopic data of cochinchinones A–B (1–2)

H-position Cochinchinone A (1) Cochinchinone B (2)

1H NMR HMBC 1H NMR HMBC

4 – – 6.40 (s, 1H) C-2, C-3, C-4a, C-9, C-9a5 7.36 (d, 1H, J = 9.0 Hz) C-7, C-8a, C-4b – –6 7.24 (dd, 1H, J = 9.0,3.0 Hz) C-7, C-4b – –8 7.59 (d, 1H, J = 3.0 Hz) C-6, C-7, C-9, C-4b 7.48 (s, 1H) C-6, C-7, C-9, C-4b1 0 3.47 (d, 2H, J = 7.0 Hz) C-1, C-2, C-3, C-20, C-30 3.35 (d, 2H, J = 7.0 Hz) C-1, C-2, C-3, C-2 0, C-30

2 0 5.29 (br t, 1H, J = 7.0 Hz) C-40, C-50 5.26 (br t, 1H, J = 7.0 Hz) C-10, C-40, C-50

4 0 1.84 (s, 3H) C-20, C-30, C-50 1.78 (s, 3H) C-20, C-30, C-50

5 0 1.76 (s, 3H) C-20, C-30, C-40 1.66 (s, 3H) C-20, C-30, C-40

100 3.57 (d, 2H, J = 7.0 Hz) C-3, C-4, C-4a, C-20, C-30 3.56 (d, 2H, J = 7.0 Hz) C-5, C-6, C-4b, C-200, C-300

200 5.27 (br t, lH, J = 7.0 Hz) C-4, C-400 5.26 (br t, 1H, J = 7.0 Hz) C-100, C-400

400 2.04 (m, 2H) C-200, C-500, C-600 1.92 (m, 2H) C-200, C-300, C-600

500 2.09 (m, 2H) C-300, C-400, C-600 2.01 (m, 2H) C-400, C-600, C-700

600 5.05 (br t, 1H, J = 7.0 Hz) C-500, C-l000 5.00 (br t, 1H, J = 7.0 Hz) C-800, C-1000

800 1.57 (s, 3H) C-600, C-700, C-1000 1.50 (s, 3H) C-600, C-700, C-1000

900 1.88 (s, 3H) C-200, C-300, C-400 1.84 (s, 3H) C-200, C-300, C-400

1000 1.64 (s, 3H) C-600, C-700, C-800 1.56 (s, 3H) C-600, C-700, C-800

1-OH 12.95 (s, 1H) C-l, C-2, C-9a 13.33 (s, 1H) C-1, C-2, C-9a

W. Mahabusarakam et al. / Phytochemistry 67 (2006) 470–474 471

side chain was at C-4. Thus, the structure of cochinchinoneA was deduced as 1.

Cochinchinone B, 1,3,6,7-tetrahydroxy-2-(3-methyl-2-butenyl)-5-(3,7-dimethyl-2,6-octadienyl)xanthone (2), wasa yellow solid, m.p. 221–222 �C. The molecular formulawas determined as C28H32O6 by HR-MS. The IR spectrumagain showed the presence of O–H (3350 cm�1) and C@O(1640 cm�1) groups. The 1H NMR spectrum (Table 1) indi-cated the presence of a hydrogen-bonded hydroxy group atd13.33 (s, 1-OH) and two isolated aromatic protons atd6.40 (s, H-4) and 7.48 (s, H-8). The resonances at d3.35(d, H-1 0), 5.26 (br t, H-2 0), 1.78 (s, H-4 0) and 1.66 (s, H-5 0) revealed the presence of a prenyl unit. The characteristicsignals of a geranyl group were observed at d3.56 (d, H-100),5.26 (br t, H-200), 1.92 (m, H-400), 2.01 (m, H-500), 5.00 (br t,H-600), 1.50 (s, H-800), 1.84 (s, H-900) and 1.56 (s, H-1000). Theprenyl group was assigned at C-2 according to the HMBCcorrelation (Table 1) of H-1 0 to C-1, C-2 and C-3 whereasthe geranyl group was placed at C-5 due to the correlationof H-100 to C-5, C-6 and C-4b. The structure 2 was there-fore indicated for cochinchinone B.

Cochinchinone C (3) was a yellow solid, m.p. 147–148 �C and ½a�29

D þ 50� (c 0.089, CHCl3). A pseudomolecu-lar ion [M � CO]+ at m/z 382.1745 was consistent for themolecular formula of C24H26O6 with the loss of 28 amu(CO). The IR spectrum also showed absorption bands ofO–H stretching at 3467 cm�1, conjugated C@O stretchingat 1642 cm�1 and unconjugated C@O stretching at1749 cm�1. The 13C NMR spectroscopic data (Table 3)exhibited carbon resonances at d180.73 (C-9) and 201.16(C-6), confirming the presence of conjugated- and unconju-gated carbonyl groups. The 1H NMR spectrum (Table 2)revealed resonances of a hydrogen-bonded hydroxy protonat d12.00 (s, 1-OH) and three aromatic protons which cou-pled as an ABM system at d6.55 (dd, J = 8.4,0.9 Hz, H-2),7.41 (t, J = 8.4 Hz, H-3), and 6.52 (dd, J = 8.4, 0.9 Hz,H-4).The spectrum further displayed the presence of an olefinic

proton (d7.51, s, H-8), a methoxy group (d3.65, s, 7-OCH3),a pair of non-equivalent methylene protons (d1.59, dd, Hb-10 and 2.39, d, Ha-10), a methine proton (d2.54, d, H-11)and a prenyl unit [d4.39 (br t, H-16), 2.64 (d, H-15), 1.37(s, H-18) and 1.01 (s, H-19)]. The prenyl unit was placedat C-5 according to the correlation of H-15 to C-4b andC-5. The HMBC correlations (Table 2) of Ha-10 to C-4b,C-8, C-11; Hb-10 to C-7, C-8, C-11 and H-11 to C-4b, C-5, C-10 suggested that CH2-10 and CH-11 were in betweenC-7 and C-4b. The signals of the methine proton H-11 andthe gem-dimethyl groups (H-13, d1.69, s and H-14, d1.33, s)revealed a 2,2-dimethyltetrahydrofuran ring. It was sug-gested as being fused at C-5, C-4b and C-11 from the cor-relations of H-13 to C-11 and of H-11 to C-4b and C-5.

Cochinchinone D (4) was shown to be a methoxy deriv-ative of the synthetically known compound (5) (Thoisonet al., 2000). It was a yellow solid, m.p. 218–219 �C. and½a�29

D � 58� (c 6.90 · 10�2 in CHCl3). The molecular ion[M � CO]+ at m/z 398.1713 was in agreement to the molec-ular formula C24H26O7 with the loss of 28 amu (CO). TheIR spectrum showed absorption bands of O–H stretchingat 3467 cm�1 and of C@O stretching at 1646 (conjugatedC@O) and 1738 (unconjugated C@O) cm�1. In the 13CNMR spectrum (Table 3), the resonances of the carbonylcarbons were at d178.00 (C-9) and 201.00 (C-6). The 1HNMR spectrum (Table 2), showed a singlet for 1-OH atd12.39 and a broad singlet for 3-OH at d8.05, whereasthe aromatic part was tetrasubstituted as evidence from sig-nals at d6.05 (d, H-4) and 6.03 (d, H-2) with meta coupling(J = 2.1 Hz). The data from the 1H NMR and HMBCspectra (Table 2) and 13C NMR spectra (Table 3) indicatedthat the non-aromatic part contained a methoxy group(d3.62, 7-OCH3), an olefinic proton H-8 (d7.44, s), a prenylunit (d4.43, H-16; 2.63,H-15; 1.40,H-18 and 1.13,H-19), apair of methylene protons Ha-10 (d2.35,d), Hb-10 (d1.59,dd), a methine proton H-11 (d2.50, d) and gem-dimethylprotons H-13 (d1.66) and H-14 (d1.31).

Table 2The 1H NMR and HMBC spectroscopic data of cochinchinones C–D (3–4)

H-position Cochinchinone C (3) Cochinchinone D (4)

1H NMR HMBC 1H NMR HMBC

2 6.55 (dd, 1H, J = 8.4, 0.9 Hz) C-1, C-4 6.03 (d, 1H, J = 2.1 Hz) C-1, C-4, C-9a3 7.41 (1H, J = 8.4 Hz) C-1, C-4a – –4 6.52 (dd, 1H, J = 8.4, 0.9 Hz) C-2, C-4a, C-9, C-9a 6.05 (d, 1H, J = 2.1 HZ) C-2, C-3, C-4a, C-9, C-9a8 7.51 (s, 1H) C-4b, C-7, C-8a, C-9 7.44 (s, 1H) C-4b, C-6, C-8a, C-910a 2.39 (d, 1H, J = 12.9 Hz) C-4b, C-8, C-11 2.35 (d 1H, J = 132 Hz) C-4b, C-7, C-8, C-1210b 1.59 (dd, 1H, J = 12.9, 9.9 Hz) C-7, C-8, C-11 1.59 (dd, lH, J = 13.2, 9.6 Hz) C-6,C-7,C-8,C-1111 2.54 (d, 1H, J = 9.9 Hz) C-4b, C-5, C-10 2.50 (d, 1H, J = 9.6 Hz) C-4b, C-7, C-12, C-1313 1.69 (s, 3H) C-11, C-12, C-14 1.66 (s, 3H) C-11, C-12, C-1414 1.33 (s, 3H) C-11, C-12, C-13 1.31 (s, 3H) C-11, C-12, C-1315 2.64 (d 2H, J = 8.1 Hz) C-4b, C-5, C-16, C-17 2.63 (d 2H, J = 7.5 Hz) C-4b, C-5, C-6, C-16, C-1716 4.39 (br t, 1H, J = 8.1 Hz) C-15, C-18, C-19 4.43 (br t, 1H, J = 7.5 Hz) C-15, C-18, C-1918 1.37 (s, 3H) C-16, C-17, C-19 1.40 (s, 3H) C-16,C-17,C-1919 1.01 (s, 3H) C-16, C-17, C-18 1.13 (s, 3H) C-16,C-17,C-181-OH 12.00 (s, 1H) C-1, C-2, C-9a 12.39 (s, 1H) C-1, C-2, C-9a3-OH – – 8.05 (br s, 1H) –7-OCH3 3.65 (s, 3H) C-7 3.62 (s, 3H) C-7

472 W. Mahabusarakam et al. / Phytochemistry 67 (2006) 470–474

In their work on bractatin and derivatives, Thoisonet al. (2000) have shown that caged-prenylated xanthonescan exist in pure enantiomeric form (with relatively largeoptical rotation) or as mixtures of enantiomers, with lowoptical rotation values. It is concluded therefore thatcochinchinone C and D are different mixtures of the twopossible enantiomers of the structures 3 and 4, respectively,in which only the relative stereochemistry is shown.

At the concentration of 50 lM (Table 4), the twelve iso-lated compounds were able to scavenge the DPPH radicalin the range of 1.7–81.0%. Compounds 2, 9 and 11 exhib-ited the most potent radical scavenging activity with IC50

values of 9.4, 19.0 and 12.3 lM, respectively. These are

more effective than butylated hydroxytoluene (BHT) withan IC50 of 28.9 lM. The greater effectiveness of compounds2, 9 and 11 than the others was possibly due to the presenceortho-dihydroxy groups which upon donating hydrogenradicals will give higher stability to their radical forms(Shahidi and Wandasundara, 1992).

Caged-prenylated xanthones have been exclusivelyfound in the genus Garcinia (Cao et al., 1998; Thoisonet al., 2000; Rukachaisirikul et al., 2000). The present workis the first report of the caged-prenylated xanthones iso-lated from the genus Cratoxylum.

3. Experimental

3.1. General method

Melting points were recorded with a digital electrother-mal melting point apparatus (Electrothermal 9100) and areuncorrected. Optical rotations were measured on anAUTOPOL� II automatic polarimeter. Ultraviolet spectrawere measured with a UV-160A spectrophotometer(SHMADZU). Infrared spectra (IR) were obtained witha FTS165 FT-IR spectrometer. 1H and 13C-Nuclear Mag-netic Resonance spectra were recorded with a FT-NMRBruker Avance 300 MHz or Varian UNITY INOVA500 MHz spectrometer, whereas high resolution mass spec-tra were obtained using a MAT 95 XL. Column and quickCC were performed on silica gel 100 and silica gel 60H(Merck), respectively. Precoated TLC sheets of silica gel60 F254 were used. Known compounds were identified bycomparison of their spectroscopic data with those in theliterature.

3.2. Plant material

The roots of C. cochinchinense (Clusiaceae) were col-lected from Amphur Bannasan, Suratthani Province inthe southern part of Thailand in February 2003. The vou-cher specimen (No. W. Nuangnaowarat 1 Suratthani: Ban-nasan 31/3/04) was identified by Dr. Kitichate Sridith andhas been deposited in the herbarium of the Department ofBiology, Faculty of Science, Prince of Songkla University,Thailand.


Chopped, dried roots of C. cochinhinense (11 kg) weresequentially extracted at room temperature with CH2Cl2(42 L) and MeOH (39 L) (3 days each). Removal of sol-vents in vacuo produced a yellow-brown, viscous, CH2Cl2extract (294 g) and a MeOH extract (292 g), respectively.An aliquot of the CH2Cl2 extract (66 g) was subjected toquick CC over silica gel 60H using hexane, hexane–CH2Cl2, CH2Cl2, CH2Cl2–Me2CO and Me2CO as eluentsto give fractions A1–A14. Fractions A4 (8.51 g) and A6(7.54 g) were further purified by crystallization in hexane–

Table 3The 13C NMR spectroscopic data of cochinchinones A–D (1–4)

C-position 1 2 C-position 3 4

1 158.27 160.00 1 162.90 160.502 108.90 110.02 2 109.57 95.493 161.13 162.28 3 138.97 167.884 105.00 93.34 4 107.41 96.954a 152.97 155.73 4a 159.44 164.504b 150.34 149.76 4b 88.76 88.505 118.87 115.32 5 84.18 83.786 124.12 150.23 6 201.16 201.007 152.43 141.60 7 84.86 84.508 108.89 105.95 8 135.25 133.258a 120.45 112.85 8a 132.14 132.259 180.90 180.11 9 180.73 178.009a 102.96 102.33 9a 106.15 100.501 0 21.58 21.28 10 29.73 29.872 0 121.58 122.63 11 49.43 49.273 0 134.92 131.40 12 83.96 83.764 0 17.94 17.79 13 30.37 30.265 0 25.86 25.72 14 29.04 28.94100 21.59 22.32 15 29.21 28.98200 121.58 121.43 16 118.48 117.86300 137.94 135.41 17 135.73 135.50400 39.72 39.66 18 25.51 25.46500 26.43 26.56 19 16.69 16.88600 123.85 124.12 7-OCH3 54.09 53.84700 131.50 131.19800 17.69 17.57900 16.27 16.231000 25.66 25.52

Table 4Radical scavenging activity of compounds 1–12 (at 50 lM)

Compounds % Scavenging of DPPH

1 20.72 79.33 1.74 5.25 5.26 1.77 20.78 5.29 75.9

10 6.911 79.312 6.9BHT 51.7

W. Mahabusarakam et al. / Phytochemistry 67 (2006) 470–474 473

CH2Cl2 to give the yellow solid 6 (738 mg). Fraction A7(18.96 g) was subjected to silica gel column and eluted withhexane–CH2Cl2, CH2Cl2, CH2Cl2–Me2CO to give frac-tions A7A (5.33 g), A7B (10.77 g) and A7C (1.94 g).Repeated chromatography of fraction A7A (2.00 g) on sil-ica gel column using hexane–Me2CO (9:1) as mobile phaseyielded the yellow solids 7 (55 mg), 1 (20 mg), 3 (15 mg)and 8 (60 mg). Fraction A7B (1.20 g) was submitted toCC over silica gel, eluted with hexane–Me2CO (17:3) togive the yellow solids 9 (6 mg), 5 (25 mg) and 4 (35 mg).Fraction A9 (4.06 g) was separated by CC over silica geland eluted with hexane–Me2CO (4:1) to give the yellow sol-ids 10 (15 mg), 2 (67 mg) and 11 (22 mg). Fraction A11(300 mg) was applied to a silica gel column usingCH2Cl2–MeOH (49:1) as eluent to yield a yellow solid 12

(40 mg).

3.3.1. Cochinchinone A (1)

Yellow solid, m.p. 119–120 �C. HREIMS m/z 448.2299for C28H32O5 (calcd. 448.2250). UV (EtOH) kmax nm(log e): 232 (4.44), 268 (4.42), 316 (4.04), 384 (3.70). IR(KBr) m (cm�1): 3413, 1641. EIMS m/z (% rel int): 449([M + H]+, 53), 448 ([M]+,15), 447 (10), 393 (18), 325(12), 323 (30), 309 (13), 281 (10), 277 (18), 270 (23), 269(100), 257 (13), 253 (10), 185 (75), 93 (60). For 1H NMRand 13C NMR spectroscopic data, see Tables 1 and 3.

3.3.2. Cochinchinone B (2)

Yellow solid, m.p. 221–222 �C. HRMS m/z 464.2189 forC28H32O6 (calcd. 464.2199). UV (EtOH) kmax nm (log e):243 (4.35), 259 (4.32), 323 (4.10), 372 (3.98). IR (KBr) m(cm�1): 3350, 1640. EIMS m/z (% rel int): 465([M + H+,18), 464 ([M]+,63), 422 (10), 421(39), 409 (76),379 (17), 353 (11), 342 (24), 339 (31), 325 (24), 297 (46),285 (45), 257 (25), 207 (13), 178 (58), 161 (32), 121 (37),108 (50), 91 (76), 79 (77), 69 (96), 57 (100). For 1H NMRand 13C NMR spectroscopic data, see Tables 1 and 3.

3.3.3. Cochinchinone C (3)

Yellow solid, m.p. 147–148 �C. HREMS [M � CO]+ m/z 382.1745 for C23H26O5 [M � CO]+ (calcd. 382.1780).Optical rotation: ½a�29

D þ 50� (c 8.9 · 10�2 in CHCl3). UV(EtOH) kmax nm (log e): 206 (4.46), 222 (4.30), 307 (3.98),346 (3.70). IR (KBr) m (cm�1): 3467, 1749, 1642. EIMSm/z (% rel int): 382 ([M � CO]+,14), 381 (52), 312 (100),285 (35), 243 (19), 68.9 (9). For 1H NMR and 13C NMRspectroscopic data, see Tables 2 and 3.

3.3.4. Cochinchinone D (4)

Yellow solid, m.p. 218–219 �C. HREIMS m/z 398.1713for (C23H26)6 [M � CO]+ (calcd. 318.1729). Optical rota-tion: ½a�29

D � 58� (c 6.90 · 10�2 in CHCl3). UV (EtOH) kmax

nm: 212 (4.47), 275 (4.09), 332 (4.06), 357 (4.10). IR (KBr)m (cm�1): 3392, 1738, 1646. EIMS m/z (% rel int): 398([M � CO]+,10), 397 (36), 329(16), 328 (100), 300 (29),259 (16), 68.9 (13). For 1H NMR and 13C NMR spectro-scopic data, see Tables 2 and 3.

3.4. Radical scavenging activity

The experiments were modified from the method ofYamasaki et al., 1994. The sample solution (3.0 mM inabsolute ethanol, 50 lL) was mixed with DPPH solution(0.05 mM, 3 mL) and allowed to stand at room tempera-ture for 30 min. The absorbance was then measured at517 nm. The results were expressed as percentage radicalscavenging, % radical scavenging = [(Acontrol � Asample)/Acontrol] · 100. The DPPH solution without sample wasused as control. The IC50 values were obtained by linearregression analysis of the dose response curves, which wereplots of % radical scavenging versus concentration. Mea-surements were performed in triplicate.

Acknowledgement

This research was supported by a scholarship to W.N.from the Postgraduate Education and Research Programin Chemistry (PERCH), funded by The Royal Thai Gov-ernment and the Graduate School, Prince of SongklaUniversity.

References

Bennett, G.J., Harrison, L.J., Sia, G.-L., Sim, K.-Y., 1993. Triterpenoids,tocotrienols and xanthones from the bark of Cratoxylum cochinchin-

ense. Phytochemistry 32, 1245–1251.Cao, S.-G, Sng, V.H.L., Wu, X.-H, Sim, K.-Y., Tan, B.H.K., Pereira, J.T.,

Goh, S.H., 1998. Novel cytotoxic polyprenylated xanthonoids fromGarcinia gaudichaudii (Guttiferae). Tetrahedron 54, 10915–10924.

Iinuma, M., Tosa, H., Tanaka, T., Yonemori, S., 1994. Two xanthonesfrom root bark of Calophyllwn inophyllum. Phytochemistry 35, 527–532.

Iinuma, M., Tosa, H., Tanaka, T., Riswan, S., 1996. Three new xanthonesfrom the bark of Garcinia dioica. Chem. Pharm. Bull. 44, 232–234.

Mahabusarakam, W., Wiriyachitra, P., Taylor, W.C., 1987. Chemicalconstituents of Garcinia mangostana. J. Nat. Prod. 50, 474–478.

Nguyen, L.H.D., Harrison, L.J., 1998. Triterpenoid and xanthone constit-uents of Cratoxylum cochinchinense. Phytochemistry 50, 471–476.

Rukachaisirikul, V., Kaewnok, W., Koysomboon, S., Phongpaichit, S.,Taylor, W.C., 2000. Caged-tetraprenylated xanthones from Garcinia

scortechinii. Tetrahedron 56, 8539–8543.Sia, G.-L, Bennett, G.J., Harrison, L.J., Sim, K.-Y., 1995. Minor

xanthones from the bark of Cratoxylum cochinchinense. Phytochem-istry 38, 1521–1528.

Sen, A.K., Sarkar, K.K., Mazumder, P.C., Banerji, N., Uusvuori, R.,Hase, T.A., 1982. The structures of garcinones A, B and C: three newxanthones from Garcinia mangostana. Phytochemistry 21, 1747–1750.

Shahidi, F., Wandasundara, P.K., 1992. Phenolic antioxidants. Crit. Rev.Food Sci. Nutr. 32, 67–103.

Stout, G.H., Stout, V.F., Welsh, M.J., 1962. The structure of celebix-anthone. Tetrahedron Lett. 13, 541–544.

Thoison, O., Fahy, J., Dumontet, V., Chiaroni, A., Riche, C., Tri, M.V.,Sevenet, T., 2000. Cytotoxic prenylxanthones from Garcinia bracteata.J. Nat. Prod. 63, 441–446.

Vo, V.V., 1997A Dictionary of Medicinal Plants in Vietnam, vol. 435. YHoc Publisher, HoChiMinh City.

Yamasaki, K., Hashimoyo, A., Kokusenya, Y., Miyamoto, T., Sato, T.,1994. Electrochemical method for estimating the antioxidative effectsof methanol extracts of crude drugs. Chem. Pharm. Bull. 42, 1663–1665.

474 W. Mahabusarakam et al. / Phytochemistry 67 (2006) 470–474

Alkaloids from Oriciopsis glaberrima Engl. (Rutaceae) q

Jean Duplex Wansi a,*, Jean Wandji b, Alain Francois Kamdem Waffo a,Happi Emmanuel Ngeufa a, Jean Claude Ndom a, Serge Fotso c, Rajendra Prasad Maskey c,

Dieudonne Njamen b, Tanee Zacharias Fomum b, Harmut Laatsch c

a Department of Chemistry, Faculty of Science, University of Douala, P.O. Box 24157, Douala, Cameroonb Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O Box 812, Yaounde, Cameroon

c Department of Organic and Biomolecular Chemistry, Georg August University, Tammannstrasse 2, D-37077 Gottingen, Germany

Received 9 March 2005; received in revised form 16 September 2005Available online 21 November 2005

Abstract

Two alkaloid derivative, oriciacridone A and B, were isolated from the stem bark of Oriciopsis glaberrima (Rutaceae). The structureswere elucidated by a detailed spectroscopic analysis. The extract exhibited in vitro significant antimicrobial activity against a range ofmicro-organisms.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Oriciopsis glaberrima; Rutaceae; Oriciacridone A and B; Antimicrobial activity

1. Introduction

Oriciopsis glaberrima Engl. (Rutaceae) is a monotypicgenus endemic to the humid rain forests of Cameroon (Let-ouzey, 1963). It is used as medicinal plant against infec-tions, hypotension, mycoses, dermatitis and many otherdiseases (Bouquet, 1969). Previous phytochemical studiesof O. glaberrima resulted in the isolation of one tretranortr-iterpenoid namely oriciopsin, and the furoquinoline alka-loid (Ayafor et al., 1982).

This paper describes the isolation and structural elucida-tion of two new alkaloids; only the antimicrobial activitiesof the isolates and the extract were examined.


Ground, air-dried, stem bark of O. glaberrima wasextracted with CH2Cl2/MeOH (1:1) at room temperature.The extract was concentrated under reduced pressure and

its anti-microbial activities against a range of micro-organ-isms were evaluated in vitro, using the agar diffusion test.Following bioassay-directed chromatographic fraction-ation, two new alkaloid, oriciacridone A (1) and oriciacri-done B (2), were isolated, together with the knownlichexanthone (3).

Oriciacridone A (1), m.p. 294 �C, ½a�25D � 45:7�, was

obtained as yellow crystals and reacted positively withFeCl3, thereby indicating the presence of a phenolic hydro-xyl group. Alkaloid 1 was shown to have the molecular for-mula of C36H32N2O9 by HR-EIMS ([M]+; m/z = 636.2108;calc. 636.21024). The IR (m = 3360, 2972, 1649, 1620,1563 cm�1) and UV (kmax = 401, 352, 323, 285, 272,269 nm) spectra suggested the presence of a 9-acridonemoiety (Takemura et al., 1998) and a xanthone skeleton(Peres and Nagem, 1997; Peres et al., 2000). The character-istic signals of two chelated hydroxyl protons at d 14.40and d 13.15 (s, each 1H) in the 1H NMR spectrum, coupledwith its molecular ion, suggested a dimeric structure. Sim-ilarly, the resonances at d 11.32 (s br, 1H) and d 10.70 (s br,1H) indicated the presence of two further D2O exchange-able protons. The 1H NMR spectrum also showed signalsof five aromatic protons at d 8.14 (dd, J = 7.8, 1.8 Hz,


doi:10.1016/j.phytochem.2005.09.031

q Part 1 in the series Oriciopsis studies (Pygmee�s plant).* Corresponding author. Tel.: +237 7817731.

E-mail address: [email protected] (J.D. Wansi).



PHYTOCHEMISTRY


N O

O O

OH

OH

O

OH

O OH

N

H 1

2

3

4

4a

5

66a

77a

8

9

10

11 1211a 12a

12b

13

14

15

1'

2'

3'

4'5'6'7'8'

9'10'

4a'5a'6a'

11'

12'

10a' 11a' 12a'

13'

14'

HH

Oriciacridone A(1)

Structures of compounds 1, 2 and 3

N O

O O

OH

OH

O

OH

O OH

N

H 1

2

3

4

4a

5

66a

77a

8

9

10

11 1211a 12a

12b

13

14

15

1'2'

3'

4'5'6'7'8'

9'10'

4a'5a'6a'

11'

12'

10a' 11a' 12a'

13'

14'

HH

OH

Oriciacridone B (2)

O

OHCH3 O

H3CO OCH3

Lichexanthone (3)

1H), 7.80 (s br, 1H), 7.78 (s br, 1H), 7.30 (m, 1H), and 5.98(s, 1H), the signals of a substituted pyran ring at d 1.62 (s,3H), 1.42 (s, 3H), 5.29 (dd, J = 3.7, 3.8 Hz, 1H), 4.30 (d,J = 3.8 Hz, 1H), and a doublet at d 9.56 (d, J = 5.2 Hz,1H). The aromatic lowfield signal at d 8.14 was deshieldedby a carbonyl group (Takemura et al., 1998). The presenceof a pyran ring was further confirmed by the 13C NMRspectrum, which showed characteristic signals at d 22.2

(C-13), 23.7 (C-14), 42.5 (C-1), 70.9 (C-2), and 76.9 (C-3)(Magiatis et al., 1999). 2D NMR techniques (HSQC andHMBC) indicated a hydroxydimethylchroman ring. Inthe HMBC spectrum (Fig. 1), the proton at d 5.29 showedcross-peaks with carbon signals at C-12a (d 141.5), C-12b(d 96.7), and C-4a (d 158.8). This finding clearly indicatedthat the hydroxydimethylchroman ring was fused in anangular fashion. All this information is in agreement withthe acridone skeleton (Bahar et al., 1982; Magiatis et al.,2001). The presence of an acridone skeleton was also con-firmed by the EI MS mass spectrum, which showed a peakat m/z 311 (C18H16NO4). Furthermore, the 1H NMR spec-trum showed signals corresponding ortho-coupled aro-matic protons at d 7.13 and 6.50 (d, J = 8.8 Hz, 1H each),an aromatic proton singlet at d 6.28 (s, 1H) and a dim-ethylchroman ring [d 1.14 (s, 6H), 1.48 (m, 2H), 2.54 (m,2H)] indicating the presence of a dihydro-6-desoxyjacareu-lin skeleton (Locksley et al., 1971). The latter was con-firmed by 13C NMR, DEPT, and 2D NMR techniques(HMBC, HSQC and COSY). The HMBC spectrumshowed cross-peaks of the chelated hydroxyl signal at d13.15 with the carbon signals at C-11a 0 (d 100.9) andC-10a (d 110.9), and between the aromatic proton singletat d 6.28 (H-6 0) and C-11a 0 (d 100.9), C-10a 0 (d 110.9). Fur-ther correlations were observed between the aromatic pro-ton at d 6.50 (H-2 0) and carbon signals at C-4 0 (d 150.6) andC-12a 0 (d 105.2), and between the proton at d 7.13 (H-3 0)and carbons C-1 0 (d 136.3), and C-4a 0 (d 133.3). Thus, 1

has a dihydro-6-desoxyjacareulin group linked to an acr-onycine skeleton.

To confirm the linkage of the two skeletons, 2D NMR(HMBC and NOESY) experiments were used. In theHMBC spectrum, cross-peaks between the proton signalat d 9.56 (H-15) and carbons C-2 0 (d 105.2) and C-2(d 70.9), and between the proton at d 5.29 (H-1) and carbonsignals at C-1 0 (d 136.3), C-12a (d 141.5), and C-4a

N O

O O

OH

OH

O

OH

O OH

N

H 1

2

3

44a

5

66a

77a

8

9

10

11 1211a 12a

12b

13

14

15

1'2'

3'4'5'6'7'

8'

9'10'

4a'5a'6a'

11'

12'

10a' 11a' 12a'

13'

14'

H

HH

H

H

Fig. 1. Significant long-range correlations observed in 1H–13C HMBC forcompound 1 in DMSO-d6.

476 J.D. Wansi et al. / Phytochemistry 67 (2006) 475–480

(d 158.8), suggested that the two substructures were linkedby nitrogen, furthermore, in the NOESY spectrum, therewere cross-peaks between the proton H-15 (d 9.56) and thearomatic proton H-2 0 (d 6.50), and between the same protonH-15 (d 9.56) and protons at d 5.29 (H-1) and d 4.30(H-2), indicating clearly the linkage position of the two frag-ments. The coupling constant of J = 3.7 Hz betweenprotons H-1 and H-2 showed that they are in a cis

relationship (Magiatis et al., 1999). From the above spectro-scopic studies, compound 1 was characterized as (�)-cis-2-

hydroxy-1-(3,4-dihydro-6-desoxyjacareulin)amino-1,2-dihydroacronycine named oriciacridone A.

Alkaloid 2, m.p. 309 �C, ½a�25D � 85:3�, isolated as a yel-

low solid, had the molecular formula C36H32N2O10 (HR-EIMS, m/z found 652.2061; calc. 652.20515) and thusone oxygen more than oriciacridone A (1). The 13CNMR spectrum revealed 36 carbon signals, which weresorted by DEPT and APT experiments into four methyl,two methylene, eight sp2 methine, two sp3 methine, and20 quaternary carbons; among the latter, two were

Table 11H (300 MHz) and 13C (75.5 MHz) assignments for oriciacridone A (1) and oriciacridone B (2) in DMSO-d6

Attribution 1 2

13C 1H [m,J (Hz)] 13C 1H [m,J (Hz)]

1 42.5 5.29 (dd, 3.7, 3.8) 42.8 5.72 (m)2 70.9 4.30 (d, 3.7) 71.5 4.13 (d, 3.9)3 76.9 – 76.2 –4 – – – –4a 158.8 – 159.0 –5 95.7 5.98 (s) 94.9 5.99 (s)6 162.7 – 163.9 –6a 104.6 – 105.2 –7 180.2 – 181.6 –7a 118.8 – 119.5 –8 124.7 8.14 (br d, 7.8) 115.5 7.57 (dd, 9.0, 3.0)9 121.9 7.30 (ddd, 6.8, 5.6, 2.3) 123.3 7.22 (d, 9.0)10 133.3 7.78 (br d, 5.7) 119.9 7.17 (d, 9.0)11 117.5 7.82 (td, 8.7, 1.5) 148.8 –11a 140.4 – 141.5 –12 – – – –12a 141.5 – 142.8 –12b 96.7 – 96.7 –13 22.2 1.42 (s) 22.9 1.39 (s)14 23.7 1.62 (s) 24.9 1.62 (s)1 0 136.3 – 136.3 –2 0 105.2 6.50 (d, 8.8) 105.7 6.58 (d, 11.4)3 0 121.9 7.13 (d, 8.8) 122.0 7.18 (d, 11.4)4 0 150.6 – 151.2 –4a 0 133.3 – 134.3 –5 0 – – – –5a 0 154.5 – 155.4 –6 0 92.6 6.28 (s) 93.2 6.30 (s)6a 0 163.2 – 163.2 –7 0 – – – –8 0 68.9 – 68.9 –9 0 42.3 1.48 (m) 43.2 1.45 (m)10 0 17.0 2.54 (m) 15.2 2.57 (m)10a 0 110.9 – 110.9 –11 0 159.3 – 159.2 –11a 0 100.9 – 100.6 –12 0 182.6 – 183.8 –12a 0 101.9 – 101.913 0 29.1 1.14 (s) 25.4 1.18 (s)14 0 29.0 1.14 (s) 27.4 1.18 (s)2-OH 4.14 (br s) 10.72 (br s)6-OH 14.40 (s) 14.60 (s)4 0-OH 11.32 (br s) 10.84 (br s)11 0-OH 13.15 (s) 13.12 (s)11-OH – 10.59 (br s)12-NH 10.70 (br s) 10.42 (br s)15-NH – 9.56 (d, 5.2) – 9.28 (m)

J.D. Wansi et al. / Phytochemistry 67 (2006) 475–480 477

carbonyls. The IR spectrum showed vibration bands at3428, 3250, and 1664 cm�1, due to hydroxyl groups, amethine, and a chelated carbonyl group, respectively.These data, together with those obtained from UV (kmax

257, 275, 278, 295, 312, 372, 404 nm), 1H NMR (two sing-lets, 1H each at d 14.60 and 13.12 due to chelated OH) and13C NMR data (Table 1), suggested that alkaloid 2 has thesame skeleton as oriciacridone A (1). Furthermore, in the1H NMR spectrum, an ABM spin system correspondingto a 1,2,3-trisubstituted benzene ring [d 7.57 (dd, J = 9.0,3.0 Hz, 1H), 7.22 (d, J = 9.0 Hz, 1H), 7.17 (d, J = 9.0 Hz,1H)], three hydroxyl signals [d 10.84, 10.59, 10.72 (s br,each 1H)], and signals corresponding to hydrox-yldimethylchroman and dimethylchroman units were alsoobserved. These data suggested that the additional oxygenatom in 2 is attached as a hydroxy group in the acronycinemoiety. Since the lowfield signal at d 7.57 of the ABM-typearomatic protons was deshielded by a carbonyl group andthe presence of H-8, H-9 and H-10 of acridone skeletonwas revealed, this indicated that the free phenolic hydroxylgroup was attached to C-11 (Basa, 1975). This location wasconfirmed by the NOESY spectrum in which a cross-peakwas observed between the hydroxyl proton H-11 (d 10.59)and the proton at H-12 (d 10.42) and by HMBC experi-ments. Therefore, the structure of alkaloid 2 was concludedto be (�)-cis-2,11-dihydroxy-1-(3,4-dihydro-6-desoxyja-careulin)amino-1,2-dihydroacronycine, named oriciacri-done B.

Compounds 1–3 were tested for their antimicrobialpotential against four bacteria (Bacillus subtilis, Streptomy-ces viridochromogenes, Staphylococcus aureus, and Esche-

richia coli), two fungi (Mucor miehei and Candida

albicans), and three microalgae (Chlorella vulgaris, Chlo-

rella sorokiniana, and Scenedesmus subspicatus) in the agardiffusion test, as shown in Table 2.

The extract exhibited in vitro significant antimicrobialactivity against C. albicans, M. miehei and S. aureus.Oriciacridone B (2) also exhibited in vitro significant

antimicrobial activity against M. miehei compared to theNystatin as reference.

3. Experimental section

3.1. General

Melting points are uncorrected; optical rotations: Per-kin–Elmer model 241 polarimeter. 1H (300 and 600 MHz)and 13C NMR spectra (75.5 and 125.7 MHz) were mea-sured on a Bruker AMX 300 and on a Varian Inova 600(599.740 MHz) spectrometer, respectively. ESI mass spec-tra were recorded on a Quattro Triple Quadrupole MassSpectrometer, Finnigen TSQ 7000 with nano-ESI-API-ion source. ESI HRMS was measured on a MicromassLCT mass spectrometer coupled with a HP 1100 HPLCwith a Diode Array Detector. Reserpin (MW = 608) andLeucin–Enkephalin (MW = 555) were used as standardsin positive and negative modes. EI-MS was recorded onVarian MAT 95 Finnigan (70 eV), high resolution withperfluorokerosine as standard. IR spectra were recordedon a Perkin–Elmer 1600 Series FT-IR spectrometer asKBr pellets. UV–VIS spectra were recorded on a Perkin–Elmer Lambda 15 UV/VIS spectrometer. Flash chroma-tography was carried out on silica gel (230–400 mesh,Merck) and silica gel (70–230 mesh, Merck) was used forcolumn chromatography. Thin layer chromatography(TLC) was performed on Polygram SIL G/UV254 (Mache-rey-Nagel & Co.).

3.2. Plant material

Stem bark of O. glaberrima was collected in January2002, at Bertoua, Cameroon. The plant was identified byNana Victor of National Herbarium. A voucher specimen(1888/HNC) documenting the collection is deposited in theNational Herbarium, Yaounde, Cameroon.

Table 2Diameter of inhibition zones of compounds 1–3 from Oriciopsis glaberrima in the agar diffusion test with 20 lg/disk (B in mm, paper disk B 9 mm)

Sample Micro-organism tested

BSa SVb SAc ECd MMe CAf CVg CSh SSi

CH2Cl2–MeOH (1/1) 10 13 12 11 17 18 15 11 13Oriciacridone A (1) 10 11 9 10 11 14 12 9 9Oriciacridone B (2) 12 11 11 9 15 12 10 9 10Lichexanthone (3) 9 10 11 9 12 11 12 10 9Nystatin 16 14 12 14 15 18 – – –

a Bacillus subtilis.b Streptomyces viridochromogenes.c Staphylococcus aureus.d Escherichia coli.e Mucor miehei.f Candida albicans.g Chlorella vulgaris.h Chlorella sorokiniana.i Scenedesmus subspicatus.


4. Extraction and isolation

Air-dried; powdered stem bark of O. glaberrima (10 kg)was extracted with a mixture of CH2Cl2/MeOH (1/1) atroom temperature during 48 h. The extract was concen-trated under reduced pressure to yield a brown viscousextract (99 g). This extract was evaluated for its antimicro-bial activity and then subjected to flash column chromatog-raphy on silica gel (500 g) (70–230 mesh, Merck) with ahexane–EtOAc–MeOH mixture of increasing polarity. Atotal of 90 sub-fractions (ca. 250 ml each) were collectedand combined on the basis of TLC analysis leading to fourmain fractions A–D. Sub-fractions 1–20, eluting with amixture of hexane–EtOAc (17:3) gave main fraction A(15 g). Fraction B (20 g) was constituted of sub-fractions21–40 eluted with a mixture of hexane–EtOAc (1:1), mainfraction C (10 g) was constituted of sub-fractions 41–66eluted with hexane–EtOAc (1:4), and fraction D (4 g) wasconstituted of sub-fractions 67–90 eluted with EtOAc–MeOH (19:1). Main fraction A was chromatographed ona silica gel column (250 g) with a hexane–EtOAc gradient.A total of 20 fractions of ca. 100 ml each were collected andcombined on the basis of TLC. Fractions 1–10 eluted witha mixture of hexane–EtOAc (9:1) yielded lichexanthone (3)(10 mg).

Main fraction C was column chromatographed over sil-ica gel with hexane–EtOAc (2:1). A total of 30 fractions ofca. 100 ml each were collected and combined on the basisof TLC. Fractions 1–10, eluted with a mixture of hex-ane–EtOAc (1:9), yielded oriciacridone A (1) (30 mg).Fractions 20–30 eluted with EtOAc yielded oriciacridoneB (2) (5 mg).

4.1. Oriciacridone A (1)

Yellow crystals; m.p. 249 �C; ½a�25D � 45:7� (DMSO, c

0.070); UV (DMSO) kmax nm (log e) 269 (4.84), 272(4.85), 285 (4.62), 323 (4.28), 352 (4.15), 401 (4.14); IR(KBr) mmax cm�1: 3360, 2972, 2362, 1920, 1731, 1649,1619,1558, 1489, 1394, 1289, 1156, 1025; 1H NMR(300 MHz, DMSO-d6) and 13C NMR (75.5 MHz,DMSO-d6) see Table 1; HR-EIMS m/z 636.2108 (calc.for C36H32N2O9, 636.21024); EI MS (70 eV) m/z (%): 636[M]+ (15), 619 (40), 580 (18), 410 (45), 363 (5), 346 (60),311 (100), 298 (80), 245 (20), 191 (30), 149 (35), 119 (28),101 (50).

4.2. Oriciacridone B (2)

Yellow crystals, m.p. 309 �C; ½a�25D � 85:3� (DMSO, c

0.075); UV (DMSO) kmax nm (log e) 239 (4.48), 257(4.60), 275 (4.40), 278 (4.40), 295 (4.12), 312 (4.13), 372(3.80), 404 (3.88); IR (KBr) mmax cm�1: 3425, 2923, 2853,2376, 2246, 1650, 1620,1558, 1502, 1471, 1380, 1360, 1279,1171, 1025, 819; 1H NMR (300 MHz, DMSO-d6) and 13CNMR (75.5 MHz, DMSO-d6) see Table 1; HR EIMS m/z652.2061 (calc. for C36H32N2O10, 652.20515); EI MS

(70 eV) m/z (%): 652 [M]+ (20), 635 (10), 620 (30), 537(40), 394 (25), 345 (15), 317 (100), 294 (80), 150 (45), 97 (18).

4.3. Lichexanthone (3)

M.p. 187–188 �C; IR (KBr), 1H NMR and EI MS datawere identical with those reported by Garcia et al. (1976).

5. Antimicrobial assay

Agar diffusion tests were performed in the usual manner(Maskey et al., 2002) with B. subtilis and E. coli (on pep-tone agar), S. aureus (Bacto nutrient broth), S. virido-chromogenes (M2 agar), the fungi M. miehei and C.

albicans (Sabouraud agar), and three microalgae (C. vulga-

ris, C. sorokiniana and S. subspicatus).Compounds 1, 2, 3 and Nystatin were dissolved in

MeOH/chloroform (87:18) at a concentration of 500 lg/ml. Paper disks (B 9 mm) were impregnated with 40 lleach using a HPLC syringe, dried for 1 h under sterile con-ditions and placed on the pre-made agar test plates.

Bacteria and fungi plates were kept in an incubator at37 �C to 12 h, micro algae plates for three days at roomtemperature in a day light incubator. The diameter of inhi-bition zones was measured.

Acknowledgments

One of the authors (J.D.W.) thanks the DAAD (Deut-scher Akademischer Austanschdienst) for a visiting grant.The authors are also grateful to Mr. R. Machinek for theNMR measurements, to Dr. H. Frauendorf for the massspectra, and to Mrs. F. Lissy for the antimicrobial screening.

References

Ayafor, F.J., Sondengam, L.B., Kimbu, F.S., Tsamo, E., Connolly, D.J.,1982. Phytochemistry 21, 2602–2603.

Bahar, M.H., Shringarpure, J.D., Kulkarni, G.H., Sabata, B.K., 1982.Structure and synthesis of atalaphylline and related alkaloids. Phyto-chemistry 21, 2729–2731.

Basa, S.C., 1975. Atalaphyllinine, a new acridone base from Atalantia

monophylla. Phytochemistry 14, 835–836.Bouquet, A., 1969. Feticheurs et Medecines Traditionnelles du Congo

Brazzaville, ORSTON, Paris, 220.Garcia, M., Ruben, F., Brown Jr., K.S., 1976. Alkaloids of three

Aspidosperma species. Phytochemistry 15, 1093–1095.Letouzey, R., 1963. Flore du Cameroun 1. Museum National d�Histoire

Naturelle, Paris.Locksley, H.D., Quillinan, A.J., Scheinmann, F., 1971. Extractives from

Guttiferae, Part XXIII. Unambiguous synthesis of 6-deoxyjacareulinand related 3,3- and 1,1-dimethylallyl and annulated xanthones. J.Chem. Soc. (C), 3804–3814.

Magiatis, P., Mitaku, S., Skaltsounis, A.L., Tillequin, F., Koch, M.,Pierre, A., Atassi, G., 1999. Synthesis and cytotoxic activity of 1-alkoxy- and 1-amino-2-hydroxy-1,2-dihydro-acronycine derivatives.Chem. Pharm. Bull. 47, 611–614.

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Magiatis, P., Mitaku, S., Skaltsounis, A.L., Tillequin, F., 2001. 1-Oxo-2-hydroxy-1,2-dihydroacronycine: a useful synthon in the acronycineseries for the introduction of amino subsistent at 6-position and for theconversion into isopropyl furanoacridones. Chem. Pharm. Bull. 49,1304–1307.

Maskey, R.P., Asolkar, R.N., Kapaun, E., Wagner-Dobler, I., Laatsch,H., 2002. Phytotoxic arylethylamides from limnic bacteria using ascreening with microalgae. J. Antibiot. 55, 643–649.

Peres, V., Nagem, T.J., 1997. Trioxygenated naturally occurring xant-hones. Phytochemistry 44, 191–214.

Peres, V., Nagem, T.J., Faustino de Oliveria, F.F., 2000. Tetraoxy-genated naturally occurring xanthones. Phytochemistry 55, 683–710.

Takemura, Y., Wada, M., Ju-Ichi, M., Ito, C., Furukawa, H., 1998. A newbimeric acridone alkaloids from Citrus pardisi MACF. Chem. Pharm.Bull. 46, 693–696.


Terpenoids and phenol derivatives from Malva silvestris

Francesca Cutillo a, Brigida D’Abrosca b, Marina DellaGreca a,*,Antonio Fiorentino b, Armando Zarrelli a

a Dipartimento di Chimica Organica e Biochimica, Universita Federico II, via Cynthia 4, I-80126 Napoli, Italyb Dipartimento di Scienze della Vita – Seconda Universita di Napoli, via Vivaldi 43, I-81100 Caserta, Italy

Received 5 September 2005; received in revised form 21 November 2005Available online 5 January 2006

Abstract

A sesquiterpene and a tetrahydroxylated acyclic diterpene as well as two known monoterpenes, 6 C13 nor-terpenes and 11 aromaticcompounds were isolated from the water extract of Malva silvestris. The structures of the compounds were determined by spectroscopicNMR and MS analysis. Effects of these compounds on germination and growth of dicotyledon Lactuca sativa L. (lettuce) were studied inthe 10�4–10�7 M concentration range.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Malva silvestris; Lactuca sativa; Phytotoxic activity; Spectroscopic analysis; Terpenes; Phenols

1. Introduction

Malva silvestris is a species widely distributed in Italyand used in traditional phytotherapy (Guarrera, 2005)and cosmetic treatments (Paufique, 2000). Fluidextract ofM. silvestris flowers and leaves are used as a valuable rem-edy for cough and inflammatory diseases of mucous mem-branes (Farina et al., 1995). The chemical composition of awater extract of M. silvestris has been investigated, andresulted in the isolation and structure elucidation of a novelsesquiterpene and a new tetrahydroxylated linear diterpeneas well as two monoterpenes, six C13 nor-terpenes and ele-ven aromatic compounds.


Fresh plants of M. silvestris were extracted with waterusing a Naviglio extractor (Naviglio, 2003). This extractoris based on the suction effect, generated by the compressionof the solvent used for extraction on solids at a pressure ofabout 8–9 bars for a particular time period, followed by

immediate decompression at atmospheric pressure. Therapid release of the liquid used for extraction from theinside of a solid matrix, because of pressure gradient, trans-ports the extractable compounds within the solid matrixtowards the outside. After extraction of the aqueous por-tion shaken with EtOAc, the organic fraction was chro-matographed on a silica gel column, the fractions werepurified by preparative thin layer chromatography andHPLC yielding 21 compounds. A test of extraction con-ducted using conventional procedure (Cutillo et al., 2005)resulted in similar amount of extract.

The compounds were identified as 4-hydroxybenzoicacid (1), 4-methoxybenzoic acid (2), 4-hydroxy-3-methoxy-benzoic acid (3), 2-hydroxybenzoic acid (4), and 4-hydroxy-2-methoxybenzoic acid (5), compounds 6 and 9as 4-hydroxybenzyl alcohol and tyrosol, compounds 7

and 8 as 4-hydroxydihydrocinnamic acid and 4-hydroxy-3-methoxydihydrocinnamic acid, and 10 and 11 as 4-hydroxycinnamic acid and ferulic acid by comparison oftheir spectral data with those of authentic samples.

Monoterpenes 12 and 13 were linalool and linalool-1-oicacid (Nicoletti et al., 1989).

The EIMS of compound 14 showed a molecular ion peakat m/z 262, and prominent peaks at m/z 245 [M � OH]+, 234[M � CO]+, and 219 [M � C3H7]+. The molecular formula


doi:10.1016/j.phytochem.2005.11.023

* Corresponding author. Tel.: +39 081 674162; fax: +39 081 674393.E-mail address: [email protected] (M. DellaGreca).



PHYTOCHEMISTRY


was determined to be C16H22O3 by HREIMS. Its IR spec-trum showed absorption bands of hydroxyl group(3300 cm�1) and phenyl group (1600 cm�1). The structureof compound 14 was established using 1H NMR and 13CNMR including COSY, NOESY, HMQC, and HMBCexperiments (Table 1). The 1H–1H COSY experimentshowed a correlation series beginning with signal of amethine at d 2.43 assigned to H-11 and coupled to two meth-yls at d 1.14 and 1.13. The signals at d 4.00 and 3.45 assignedto methylene H-14 were correlated with H-10 at d 3.45, whichin turn was coupled to the two H-9 protons at d 1.97 and 1.49.These were correlated with H-8 at d 2.12 and 1.30. Presentalso, were three singlets attributed to two methyls and amethine. The 13C NMR spectrum of 14 showed 16 carbonsignals due to four methyls, three methylenes, and threemethines. An HMQC experiment allowed to assign the pro-tons to the corresponding carbons. In the HMBC spectrumH-5 was correlated with C-1, C-3, and C-7, while the meth-oxyl group at d 3.81 and the methyl at d 2.32 were correlatedwith the C-3. The multiplet at d 3.45 (H-10) was correlatedwith C-2 and C-6. While H-14 was correlated with C-1, C-7, C-9, and C-10, thus completely dating the structure of14. The analysis of NOESY spectrum evidenced NOEs of

the methyls at d 1.14 and 1.13 with H-5 methine, and themethyl at d 2.32 with the methoxyl at d 3.81 (Fig. 1).

Compounds 15 and 16 were identified as (6R,7E,9S)-9-hydroxy-4,7-megastigmadien-3-one and blumenol A(Cutillo et al., 2005) and compound 17 as (+)-dehydro-vomifoliol (Mori, 1974) on the basis of their spectral data.

Compounds 18–20 had spectral data identical with thosereported for (3R,7E)-3-hydroxy-5,7-megastigmadien-9-one,(3S,5R,6S,7E,9R)-5,6-epoxy-3,9-dihydroxy-7-megastigmeneand (3S,5R,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigm-ene isolated from Chenopodium album and Cestrum parqui

(DellaGreca et al., 2004; D’Abrosca et al., 2004a).Compound 21 showed a molecular ion peak at m/z 340,

and peaks at m/z 325 [M � CH3]+, 322 [M � H2O]+, and297 [M � C3H7]+. The molecular formula was determinedto be C20H36O4 by HREIMS. The four oxygen functionswere ascribed to two secondary hydroxyl groups and theremaining two were attributed to tertiary hydroxyl groups(Table 2). The structure of 21 was characterized by 1HNMR and 13C NMR including COSY, NOESY, HMQC,and HMBC experiments. Five singlet methyls, ten aliphaticprotons of five methylenes, two methines bearing oxygen,five olefinic protons, two as broad triplets, and three asdouble doublets were present in the 1H NMR spectrumof 21. The 13C NMR spectrum showed 19 carbon signalsidentified as five methyls, six methylenes, and five methines.All the carbons were correlated to the corresponding pro-tons on the basis of an HMQC experiment. The tertiaryhydroxyl groups were positioned at C-3 and C-15 on thebasis of an HMBC experiment that showed correlationsbetween the C-3 carbon with the H-1, H-2, H-4 protons,and C-15 with the H-14, H-16, and H-17 protons. Further-more, NOESY correlations of H-8 with H-6, H-10, andCH3-19, and H-14 with CH3-16 and CH3-17 confirmedthe structure of diterpene 21 (Fig. 1).

The absolute configurations at the C-8 and C-14 second-ary carbinol carbons have been established by Mosher’smethod (Dale and Mosher, 1973) by converting compound21 into the diasteromeric MTPA diesters. The chemical shiftdifferences of protons, at b position of C-8 and C-14 chiralcarbons, were assigned by a 1H–1H COSY experiment(Ohtani et al., 1991). The chemical shifts comparison of thesignals due to H-9 and H-6/H-19 protons in both the (R)and the (S) MPTA derivatives and the calculation of the cor-responding differences, expressed as DdR–S, were in agree-ment with the S configuration for C-8. For the C-14

Table 1NMR spectral data of compound 14 in CDCl3

Position dHa NOESY dC HMBCb

1 125.1 (q)c

2 142.9 (q)3 144.0 (q)4 127.0 (q)5 6.69 s 12, 13, 15 115.8 (t) 1, 3, 76 139.5 (q)7 74.7 (q)8 2.12 m, 1.30 m 27.3 (s) 6, 7, 99 1.97 m, 1.49 m 23.6 (s) 1

10 3.45 m 27.8 (t) 2, 611 2.43 q (6.8) 31.2 (t) 6, 7, 12, 1312 1.14 d (6.8) 5 18.5 (p) 7, 1113 1.13 d (6.8) 5 17.8 (p) 7, 1114 4.00 dd (8.3, 2.4),

3.45 m

67.9 (s) 1, 7, 9, 10

15 2.32 s 5, 12, 13, OMe 16.1 (p) 3, 4, 5OMe 3.81 s 15 60.7 (p) 3

a 1H chemical shift values (d ppm from SiMe4) followed by multiplicityand then the coupling constants (J in Hz).

b HMBC correlations from H to C.c Letters, p, s, t and q, in parentheses indicate, respectively, the primary,

secondary, tertiary and quaternary carbons, assigned by DEPT.

OMe

OH

O

14

12

3

468

9

10

11

12

14

15

OHOHOH

21

1

47

10

1215

16

18 19 20

OH

Fig. 1. Structure of terpenes isolated from M. silvestris.

482 F. Cutillo et al. / Phytochemistry 67 (2006) 481–485

carbon, the positive DdR–S for the H-16/H-17 and negativevalue for H-13 were found, indicating R configuration forC-14. Therefore, the structure of 21 was deduced tobe (6E,8S,10E,14R)-3,7,11,15-tetramethylhexadeca-1,6,10-trien-3,8,14,15-tetraol.

The phytotoxicity of compounds 1, 3, 7–10, and 15–20

on the seeds of Lactuca sativa was previously reported (Del-laGreca et al., 2004; D’Abrosca et al., 2004a,b). Com-pounds 2, 5, and 11–14 were tested for their activities onthe seeds of L. sativa. Aqueous solutions, ranging between10�4 and 10�7 M, were tested on germination, root lengthand shoot length of treated lettuce seeds (Fig. 2). Com-pound 2 and sesquiterpene 14 reduced the germination by20% at 10�4 M respect to the control. Compounds 5, 11,and 13 were inactive at all tested concentrations. Linaloolshowed about 80% inhibition on germination at the higherconcentration tested, while no effects were observed on rootand shoot length. Among compounds tested, only 2 reducedroot length significantly. Activities of 15–30% on the shootlength at a concentration of 10�4 M have been observed forcompounds 2, 5, and 11–13 as compared to the control.

3. Experimental

3.1. General experiment procedures

1H and 13C NMR spectra were run on a Varian INOVA500 NMR spectrometer at 500 and 125 MHz, respectively,

in CDCl3 or CD3OD at 25 �C. MS spectra were obtainedwith a HP 6890 spectrometer equipped with a MS 5973N detector. IR spectra were recorded on a Jasco FT/IR-430 instrument. UV–Vis spectra were recorded in CHCl3or MeOH on a Perkin–Elmer Lambda 7 spectrophotome-ter. HPLC was performed on an Agilent 1100 by usingan UV detector. Silica gel 60 (230–400 mesh, Merck) wasused for CC, and preparative TLC was performed on silicagel (UV-254 precoated) plates with 0.5 and 1.0 mm thick-ness (Merck). Preparative HPLC was performed usingRP-18 (LiChrospher 10 lm, 250 · 10 mm i.d., Merck)column.

3.2. Plant material

Aerial parts of M. silvestris were collected near Caserta(Italy) in the spring of 2003 and identified by ProfessorAntonino Pollio of the Dipartimento di Biologia Vegetaleof University of Naples. Voucher specimens (HERB-NAQA650) are deposited at the Dipartimento di BiologiaVegetale of University Federico II of Naples.


Fresh leaves (10.0 kg) of the plant were extracted withH2O at room temperature using the Naviglio extractor.The water was reduced in volume and partitioned betweenEtOAc and H2O. The organic extract (19 g) was subjectedto silica gel column chromatography, by using CHCl3 andsuccessively increasing the EtOAc concentration by 25%,50% and 80% in CHCl3. Fractions of 200 ml were collectedand fractions with similar TLC profiles were combined.The first fraction eluted with 100% CHCl3 was purifiedby flash silica gel column chromatography with hexane–ethyl ether (1:1) to give fractions containing compounds14–20. Fraction containing crude 14 was purified by reversephase C-18 HPLC with MeOH–MeCN–H2O [(1:6:3),5 mg]: ½a�25

D �2.0� (CHCl3; c 0.5), MS: m/z 262 [M]+; HRE-IMS m/z 262.1498 (Calcd. for C16H22O3, 262.1569); NMRdata: see Table 1. Compounds 15 (10 mg), 17 (14 mg), and18 (19 mg) were purified by preparative TLC with CHCl3–Me2CO (7:3). The fraction containing crude 20 (5 mg) waspurified by preparative TLC with CH2Cl2–MeOH–H2O(11:10:9). Compounds 16 (20 mg) and 19 (20 mg) werepurified by preparative TLC with CHCl3–MeOH (19:1).

The second fraction eluted with 100% CHCl3 wasextracted with 2 N NaOH. After neutralization this fractionwas extracted with EtOAc to give 600 mg of residual mate-rial. Column chromatography on silica gel gave a fractioncontaining 4, 8 and 13. Compound 4 (20 mg) was purifiedby preparative TLC with CH2Cl2–MeOH–H2O (33:30:35)lower layer. Compounds 8 (40 mg) and 13 (10 mg) werepurified by C-18 HPLC with H2O–MeCN–MeOH (7:2:1).

The fifth fraction eluted with 50% EtOAc was purifiedby flash column chromatography on Si gel using CH2Cl2and successively increasing the Me2CO concentration from0% to 50% in CH2Cl2. Fractions eluted with 100% CH2Cl2

Table 2NMR spectral data of compound 21 in CD3OD

Position dHa NOESY dC HMBCb

1 5.19 dd, 5.03 dd

(17.0, 10.5, 1.5)112.6 (s)c 2, 3

2 5.94 dd (17.0, 10.5) 146.7 (t) 33 74.3 (q)4 1.52 dd (8.0, 7.6) 43.5 (s) 2, 3, 5, 6, 205 2.08 ddd (8.0, 7.6, 7.0) 23.8 (s) 4, 6, 76 5.34 brt (7.0) 4, 8 127.9 (t) 4, 5, 8, 197 138.4* (q)8 3.93 t (6.8) 6, 10, 19 79.2 (t) 6, 7, 9, 10, 199 2.24 m 35.2 (s) 7, 8, 10, 11

10 5.16 brt (7.0) 8, 12 122.4 (t) 12, 1811 138.2* (q)12 2.24 m, 2.05 m 38.5 (s) 10, 11, 13, 1813 1.70 m, 1.34 m 31.2 (s) 1214 3.23 dd (10.4, 2.0) 12, 16, 17 79.5 (t) 1215 74.3 (q)16 1.16 s 14 25.5 (p) 14, 15, 1717 1.15 s 14 26.1 (p) 14, 15, 1618 1.63 s 16.9 (p) 10, 1219 1.60 s 8 11.9 (p) 6, 7, 820 1.25 s 28.1 (p) 2, 3, 4

a 1H chemical shift values (d ppm from SiMe4) followed by multiplicityand then the coupling constants (J in Hz).

b HMBC correlations from H to C.c Letters, p, s, t and q, in parentheses indicate, respectively, the primary,

secondary, tertiary and quaternary carbons, assigned by DEPT.* Assignments may be interchanged.

F. Cutillo et al. / Phytochemistry 67 (2006) 481–485 483

were rechromatographed on silica gel under the same con-ditions. The subfraction eluted with 10% Me2CO was puri-fied by C-18 HPLC with MeOH–H2O (4:3) to givecompounds 1 (5 mg), 2 (3 mg), 7 (20 mg), 10 (5 mg), and11 (5 mg). The subfraction eluted with 15% Me2CO waspurified by C-18 HPLC with MeOH–H2O (4:3) to givecompounds 3 (2 mg), and 5 (2 mg). The subfraction eluted

with 50% Me2CO was purified by C-18 HPLC withMeOH–MeCN–H2O (4:1:5) to give compound 6 (5 mg).The fraction eluted with 50% Me2CO was rechromato-graphed on silica gel using CH2Cl2 and successivelyincreasing the Me2CO concentration by 20%, 40%, and100% in CH2Cl2. The fraction eluted with 20% Me2COwas purified by C-18 HPLC with MeOH–MeCN–H2O

Fig. 2. (A) Effect of compounds 2, 5, and 11–14 on germination of L. sativa L. Value presented as percentage differences from control and are notsignificantly different with P > 0.05 for Student’s t test: (a) P < 0.01; (b) 0.01 < P < 0.05. (B) Effect of compounds 2, 5, and 11–14 on root length of L.

sativa L. Value presented as percentage differences from control and are not significantly different with P > 0.05 for Student’s t test: (a) P < 0.01; (b)0.01 < P < 0.05. (C) Effect of compounds 2, 5, and 11–14 on shoot length of L. sativa L. Value presented as percentage differences from control and are notsignificantly different with P > 0.05 for Student’s t test: (a) P < 0.01; (b) 0.01 < P < 0.05.

484 F. Cutillo et al. / Phytochemistry 67 (2006) 481–485

(4:1:5) to give 12 (20 mg). The fraction eluted with 40%Me2CO was purified by preparative TLC with EtOAc–Me2CO (19:1) to give 9 (12 mg). The fraction eluted with100% Me2CO was purified by C-18 HPLC with MeOH–MeCN–H2O (3:2:5) to give 21 (2 mg): ½a�25

D +21.0� (MeOH;c 0.05), MS: m/z 340 [M]+; HREIMS m/z 340.2598 (Calcd.for C20H36O4, 240.2614); NMR data: see Table 2.

3.4. Bioassays

Seeds of L. sativa L. (cv Napoli V.F.) collected during2003, were obtained from Ingegnoli S.p.a. All undersizedor damaged seeds were discarded and the assay seeds wereselected for uniformity. Bioassays used Petri dishes (50 mmdiameter) with one sheet of Whatman No. 1 filter paper assupport. In four replicate experiments, germination andgrowth were conducted in aqueous solutions at controlledpH, using MES (2-[N-morpholino]ethanesulfonic acid,10 mM, pH 6). Test solutions (10�4 M) were prepared inMES and the rest (10�5–10�7 M) were obtained by dilu-tion. Parallel controls were performed. After adding 25seeds and 5 ml test solutions, Petri dishes were sealed withParafilm� to ensure closed-system models. Seeds wereplaced in a growth chamber KBW Binder 240 at 25 �C inthe dark. Germination percentage was determined dailyfor five days (no more germination occurred after thistime). After growth, plants were frozen at �20 �C to avoidsubsequent growth until the measurement process. Dataare reported as percentage differences from control in thegraphics and tables. Thus, zero represents the control; posi-tive values represent stimulation of the control; positivevalues represent stimulation of the parameter studied andnegative values represent inhibition.

3.5. Statistical treatment

The statistical significance of differences between groupswas determined by a Student’s t test, calculating mean val-ues for every parameter (germination average, shoot androot elongation) and their population variance within aPetri dish. The level of significance was set at P < 0.05.

Acknowledgement

NMR experiments have been performed at CentroInterdipartimentale di Metodologie Chimico-Fisiche ofUniversity Federico II of Naples on a 500 MHz spectrom-eter of Consortium INCA Lab.

References

Cutillo, F., DellaGreca, M., Previtera, L., Zarrelli, A., 2005. C13 nor-isoprenoids from Brassica fruticulosa. Nat. Prod. Res. 19, 99–103.

D’Abrosca, B., DellaGreca, M., Fiorentino, A., Monaco, P., Temussi, F.,2004a. Structure elucidation and phytotoxicity of C13 nor-isoprenoidsfrom Cestrum parqui. Phytochemistry 65, 497–505.

D’Abrosca, B., DellaGreca, M., Fiorentino, A., Monaco, P., Zarrelli, A.,2004b. Low molecular weight phenols from bioactive aqueous fractionof Cestrum parqui. J. Agric. Food Chem. 52, 4101–4108.

Dale, J.A., Mosher, H.S., 1973. Nuclear magnetic resonance enantiomerreagents. Configurational correlations via nuclear magnetic resonancechemical shifts of diastereomeric mandelate, O-methylmandelate, anda-methoxy-a-trifluoromethylphenylacetate (MTPA) esters. J. Am.Chem. Soc. 95, 512–519.

DellaGreca, M., Di Marino, C., Zarrelli, A., D’Abrosca, B., 2004.Isolation and phytotoxicity of apocarotenoids from Chenopodium

album. J. Nat. Prod. 67, 1492–1495.Farina, A., Doldo, A., Cotichini, V., Rajevic, M., Quaglia, M.G., Mulinacci,

N., Vincieri, F.F., 1995. HPTLC and reflectance mode densitometry ofanthocyanins in Malva silvestris L.: a comparison with gradient-elutionreversed-phase HPLC. J. Pharmaceut. Biomed. Anal. 14, 203–211.

Guarrera, P.M., 2005. Traditional phytotherapy in central Italy (Marche,Abruzzo, and Latium). Fitoterapia 76, 1–25.

Mori, K., 1974. Carotenoids and degraded carotenoids. IV. Syntheses ofoptically active grasshopper ketone and dehydrovomifoliol as asynthetic support for the revised absolute configuration of (+)-abscisicacid. Tetrahedron 30, 1065–1072.

Naviglio, D., 2003. Naviglio’s Principle and presentation of an innovativesolid–liquid extraction technology: extractor Naviglio. Anal. Lett. 36,1647–1659.

Nicoletti, M., Tommassini, L., Serafini, M., 1989. Two linalool-1-oic acidsfrom Kickxia spuria. Fitoterapia 60, 252–254.

Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-fieldNMR application of Mosher’s method. The absolute configurations ofmarine terpenoids. J. Am. Chem. Soc. 113, 4092–4096.

Paufique, J.J., 2000. Method for extracting a active principle based onMalva sylvestris, the active principle obtained, and cosmetic treatmentusing it. Patent Application: FR 2000-11973 20000920.

F. Cutillo et al. / Phytochemistry 67 (2006) 481–485 485

Hydroquinone diglycoside acyl esters from the stems ofGlycosmis pentaphylla

Junsong Wang, Yingtong Di, Xianwen Yang, Shunlin Li, Yuehu Wang, Xiaojiang Hao *

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,

Heilong tan, Kunming 650204, PR China

Received 1 August 2005; received in revised form 30 October 2005Available online 19 January 2006

Abstract

Four hydroquinone diglycoside acyl esters, glypentosides A–C (1–3) and seguinoside F (4), were isolated from the stems of Glycosmis

pentaphylla. Glypentosides A–B (1–2) were identified as compounds and designated as methoxyquinol 4-O-[(5-O-trans-p-coumaroyl)-b-D-apiofuranosyl-(1! 2)-b-D-glucopyranoside] (1) and 4-demethylantiarol 4-O-[(3-methoxy-4-hydroxy-benzoyl)-b-D-apiofuranosyl-(1! 2)-b-D-glucopyranoside] (2). Glypentoside C (3) is a hydroquinone diglycoside acyl ester with a neolignan moiety in the acyl unit.Their structures were elucidated by the combination of one- and two-dimensional NMR analysis, mass spectrometry and chemicalevidences.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Glycosmis pentaphylla; Rutaceae; Glypentosides A–C; Hydroquinone glycosides; Acyl esters; Apiosyl-(1! 2)-glucosides

1. Introduction

The genus Glycosmis of the family Rutaceae is repre-sented in China by nearly 11 species (Huang, 1997). Glycos-

mis pentaphylla (Retz.) DC. is a shrub or small (1.5–5 m)tree widely distributed from India, Malaysia and southernChina to the Philippine Islands where it occurs in tropicalforests at low altitudes. It has been used as a folk medicinein the treatment of fever, liver complaints and certain otherdiseases (Sastri, 1956). Phytochemical researches of thisspecies were mainly focused on hydrophobic alkaloids,including those of the quinolone (Bhattacharyya andChowdhury, 1985), quinazoline (Muthukrishnan et al.,1999; Sarkar and Chakraborty, 1979), acridone (Quaderet al., 1999) and carbazole (Jash et al., 1992; Chowdhuryet al., 1987) types, of leaves, root and stem bark. No studyon glycosidic constituents of G. pentaphylla has been

reported. In this paper, we present results of an study onthe polar constituents of the stem wood of the plant.


The EtOAc-soluble fraction of the MeOH extract wassubjected to a succession of chromatographic proceduresand finally by preparative ODS-HPLC to give three newhydroquinone diglycosides, namely glypentoside A (1),glypentoside B (2) and glypentoside C (3), besides theknown compound seguinoside F (4). The known com-pound was identified by comparing its spectral data withthose previously reported (Zhong et al., 1998).

Compound 1 was isolated as amorphous powder. Itsmolecular formula C27H32O14 was deduced from the nega-tive HRFABMS spectrum and 13C NMR spectral data.The IR spectrum showed absorption bands due to hydro-xyl (3420 cm�1) and carbonyl groups (1690 cm�1). The1H NMR signals due to aromatic and olefinic protons atd 7.54 (1H, d, J = 15.7 Hz), 7.37 (2H, d, J = 8.4 Hz), 6.79


doi:10.1016/j.phytochem.2005.11.025

* Corresponding author. Tel.: +86 871 522 3263; fax: +86 871 515 0227.E-mail address: [email protected] (X. Hao).



PHYTOCHEMISTRY


(2H, d, J = 8.4 Hz), 6.20 (1H, d, J = 15.7 Hz), as well asone ester carbonyl carbon at d 168.8, suggested the pres-ence of one trans-p-coumaroyl moiety. The 1H and 13CNMR spectra of 1 (Table 1) also revealed the presence ofglucopyranose and apiofuranose moieties. The b-anomericconfiguration for the glucopyranose was determined from alarge coupling constant value (7.3 Hz) of the anomeric pro-ton (Agrawal, 1992; Ishii and Yanagisawa, 1998). The b-anomeric configuration for the apiofuranose was indicatedfrom the anomeric signals at dC 110.5 (Kitagawa et al.,1993) with dH 5.48 (1H, d, J = 1.8 Hz) (Mbaırarouaet al., 1994; Otsuka et al., 1994). On acid hydrolysis, 1

afforded D-glucose and D-apiose as component sugars,which was identified by TLC and GLC analysis. The apio-syl-(1! 2)-glucosyl linkage of the glycosidic moiety wasassigned from the cross-peaks observed between apioseH-1 and glucose H-2 in the NOESY spectrum. Also inthe HMBC spectrum (Fig. 1) of 1, a correlation was evi-dent between apiose H-1 (d 5.48) and glucose C-2 (d78.8). The position was confirmed by the chemical shift(d 78.8) of glucose C-2, as compared with a nonsubstitutedC-2, which is ca. d 74.0. All chemical shifts of this sugarmoiety are in good agreement with the literature data

(Zhong et al., 1998). The significant deshielding of H-5 ofapiose (4.26 and 4.37 ppm) and the HMBC cross-peakbetween the proton at 4.37 ppm and the carbonyl carbonat 168.8 ppm confirmed that the coumaroyl unit wasattached to position 5 of apiose.

The 13C NMR spectrum of 1 showed, for the aglyconportion, seven signals. These were assigned to a methoxygroup, three to aromatic CH, and three to phenolic func-tions. The 1H NMR spectrum contained the signals forthree aromatic protons at d 6.70 (1H, d, J = 2.4 Hz), 6.62(1H, d, J = 8.2 Hz), and 6.48 (1H, dd, J = 2.4, 8.2 Hz),along with a signal for a methoxy group at d 3.86, whichcorrelated in the HMBC spectrum with a signal at dC

149.2, corresponding to a typical methoxyquinol. The siteof glycosidation was revealed to be C-4 by HMBC experi-ment, which showed a long-range correlation between C-4(d 152.6) and the anomeric proton (d 4.80) of glucose,which was further supported by the NOE cross-peaks(Fig. 2) observed from the anomeric proton (d 4.80) of glu-cose to two aromatic protons at dH 6.48 (1H, dd, J = 2.4,8.2 Hz) and 6.70 (1H, d, J = 2.4 Hz). Based on the aboveresults, the structure of glycopentoside A (1) was estab-lished as methoxyquinol 4-O-(5-O-trans-p-coumaroyl)-b-D-apiofuranosyl-(1! 2)-b-D-glucopyranoside.

Table 1The 1H NMR (400 MHz) and 13C NMR (100 MHz) data of 1 and 2 inCD3OD (J in Hz within parentheses)

No. 1 2

dH (J in Hz) dC dH (J in Hz) dC

1 142.9 s 155.5 s

2 149.2 s 5.99 s 94.5 d

3 6.70 d (2.4) 103.4 d 154.8 s

4 152.6 s 128.5 s

5 6.48 dd (2.4, 8.2) 109.6 d 154.8 s

6 6.62 d (8.2) 116.1 d 5.99 s 94.5 d

10 4.80 d (7.3) 102.0 d 4.94 d (7.6) 102.7 d

20 3.58–3.65 78.8 d 3.62–3.72 78.8 d

30 3.58–3.65 78.5 d 3.62–3.72 78.5 d

40 3.33–3.36 71.7 d 3.33–3.37 71.7 d

50 3.33–3.36 78.1 d 3.33–3.37 78.1 d

60 3.58–3.65, 3.85a 62.6 t 3.62–3.72, 3.82a 62.6 t

100 5.48 d (1.8) 110.5 d 5.50 d (2.2) 110.5 d

200 4.02 d (1.8) 78.7 d 4.00 d (2.2) 78.7 d

300 79.2 s 79.6 s

400 Ha 3.88 d (9.5)Hb 4.28 d (9.5)

75.4 t Ha 3.90 d (9.8)Hb 4.38 d (9.8)

75.7 t

500 Ha 4.26 d (11.2)Hb 4.37 d (11.2)

67.6 t Ha 4.36 d (10.8)Hb 4.43 d (10.8)

67.8 t

1000 127.1 s 122.4 s

2000 7.37 d (8.4) 131.2 d 7.44 d (1.8) 113.6 d

3000 6.79 d (8.4) 116.9 d 152.9 s

4000 161.3 s 148.6 s

5000 6.79 d (8.4) 116.9 d 6.78 d (8.2) 115.9 d

6000 7.37 d (8.4) 131.2 d 7.47 dd (8.2,1.8) 125.2 d

a 168.8 s 168.1 s

b 6.20 d (15.7) 114.8 d

c 7.54 d (15.7) 146.9 d

OMe 3.86 s 56.4 q 3.83 s (MeO-3000) 56.3 q (MeO-3000)3.70 s (MeO-3,5) 56.7 q (MeO-3,5)

a Signal pattern unclear due to overlapping.

OMe

OH

O

HO

23

5 6

5'''β

γα

1

1'2'''

3'''

OMe

OH

O

O

HO

MeO

1

2

1'

6' 4

1''

5

OMe

OH

OH

O

O

OH

OH OH

O

O

O

OH

OH

O

O

OH

OH OH

O

O

O

1'''

6'''

1''

5''

46'

4'''

5''1'''2'''

3'''

4'''

5'''6'''

7'''

8'''

9''''

9'''

8''''7''''

1''''

2''''3''''4''''

5''''

6''''OH 3

6

1

3

OMe

OH

O

HOOH

OH

O

O

OH

OH OH

O

O

O

Fig. 1. Selected HMBC correlations of 1.

J. Wang et al. / Phytochemistry 67 (2006) 486–491 487

Glycopentoside B (2) was isolated as amorphous pow-der, whose elemental composition was determined to beC27H34O16. Its IR spectrum showed hydroxyl groups(3424 cm�1), a conjugated ester group (1706 cm�1), andaromatic ring (1605; 1513 cm�1). Comparison of its 1HNMR and 13C NMR data (Table 1) with those of com-pound 1 suggested the same sugar portion. Furthermore,the signals attributable to a ABX-coupling system wereobserved at d 7.44 (1H, d, J = 1.8 Hz), 6.78 (1H, d,J = 8.2 Hz), and 7.47 (1H, dd, J = 1.8, 8.2 Hz), in combina-tion with the signal for a carbonyl carbon at d 168.1 in the13C NMR spectrum, suggested a 1,3,4-substituted benzoylmoiety in 2. HMBC correlations (Fig. 3) were observedbetween the proton signals at d 7.44 (H-2000) and 7.47 (H-6000) and the carbonyl carbon, as well as methoxy signals

at d 3.83 and the carbon signal at 152.9, which confirmedthe presence of a vanillic acid unit in the compound. TheHMBC spectrum of 2 confirmed that the ester linkage ison the hydroxyl group on C-5 of apiose, since significantcross-peaks were observed between dC 168.1 and dH 4.36and 4.43.

In the 1H NMR spectrum of 2, the only features for theaglycon moiety were a signal at d 5.99 (2H, s) and a signalfor a methoxy group at d 3.70 (6H, s). The shielded aro-matic protons allowed us to propose the presence of iso-lated aromatic protons in symmetrical relationship withthe rest of the molecule, positioned in an electron donorenvironment. The 13C NMR spectrum of 2 exhibited, forthe aglycon moiety, five signals. These were indicative fortwo methoxy group, two aromatic CH, and four phenolicfunctions.

The location of the methoxy groups at C-3,5 and of thedisaccharide chain at C-4 were deduced from the HMBCcorrelations between the proton signal at d 3.70 (OMe)and the carbon resonance at d 154.8 (C-3,5), and betweenthe anomeric signal of the glucose unit at d 4.94 and thecarbon resonance at d 128.5 (C-4). Thus, the structure 4-demethylantiarol 4-O-(3-methoxy-4-hydroxy-benzoyl)-b-D-apiofuranosyl-(1! 2)-b-D-glucopyranoside was assignedto 2.

Glycopentoside C (3) was isolated as an amorphouspowder. The molecular formula was established asC38H44O18 by HRFABMS. In comparison of the 1H and13C NMR spectra (Table 2) of 3 with those of 1, the signalsdue to sugars and the aglycon were superimposable. Onacid hydrolysis, 3 afforded D-glucose and D-apiose as com-ponent sugars. These suggested that 3 was also methoxyqu-inol apiosyl-(1! 2)-glucoside acyl ester. The 1H NMR

OMe

OH

O

HO

OMe

4

OH

OH

O

O

OH

OH OH

O

O

O

OMe

OH

O

HO

OH

OH

O

O

OH

OH OH

O

O

O

OHOOH

OH

O

O

O

OH

OMe

OH OH

O O

HO

2

1

1'

23

5 6

6'

MeO

MeO

4

1''

5''1'''

2'''4'''

5'''6'''

3'''

α

Fig. 2. Key NOEs correlations of 1.

OH

OHOH

O

O

O

OH

OMe

OH OH

O

O

O

HO

MeO

MeO


Table 2The 1H NMR (400 MHz) and 13C NMR (100 MHz) data of 3 in CD3OD(J in Hz within parentheses)

No dH (J in Hz) dC No dH (J in Hz) dC

1 142.9 s 3000 145.8 s

2 149.2 s 4000 152.1 s

3 6.68 d (2.2) 103.2 d 5000 131.7 s

4 152.7 s 6000 7.08 s 119.3 d

5 6.46 d (8.2, 2.2) 109.5 d 7000 7.55 d (15.8) 147.1 d

6 6.62 d (8.2) 116.1 d 8000 6.25 d (15.8) 115.5 d

10 4.81 d (7.8) 102.0 d 9000 168.7 s

20 3.54–3.65 78.8 d 10000 134.1 s

30 3.54–3.65 78.3 d 20000 6.94 d (2.0) 110.6 d

40 3.34 m 71.7 d 30000 149.2 s

50 3.34 m 78.1 d 40000 147.8 s

60 3.86a, 3.54–3.65 62.6 t 50000 6.77 d (8.2) 116.2 d

100 5.53 d (2.0) 110.5 d 60000 6.82 dd (2.0, 8.2) 119.9 d

200 4.01 d (2.0) 78.7 d 70000 5.57 d (6.4) 89.9 d

300 79.2 s 80000 3.54 m 54.7 d

400 Ha 3.78 d (10.2)Hb 4.24 d (10.2)

75.4 t 90000 3.83a 64.6 t

500 Ha 4.20 d (11.0)Hb 4.28 d (11.0)

67.6 t 2-OMe 3.81 s 56.4 q

1000 129.6 s 3000-OMe 3.88 s 56.8 q

2000 7.04 s 113.6 d 30000-OMe 3.75 s 56.4 q

a Signal pattern unclear due to overlapping.

488 J. Wang et al. / Phytochemistry 67 (2006) 486–491

spectrum revealed the remaining signals of five aromaticprotons enclosed in two aromatic systems: an AX systemcorresponding to a 1,2,3,5-tetrasubstituted ring and anAMX system corresponding to a 1,2,4-trisubstituted ring.The aromatic region of the 1H NMR spectrum of 3 showedone AB set of signals at d 6.25 and 7.55 (1H, d,J = 15.8 Hz). The coupling constants of the vinylic systemindicated that they have a trans configuration. HMBC cor-relations (Fig. 4) between the proton resonances of thevinylic system with the carbon resonance of the estergroup, suggested that compound 3 have one a,b-unsatu-rated ester moieties in its structure. The presence of thesemoieties was also confirmed by the presence of a band at1699 cm�1 in the IR spectrum of 3. In the 1H NMR spec-trum of 3, an ABC set of signals can also be found. Theyare due to proton resonances of a methine group [dH 3.54(m); dC 54.7], one benzylic methine group [dH 5.57 (d,J = 6.4 Hz); dC 89.9] and also a methylene group (dH

3.83; dC 64.6). The 13C NMR spectrum of 3 displayed char-acteristic signals for three methoxy groups. Besides thesesubstituent signals, 18 skeletal carbon resonances appearedin the 13C NMR spectrum of the acyl unit of 3, which, incombination with its 1H NMR data, suggested that the acylmoiety of 3 is a dihydrobenzo[b]furan neolignan (Chakrav-arty et al., 1996; Li et al., 1997). HMBC correlations(Fig. 4) led to the planar structure of the acyl moiety.The NOE interactions (Fig. 5) between OMe/H-3, OMe/H-2000 and OMe/ H-20000 allowed the locations of the meth-oxy at C-2, C-3000 and C-30000. Since the coupling constant(J = 6.4 Hz) of H-70000 was similar to J = 6.2 Hz of H-7 intrans-dehydrodiconiferyl alcohol (Wang et al., 1992), therelative configuration of C-70000 and C-80000 in 3 was deter-

mined as trans form which was also confirmed by a lackof correlation between H-70000 and H-80000 and a pronouncedcoupling between the two protons at C-90000 and C-70000 fromthe NOESY spectrum (Fig. 5).

The absolute configuration of the dihydrofuran ring wasdetermined using CD spectroscopic evidence. The CDspectrum of 3 showed a negative Cotton effect at 287 nm,providing evidence that the configuration in 3 must be70000S, 80000R (Lynn et al., 1987; Wang and Jia, 1997). Thelinkage of acyl moiety with sugar moiety was solved byanalysis of the HMBC spectrum. In the HMBC spectrum,the carbonyl group (dC 168.7) not only showed correlationwith protons assigned to double bond but also had correla-tion with methylene protons at d 4.20 (1H, d, J = 11.0) and4.28 (1H, d, J = 11.0 Hz), suggesting the acyl moiety is con-nected to the hydroxyl group on C-5 of apiose. This con-firmed the structure of glycopentoside C is shown as thatof 3.

The four hydroquinone diglycoside acyl esters identifiedin this investigation of glycosidic metabolites from thegenus Glycosmis is the first report of a compound of thistype in the Rutaceae. However, since no studies on thepolar constituents of the stems of other species have beencarried out, whether this has taxonomic value remainsunsure.

These phenolic glycoside esters may store in the plant asprecursors of ‘post-inhibitin’ and has a defensive ecologicalrole. The sugar moiety appears to stabilize the molecules,preventing dehydrogenation to give hydroquinones whichhave been reported to show antimicrobial (Jin and Sato,2003; Ma et al., 1999; Perry and Brennan, 1997) and cyto-toxic activities (Perry and Brennan, 1997) and to be allelo-pathic agents (Weidenhamer and Romeo, 2004). However,glycoside form of these hydroquinone generally exhibitedlow activities (Jin and Sato, 2003; Ma et al., 1999; Perryand Brennan, 1997; Weidenhamer and Romeo, 2004).Active phenolic toxins are released from the correspondingglycosides by enzymic hydrolysis caused by microbial inva-sion or herbivore attack on foliage against the invasion ofpathogens under environmental conditions (Harborne,1988).

3. Experimental

3.1. General procedures

1H, 13C, and 2D NMR spectra were recorded on a Bru-ker AM 400 NMR and a DRX-500 spectrometer withTMS as internal standard. MS data were obtained on aVG AutoSpec 3000 spectrometers. UV spectra wereobtained on a Shimadzu double-beam 210A spectropho-tometer. The IR (KBr) spectra were obtained on aBio-Rad FTS-135 spectrometer. HPLC separations wereperformed on a HP 1100 apparatus equipped with Diodearray UV detector and XTERRA� C18 (Waters, 10 lm,15 · 200 mm, flow rate: 15 mL/min) column. GC–MS

OMe

OH

O

O

HO

MeO

OMe

OH

OHO

O

OH

OH OH

O

O

O

OH


OMe

OH

O

O

HO

MeO

OMe

OH

OHO

O

OH

OH OH

O

O

O

OH

Fig. 5. Key NOEs correlations of 3.


was run on a FISONS MD-800 instrument. The CD spec-tra were measured with a JASCO-715 spectropolarimeter.Optical rotations were measured with a JASCO DIP-370digital polarimeter in MeOH solution.

3.2. Plant material

The stems of G. pentaphylla were collected in Xishu-angbanna, Yunnan, China., in March 2003. The plantmaterial was identified by Prof. De-Ding Tao, and a vou-cher specimen (BN20030412) was deposited in the Herbar-ium of Kunming Institute of Botany, Chinese Academy ofSciences.


The air-dried material (10 kg) was finely pulverized andextracted by percolation with MeOH for one month atroom temperature. The combined extracts were filteredand concentrated under vacuum to obtain a crude extract(350 g). The extract was partitioned between water andCHCl3 and then further extracted with EtOAc. TheEtOAc-soluble fraction (25 g) was fractionated by columnchromatography over D101 porous resin using gradientaqueous ethanol to give six fractions (fractions I–VI).

Fraction II (5 g) was subjected to column chromatogra-phy over D101 porous resin and eluted with 5–30% Me2COin H2O, yielding fractions II-1–II-5. Fraction II-2 (956 mg)was subjected to medium pressure chromatography(MPLC) over C18 Si gel and eluted with MeOH–H2O(1:9–5:5) under gradient conditions yielding fractions II-2-1–II-2-6. Fraction II-2-4 (66 mg) was applied to Sepha-dex LH-20 with MeOH. The major component eluted asa yellow band (32 mg) and was further purified by RP-18preparative HPLC with MeOH–H2O (2:8), yielded com-pound 2 (7 mg) at 26.4 min. Fraction II-3 (3 g) was appliedto RP-18 MPLC and eluted with Me2CO–H2O (1:9–3:7)under gradient conditions yielding fractions II-3-1–II-3-6.Fraction II-3-1 (1.3 g) was again applied to RP-18 MPLCand eluted with MeOH–H2O (1:9–5:5) yielding fractionsII-3-1-1–II-3-1-6. Fraction II-3-1-1 (600 mg) and FractionII-3-1-5 (90 mg) were chromatographed on Sephadex LH-20 with MeOH and further purified by successive RP-18preparative HPLC with 40% MeOH to obtain 1 (5 mg, tR

26.1 min), and with 30% MeOH to afford 3 (10 mg, tR

41.4 min), respectively.

3.3.1. Methoxyquinol 4-O-[(5-O-trans-p-coumaroyl)-b-D-

apiofuranosyl-(1! 2)-b-D-glucopyranoside]

(Glycopentoside A), 1Amorphous powder; ½a�25

D : �7.5� (c 0.33, MeOH); UV(MeOH) kmax nm (loge): 313 (4.12), 289 (4.15), 206 (4.55);IR mmax (KBr) cm�1: 3420, 2937, 2076, 1690, 1605, 1514,1454, 1361, 1271, 1200, 1168, 1110, 1074, 1030, 945, 833;1H and 13C NMR spectral data: see Table 1; HRFABMS(negative-ion mode) m/z: 579.1726 [M � H]� (C27H31O14

requires 579.1714).

3.3.2. 4-Demethylantiarol 4-O-[(3-methoxy-4-hydroxy-

benzoyl)-b-D-apiofuranosyl-(1! 2)-b-D-glucopyranoside]

(Glycopentoside B), 2Amorphous powder; ½a�25

D :�60.7� (c 0.48, MeOH); UV(MeOH) kmax nm (loge): 267 (4.03), 206 (4.62); IR mmax

(KBr) cm�1: 3424, 2940, 2850, 2075, 1706, 1605, 1513,1465, 1429, 1336, 1284, 1219, 1119, 1073, 1029, 817; 1Hand 13C NMR spectral data: see Table 1; HRFABMS (neg-ative-ion mode) m/z: 613.1773 [M � H]� (C27H33O16

requires 613.1769).

3.3.3. Glycopentoside C, 3Amorphous powder; ½a�25

D :�49.7� (c 0.84, MeOH); UV(MeOH) kmax nm (loge): 329 (4.34), 290 (4.14), 224 (4.44),205 (4.70); IR mmax (KBr) cm�1: 3424, 2937, 2885, 2059,1699, 1630, 1606, 1514, 1454, 1432, 1336, 1274, 1144,1072, 1030, 943, 837, 803; 1H and 13C NMR spectral data:see Table 2; HRFABMS (negative-ion mode) m/z:787.2462 [M � H]� (C38H43O18 requires 787.2449). CD(MeOH): [h]287 = �12400�.

3.4. Acid hydrolysis, TLC and GC analysis of 1–3

Each solution of 1–3 (each 2 mg), in 1 M HCl (dioxane–H2O, 1:1, 2 mL) was heated at 95 �C for 2 h in a waterbath. After removing the solution under a stream of nitro-gen, the residue was suspended with H2O and extractedwith EtOAc three times. The aqueous layer was then neu-tralized with NaHCO3 and concentrated to dryness underreduced pressure.

The residue was compared with standard sugars by co-thin layer chromatography (CHCl3–MeOH–H2O–HOAc,16:9:2:2; detection with spray agent: 4% a-naphthol–EtOH–5% H2SO4). Hexoses gave purple spots and pen-toses blue spots. The Rf values of each sugar are as follows:glucose, 0.42 and apiose, 0.52.

The residue was dried and dissolved in pyridine(0.5 mL). Then trimethylchlorosilane (0.5 mL) was addedand the reaction mixture was kept at ambient temperaturefor 20 min. After concentrated to dryness under reducedpressure, the residue was dissolved in diethyl ether and thendirectly subjected to GC–MS [column: 30 m · 0.32 mm(30QC2/AC5)] analysis under the following conditions:electron-impact (EI) mode (70 eV), temperature program-ming from 180 to 240 �C at 5 �C/min; carrier N2 gas. Inthe acid hydrolysate of 1–3, D-glucose and D-apiose wereconfirmed by comparison of the retention times of theirTMSi derivatives with those of D-glucose and D-apiosederivatives prepared in a similar way, which showed reten-tion times of 6.86 and 3.08 min, respectively.

Acknowledgments

The authors gratefully acknowledge financial supportfrom the National Natural Science Foundation of China(NSFC) for Distinguished Young Scientists to X.-J. Hao

490 J. Wang et al. / Phytochemistry 67 (2006) 486–491

(39525025, 30070087). We are grateful to members of theanalytical group in the Laboratory of Phytochemistry,Kunming Institute of Botany for the spectralmeasurements.

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Unusual chromenes from Peperomia blanda

Leosvaldo S.M. Velozo a, Marcelo J.P. Ferreira b, Maria Isabel S. Santos c,Davyson L. Moreira a, Vicente P. Emerenciano b,*, Maria Auxiliadora C. Kaplan a

a Nucleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazilb Instituto de Quımica, Quımica Fundamental, Universidade de Sao Paulo, Caixa Postal 26077, CEP: 05513-970, Sao Paulo, SP, Brazil

c Departamento de Produtos Naturais e Alimentos, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil

Received 23 June 2005; received in revised form 9 September 2005Available online 3 February 2006

Abstract

From the methanol extract of the aerial parts of Peperomia blanda (Piperaceae), two chromenes were isolated and characterizedmainly through application of 2D-NMR spectroscopy. The structures were 2S-(4-methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7-dimethyl-2H-chromene and 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7-dimethyl-2H-chromene named as blandachromenes Iand II, respectively.� 2006 Published by Elsevier Ltd.

Keywords: Peperomia blanda; Piperaceae; Chromenes; Aerial parts

1. Introduction

The Peperomia genus belongs to the Piperaceae familythat comprises some 600 species (Mabberley, 1993) widelydistributed in southeast Brazil. Chemical studies carriedout on Piperaceae species have revealed the occurrence ofa variety of compounds including essential oils, pyrones,lignoids, polyphenols, unsaturated amides and alkaloids(Parmar et al., 1997, 1998; Moreira et al., 1998a,b; Baldo-qui et al., 1999). These species have been extensively inves-tigated as a source of new natural products with potentialantimicrobial, antitumor and insecticidal activities (Cost-antin et al., 2001; Min et al., 2004; Konishi et al., 2005; Sac-chetti et al., 2005). In contrast to the extensive studies ofthe Piper compounds (Parmar et al., 1997, 1998), few phy-tochemical studies of Peperomia have been reported. Previ-ous phytochemical investigations on different species ofPeperomia have shown the presence of flavonoids (Aqilet al., 1993), benzopyran derivatives (Seeram et al., 1998;

Mbah et al., 2002; Salazar et al., 2005), secolignans (Chenet al., 1989; Monache and Compagnone, 1996; Govinda-chari et al., 1998), terpenes, arylpropanoids, phenolic com-pounds (Tanaka et al., 1998; Moreira et al., 1999; Baymaet al., 2000; Li et al., 2003) and essential oils (Bessiereet al., 1994; Silva et al., 1999; Zoghbi et al., 2005). Somebiological activities were found in compounds isolatedfrom the Peperomia genus, e.g., prenylated phenols, withantiparasitic activity, from P. galioides (Mahiou et al.,1995, 1996), as well as analgesic activity with mice thatencountered extracts of aerial parts from P. pellucida(Aziba et al., 2001). As part of our studies on Brazilian Pip-eraceae species, we have performed a phytochemical exam-ination of the aerial parts of P. blanda collected in theBrazilian Atlantic Forest. In this paper, we describe the iso-lation and structural elucidation of two chromenes fromthe non-polar fraction of P. blanda.


The hexane solubles of a methanol extract, obtainedfrom the aerial parts of P. blanda, afforded a mixture of

0031-9422/$ - see front matter � 2006 Published by Elsevier Ltd.

doi:10.1016/j.phytochem.2005.12.012

* Corresponding author. Tel.: +55 11 30912056; fax: +55 11 38155579.E-mail address: [email protected] (V.P. Emerenciano).



PHYTOCHEMISTRY


three sterols – stigmasterol, sitosterol and campesterol –and two unusual chromenes (compounds 1 and 2, Fig. 1),which were isolated and identified.

Compound 1 (Blandachromene I) was obtained as a yel-lowish oil and its molecular formula was assigned asC18H22O3 by HREIMS showing an [M+] ion at m/z286.1536. The mass spectral fragmentation pattern showeda base peak at m/z 203.0695 [C12H11O3]+ and a signal atm/z 271 due to the fragment [M-15]+ that resulted fromloss of a methyl group. Its IR spectrum showed a broadband at 3350 cm�1 and an absorption at 1738 cm�1 whichindicated the presence of a hydroxyl group and an aldehydecarbonyl group, respectively. The absorptions at 1651 and1584 cm�1 suggested an aromatic ring. The 1H NMR spec-trum (Table 1) showed only one resonance due to an aro-matic proton at d 6.95 indicating a pentasubstitutedaromatic ring that was correlated with carbon signals ofC4, C4a, C7, C8a and C9. An AB system (d 5.65 and6.55, Ja,b = 10.0 Hz) suggested a cis-olefin with the signalat d 5.65 being attributed to H3 which was correlated withC2, C4, C4a, C1 0 and C100. On the other hand, the reso-nance at d 6.55 exhibited correlations with C2, C4a, C5and C8a confirming the location of the aromatic proton

in C5. The presence of an aldehyde carbonyl group is indi-cated from the singlet at d 10.3 and its correlations whichwere observed with C5, C6 and C7, demonstrating thatthe aldehyde group should be attached to C6. The aromaticmethyl group (C10) at d 2.40 was located at C7 based onobserved correlations with C6, C7 and C8. Additional sig-nals included an olefinic proton at d 5.10 (1H, m), four cou-pled aliphatic protons at d 2.05 (2H, m, H-2 0) and d 1.70(2H, t, H-1 0), besides three singlets corresponding tomethyl groups at d 1.35, 1.50 and 1.60. The correlationswith C2, C3, C1 0 observed for the first methyl group con-firmed its position to an oxygen-bearing carbon. The otherdata suggested a 4-methyl-3-pentenyl substitution in posi-tion C2 of the chromene skeleton. The 13C NMR spectro-scopic data of 1 (Table 1) are in agreement with theproposed assignments and confirm the presence of an alde-hyde carbonyl group (d 190.0), an oxygen-bearing aromaticcarbon (C-8) at d 160.0 and a methyl (C-10) at d 22.0substituted aromatic carbon (C-7) at d 142.5. Based onthe HMBC analysis (Table 1), the methyl and the 4-methyl-3-pentenyl units were attached to C2 (d 79.9) of apyrane ring. Comparative phytochemistry using 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran

4'

1''

1'

10

9

82

4

O

O

OH

4

28

9

10

1'

1''

4'

OH

O

O

1 2

Fig. 1. Chromenes isolated from Peperomia blanda.

Table 1NMR spectroscopic data of chromenes 1 and 2 isolated from Peperomia blanda (500 MHz for 1H NMR and 125 MHz for 13C NMR, DMSO-d6)

No. 1 2

1H NMR 13C NMR HMBC 1H NMR 13C NMR HMBC

2 79.9 80.03 5.65 d (J = 10.0 Hz) 127.5 C2, C4, C4a, C1 0, C100 5.65 d (J = 10.0 Hz) 127.5 C2, C4, C4a, C1 0, C100

4 6.55 d (J = 10.0 Hz) 116.5 C2, C4a, C5, C8a 6.60 d (J = 10.0 Hz) 115.0 C2, C4a, C5, C8a4a 106.1 106.45 6.95 s 111.3 C4, C4a, C7, C8a, C9 160.06 116.0 113.07 142.5 145.08 160.0 6.25 s 111.0 C4a, C6, C7, C8a, C108a 158.0 160.09 10.30 s 190.0 C5, C6, C7 10.0 s 196.2 C5, C6, C710 2.40 s 22.0 C6, C7, C8 2.45 s 18.0 C6, C7, C81 0 1.70 t (J = 6.9 Hz) 40.0 C2, C20, C3 0 1.65 t (J = 6.9 Hz) 40.0 C2,C20, C30

2 0 2.05 m 22.5 C2, C10, C3 0, C40 2.00 m 22.5 C2, C10, C3 0, C40

3 0 5.10 m 124.2 C1 0, C20, C5 0, C60 5.05 m 124.0 C1 0, C20, C5 0,C60

4 0 131.2 131.55 0 1.60 s 25.7 C3 0, C40, C6 0 1.60 s 25.8 C3 0, C40, C6 0

6 0 1.50 s 17.5 C3 0, C40, C5 0 1.50 s 17.5 C3 0, C40, C5 0

100 1.35 s 26.0 C2, C3, C10 1.37 s 27.0 C2, C3, C10

OH 12.6 C4a, C5, C6

L.S.M. Velozo et al. / Phytochemistry 67 (2006) 492–496 493

(Ferraz et al., 2001), lhotzchromene (Moreira et al., 1998a),clusifoliol (Seeram et al., 1998), methyl 8-hydroxy-2,2-dimethyl-2H-chromene-6-carboxylate (Orjala et al., 1993)and galopiperone (Mahiou et al., 1996) as models, furthersupports the proposed structure for 1 as 2-(4-methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7-dimethyl-2H-chromene.The optical rotation, ½a�25

D + 26.0 was very similar to thatobserved for sargatriol (Kikuchi et al., 1983), although thiscompound is not appropriate for such direct comparisonsince its side-chain has two additional chiral centers. Nev-ertheless, the CD curve for blandachromene I (Fig. 2) wasfully opposite to that observed for the sargatriol and dau-richromenes (Iwata et al., 2004) including the positive Cot-ton effect at 260–280 nm. Therefore, despite differences inthe substitution pattern in the aromatic ring of blanda-chromene I, the S configuration at C-2 was suggested.

Compound 2 (Blandachromene II), also identified as achromene, was obtained as a yellowish oil. The UV spec-trum revealed a phenolic compound with an extendedchromophore. The molecular ion [M]+ at m/z 286.1478 iscompatible with the molecular formula of C18H22O3. ItsIR spectrum showed a broad band at 3441 cm�1 typicalof a hydroxyl group chelated to a conjugated carbonylaldehyde group at 1725 cm�1. The major difference in the1H NMR spectrum of 2 as compared to 1, was a hydroxylgroup signal at d 12.6, due to a hydrogen bonded phenolichydroxyl. This signal was correlated with carbon signals ofC4a, C5 and C6, while the aldehyde resonance at d 10.0 hasHMBC correlations (C5, C6 and C7) that confirmed thehydroxyl and aldehyde groups should be attached to C5

and C6 positions, respectively. The aromatic hydrogen atd 6.25, correlated with C4a, C6, C7, C8a and the methylcarbon (C10), was located at C8. The aromatic methylgroup (C10) was located at C7 based on observed correla-tions with C6, C7 and C8. The signal of H4 was correlatedwith C2, C4a, C5 and C8a, and finally, the correlationsbetween H3 and C1 0 and C100 confirmed the location ofall substituents in the aromatic ring. The 13C NMR spec-trum assignments of 2 are in agreement with the proposedstructure and also confirm the presence of the aldehyde car-bonyl group with a signal at d 196.2, the aromatic carbon(C-5) bearing an oxygen-substituent at d 160.0 and themethyl substituted aromatic carbon (C-7) at d 145.0. Thesespectroscopic data are in agreement with that for a racemicmixture previously described (Dike and Merchant, 1978).The placement of the hydroxyl group was confirmed byanalysis of the NOE effects. The NOE effects (7-Me/H-8and CHO) confirmed the structure for the compound 2

as 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7-dimethyl-2H-chromene. The optical rotation, ½a�25

D + 22.0was observed for the compound 2 and the CD spectra ofthe substance showed the same pattern obtained for thecompound 1 (Fig. 2); thus the S configuration at C-2 wasalso suggested.

3. Conclusions

Both chromenes isolated from P. blanda are rare exam-ples of the occurrence of benzaldehyde derivatives in the

180 200 220 240 260 280 300 320

nm

-4

-3

-2

-1

0

1

2

3

4

5

6

Fig. 2. CD spectra of compounds 1 and 2 (in CHCl3) d – Compound 1; h – Compound 2.

494 L.S.M. Velozo et al. / Phytochemistry 67 (2006) 492–496

Piperaceae species, since their members usually producecompounds more oxidized, for example benzoic acid deriv-atives (Moreira et al., 1998a,b; Baldoqui et al., 1999; Sala-zar et al., 2005). These chromenes were biosynthesizedprobably through the polyketide route and reaffirm thepossible hypothesis of horizontal gene transfer suggestedin P. villipetiola (Salazar et al., 2005).

4. Experimental

4.1. General procedures

Optical rotations were determined using the Perkin–Elmer 243B; polarimeter UV and IR spectra were mea-sured on a Shimadzu UV-1601 and a Perkin–Elmer 599B,respectively. CD spectra were measured in methanol witha JASCO ORD/UV-6 spectropolarimeter. GC-MS analysiswas performed in a GC-MS-QP5000 Shimadzu using fusedcapillary column (DB-1 30 m · 0.20 mm), H2 as carrier gasand temperature programing from 40 to 240 �C (5 �C/min).High-resolution mass spectra were recorded on a Micro-mass spectrometer, VG-Autospec model; 1H- and 13CNMR were obtained on a Varian Gemini 200 NMR spec-trometer operating at 200 MHz for 1H NMR and50.2 MHz for13C NMR in CDCl3, using TMS as internalstandard. The 1H and 13C NMR data were recorded tooon a Bruker – Avance DRX 500 spectrometer (500 MHzfor 1H NMR and 125 MHz for 13C NMR) with TMS asinternal standard, using DMSO-d6 as solvent.

4.2. Plant material

Aerial tissue of P. blanda (Jacq.) Humb. Bonpl. andKunth were collected near Parati, Rio de Janeiro State,Brazil. The identification of the plant was done by Prof.Elsie F. Guimaraes, Botanical Garden of Rio de Janeiro.A voucher sample (RB: 325983) was deposited in the Her-barium of Rio de Janeiro Botanical Garden, Rio deJaneiro, Brazil.


The dried aerial parts of the plant were exhaustivelyextracted with MeOH. The MeOH extract was suspendedin a MeOH–H2O (7:3) mixture and extracted in successivesteps using hexane, CH2Cl2, EtOAc and n-BuOH. The hex-ane-soluble part was submitted to silica gel CC eluting withhexane/EtOAc mixtures of increasing polarity. The frac-tion eluted with hexane/EtOAc 5–10% was purifiedthrough a Sephadex LH-20 column yielding compounds1 (18 mg) and 2 (22 mg) that were shown to be pure byTLC and GC/MS analysis. The sterols – stigmasterol,sitosterol and campesterol – were isolated as a mixture of172 mg and identified from the literature (Blunt and Sto-thers, 1977).

4.4. 2S-(4-Methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7-

dimethyl-2H-chromene (1)

Yellowish oil. ½a�25D +26 (MeOH, c 0.1); UV kMeOH

max nm(log e): 211 (2.36), 266 (1.99); IR mfilm

max: 3350, 2962, 2924,2855, 1738, 1651, 1584, 1385; CD (CHCl3) kmax De287

+4.92, De254 �2.87; HREIMS, m/z: 286.1536 (C18H22O3

requires 286.1506); GC-MS m/z (rel. int.): 286 [M+] (3),271 [M�CH3]+ (2), 203 [M�C6H11]+ (100), 173 (4), 145(3), 128 (3), 115 (7), 91 (11), 69 (33); For 1H and 13CNMR spectra (DMSO-d6), see Table 1; 1H NMR (CDCl3):d 1.44 (3H, s, H-1), 1.56 (3H, s, H-6), 1.62 (3H, s, H-5), 1.77(2H, t, J = 6.9 Hz, H-1), 2.10 (2H, m, H-2), 2.52 (3H, s, H-10), 5.09 (1H, m, H-3), 5.57 (1H, d, J = 10.0 Hz, H-3), 6.98(1H, s, H-5), 6.64 (1H, d, J = 10.0 Hz, H-4), 10.42 (1H, s,CHO); 13C NMR (CDCl3): d 79.7 (s, C-2), d 127.3 (d, C-3), d 116.6 (d, C-4), d 106.8 (s, C-4a), d 111.0 (d, C-5), d116.1 (s, C-6), d 143.1 (s, C-7), d 159.6 (s, C-8), d 155.6(s, C-8a), d 190.5 (d, C-9), d 22.0 (q, C-10), d 41.0 (t, C-1 0), d 22.6 (t, C-2 0), d 123.6 (d, C-3 0), d 131.9 (s, C-4 0), d25.5 (q, C-5 0), d 17.5 (q, C-6 0), d 26.1 (q, C-100).

4.5. 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7-

dimethyl-2H-chromene (2)

Yellowish oil. ½a�25D +22 (MeOH, c 0.1); UV kMeOH

max nm (loge): 203 (0.72), 276 (1.03), 319 (0.49); IR mfilm

max: 3441, 2969, 2875,1725, 1653, 1568, 1379; CD (CHCl3) kmax De292 +5.63, De260

�3.78; HR-MS, m/z: 286.1478 (C18H22O3 requires286.1506); GC-MS m/z (rel. int.): 286 [M+] (3), 271[M�CH3]+ (5), 203 [M�C6H11]

+ (100), 173 (9), 145 (5),115 (13), 91 (17), 69 (66), 55 (84); for 1H and 13C NMR spec-tra (DMSO-d6), see Table 1; 1H NMR (200 MHz, CDCl3):1.44 (3H, s, H-1), 1.59 (3H, s, H-6), 1.68 (3H, s, H-5), 1.76(2H, t, J = 6.9 Hz, H-1), 2.08 (2H, m, H-2), 2.50 (3H, s, H-10), 5.11 (1H, m, H-3), 5.51 (1H, d, J = 10.0 Hz, H-3), 6.19(1H, s, H-8), 6.72 (1H, d, J = 10.0 Hz, H-4), 10.0 (1H, s,CHO); 13C NMR (CDCl3): d 80.5 (s, C-2), d 126.1 (d, C-3),d 115.7 (d, C-4), d 106.5 (s, C-4a), d 160.8 (s, C-5), d 113.0(s, C-6), d 143.5 (s, C-7), d 110.8 (d, C-8), d 160.4 (s, C-8a),d 192.7 (d, C-9), d 18.2 (q, C-10), d 41.6 (t, C-1 0), d 22.5 (t,C-20), d 123.6 (d, C-3 0), d 131.8 (s, C-4 0), d 25.5 (q, C-50), d17.5 (q, C-60), d 27.2 (q, C-100).

Acknowledgements

We are grateful to Eduardo M.B. da Silva and Marco A.Schiavon for obtaining, respectively, the NMR and HRMSspectra and to Luıs Carlos Giordano for helping us withcollecting the plant material. Financial support fromCNPq, FAPERJ and FAPESP are also acknowledged.

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496 L.S.M. Velozo et al. / Phytochemistry 67 (2006) 492–496

Cytotoxic and aromatic constituents from Salvia miltiorrhiza

Ming-Jaw Don a, Chien-Chang Shen a,b, Wan-Jr Syu c, Yi-Huei Ding a, Chang-Ming Sun a,b,*

a National Research Institute of Chinese Medicine, No 155-1, Section 2, Li-Nong Street, Shih-Pai, Taipei 112, Taiwanb Department of Biochemistry, National Yang-Ming University, Taipei 112, Taiwan

c Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan

Received 28 May 2005; received in revised form 1 October 2005Available online 20 December 2005

Abstract

As part of an ongoing study of traditional Chinese medicinal plants, the root tissue of Salvia miltiorrhiza was further investigated forits chemical constituents. Five naturally occurring products along with 13 known constituents were isolated from an ethyl acetate-solubleportion of its ethanol extract. Their structures were elucidated by means of spectroscopic methods. Some selected compounds were alsoevaluated for biological activity.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Salvia miltiorrhiza; Labiatae; Danshen; Cytotoxicity

1. Introduction

The traditional Chinese medicine Salvia miltiorrhiza

Bunge (Labiatae) has drawn attention by natural productchemists and medicinal clinicians as it has been used fortreatment of menstrual disorder, menostasis, menorrhalgia,insomnia, arthritis, and coronary heart diseases, particu-larly angina pectoris and myocardial infarction (JiangsuNew Medical College, 1988; Chen, 1984; Chang and But,2001). Numerous diterpenoid tanshinones have also beenisolated from S. miltiorrhiza (Kakisawa et al., 1969; Changet al., 1990) and many of them were shown to have variousbiological activities including antitumor (Chang and But,2001; Ryu et al., 1997b; Yang et al., 1981; Wu et al.,1991) and antimicrobial (Honda et al., 1988; Gao et al.,1979) activities. Previously, we reported a novel compoundwith antitumor activity from S. miltiorrhiza (Wang et al.,2004). In the present paper, we describe the isolation andstructural determination of several natural products from

the EtOAc fraction of the ethanolic extract of this plant.Some selected compounds have also been evaluated fortheir biological activity.


The further chemical investigation of S. miltiorrhiza wasfocused on the ethyl acetate-soluble portion of an ethanolicextract of the dried roots. Further fractionation byrepeated column chromatography of the EtOAc extractresulted in the isolation of 18 components including fivenew naturally occurring products (1–5) along with 13known compounds.

Compound 1 was obtained as a yellow oil with a molec-ular formula of C18H20O4 as determined by HREIMS. The1H NMR spectrum of 1 showed five aromatic protons at d8.33, 7.45, 7.40, 7.67, and 7.39, one aliphatic methine sig-nal, two methylene groups (one oxygenated), and threemethyl protons at d 2.65, 2.02, and 1.07 (Table 1). Further-more, one OH group signal with intramolecular hydrogenbonding was observed at d 14.00. The 13C NMR andDEPT spectra of 1 indicated the presence of 18 carbon


doi:10.1016/j.phytochem.2005.11.005

* Corresponding author. Tel.: +886 2 28201999; fax: +886 2 28264276.E-mail address: [email protected] (C.-M. Sun).



PHYTOCHEMISTRY


signals (Table 2), which were assigned to three methyl, twomethylene (one oxygenated), one saturated methine, oneketone (d 205.1), one ester (d 171.0), and 10 aromatic (fiveprotonated and five quaternary) carbons. The connectivi-ties of the 1H and 13C signals were determined by analysisof its HMQC spectrum, whereas 2D COSY correlations(H-1/H-2; H-6/H-7) and HMBC correlations (H-1/C-3,C-5, C-9; H-18/C-3, C-4, C-5; H-6/C-8, C-10; H-7/C-5,C-9) (Fig. 1) revealed the presence of a naphthalene ring.In addition, 2D COSY correlations (H-13/H-12, H-14,H-17) and HMBC correlations (H-13/C-11; H-14/C-15;H-16/C-15) suggested the presence of a 4-acetoxy-3-meth-ylbutanoyl moiety. The moiety was also supported fromanalysis of the EIMS data which showed a molecularion peak at m/z 300 and major fragment peaks at m/z240, 225 and 185, suggesting [M�CH3COOH]+,[M�CH3COOH�CH3]+ and [M�CH3CO2C4H8]+, respec-tively. Furthermore, the IR spectrum exhibited one car-bonyl absorption of a saturated aliphatic ester at1739 cm�1and the other carbonyl group with intramolecu-lar hydrogen bonding and conjugation to an aromatic ringat 1623 cm�1. The HMBC correlations of the OH group(dH 14.00) with C-8 and C-10 and H-7 with C-11 (Fig. 1)indicated that the 4-acetoxy-3-methylbutanoyl and hydro-xyl groups were located on C-8 and C-9, respectively.

The structure of 1 was therefore assigned as 4-(1-hydroxy-5-methylnaphthalen-2-yl)-2-methyl-4-oxobutyl ace-tate and compound 1 was given the trivial namesalvianonol.

Compound 2 was obtained as orange crystals with amolecular formula of C18H14O4 as determined by HRE-IMS. The 1H NMR spectrum exhibited five aromatic pro-tons at d 8.30, 7.95, 7.78, and 7.45–7.46, one aliphaticmethylene group at d 4.47 and 4.76, one aliphatic methineproton at d 3.37, and two methyl signals at d 2.62 and 1.42(Table 1). The 13C NMR and DEPT spectra of 2 showed 18carbon signals (Table 2), which were assigned to twomethyl, one oxygenated methylene, one saturated methine,five protonated aromatic, and nine sp2 quaternary carbons.The connectivities of the 1H and 13C signals were deter-mined by analysis of its HMQC spectrum. Analysis ofthe COSY, HMQC and HMBC data indicated that 2 waspartially similar to 1 having a naphthalene ring. The COSYspectrum showed that the methine proton (H-13) was cor-related with the methylene group (H-14) and the methylprotons (H-17). In the HMBC spectrum, the correlations(H-17/C-12, C-14; H-13/C-11, C-16; H-14/C-12, C-15; H-7/C-9, C-11) were observed (Fig. 1), which suggested thata 5,6-dihydropyran-2-one moiety was fused to the bezo-chromen-4-one ring. In addition, the IR spectrum of 2

Table 11H NMR (500 MHz) spectroscopic data of 1–5 in CDCl3

Position d(J)a

1 2 3 4 5

1 8.33 d (7.5) 8.30 m ax 1.54 t (11.5) 2.28 s

eq 2.53 m

2 7.40 t (7.5) 7.45–7.46 m 5.17 m 2.90 t (7.0)3 7.45 d (7.5) 7.45–7.46 m eq 1.86 dd (3.5, 13.5) 2.03 t (7.0) 6.56 d (16.0)

ax 1.33 t (13.5)4 7.19 d (16.0)5 1.86 dd (3.5, 13.5)6 7.39 d (9.0) 7.78 d (9.0) ax 2.58 dd (13.5, 18.0) 7.43 d (9.0) 6.59 d (3.0)

eq 2.68 dd (3.5, 18.0)7 7.67 d (9.0) 7.95 d (9.0) 7.95 d (9.0) 6.35 d (3.0)9 4.61 s

11 6.71 s

12 a 2.89 dd (8.0, 16.0)b 3.18 dd (6.0, 16.0)

13 2.62 m 3.37 m

14 a 4.01 dd (6.5, 11.0) a 4.47 dd (1.0, 11.5) 7.90 s 7.25 s

b 4.08 dd (5.5, 11.0) b 4.76 dd (3.5, 11.5)15 3.18 m 3.13 sept (7.0)16 2.02 s 1.20 d (7.0)b 1.27 d (7.0)17 1.07 d (6.5) 1.42 d (7.5) 1.21 d (7.0)b 1.27 d (7.0)18 2.65 s 2.62 s 0.97 s 1.42 s

19 1.04 s 1.42 s

20 1.26 s

22 2.05 s

Palmitate

2 2.67 t (7.5)3 1.83 m

16 0.86 t (7.0)

a J = Coupling constant in Hertz.b Interchangeable.

498 M.-J. Don et al. / Phytochemistry 67 (2006) 497–503

showed the presence of a lactone (1745 cm�1) and a conju-gated ketone (1644 cm�1) absorptions. The structure of 2

was therefore assigned as 4,8-dimethyl-8,9-dihydro-10,12-dioxa-benzo[a]anthracene-7,11-dione and compound 2

was given the trivial name salviamone.Compound 3 was obtained as an orange oil with a

molecular formula of C22H30O4 as determined by HRE-IMS. The 13C NMR and DEPT spectra of 3 exhibited 22carbon signals (Table 2), which were assigned to six methyl,three methylene, three aliphatic methine, two aliphatic qua-ternary, two carbonyl (d 171.1 and 198.4), and six aromatic(two protonated and four quaternary) carbons. The 1HNMR spectrum exhibited two aromatic protons (d 6.71and 7.90), two doublets of two methyl signals (d 1.20 and1.21), four singlets of four methyl groups, three aliphaticmethine protons, and three methylene signals, which waspartially similar to those of 2a-hydroxysugiol (Gonzalez

et al., 1988). The HMBC spectrum showed correlationsof the carbonyl group (dC 171.1) with the methine (H-2,dH 5.17) and methyl groups (Me-22, dH 2.05), which indi-cated that an acetoxyl group was located on C-2. The rel-ative stereochemistry of the acetoxyl group wasdetermined on the basis of a NOESY experiment, whichshowed correlations of H-2 with Me-19 and Me-20 andsuggested that the acetoxyl group was located in an equa-torial orientation at C-2. In addition, the IR spectrumshowed the presence of a hydroxyl group at 3286 cm�1

and the carbonyl groups of saturated aliphatic ester andconjugated ketone at 1733 and 1652 cm�1, respectively.Thus, compound 3 was established as 2a-acetoxysugiol.

Compound 4 was obtained as a yellow oil with a molec-ular formula of C35H52O4 as determined by HREIMS. TheEIMS of 4 gave a molecular ion peak at m/z 536 and amajor fragment peak at m/z 298, suggesting a [M�palmi-toyl moiety + H]+. In the 1H NMR spectrum, the palmi-toyl signals were observed at d 0.86, 1.42, 1.83, and 2.67.The presence of the palmitoyl moiety was further sup-ported by analysis of the 13C NMR spectrum and compar-ison of the NMR spectroscopic data with those of methylpalmitate (Vandevoorde et al., 2003). In additional to thepalmitoyl signals, the 1H NMR spectroscopic data (Table1) showed an AB pattern for two ortho-aromatic protonsat d 7.43 and 7.95, one aromatic signal as a singlet at d7.25, a geminal dimethyl group at d 1.42 (6H), two methy-lene groups at d 2.03 and 2.90, and an isopropyl group at d1.27 (6H) and 3.13 (1H), which were similar to those ofarucadiol (Majetich et al., 1997). The IR spectrum of 4indicated presence of a carbonyl stretch of an ester athigher frequency at 1762 cm�1, suggesting the presence ofaromatic group conjugation with an alcohol (OH) moiety.In addition, a carbonyl absorption was observed at lowerfrequency at 1642 cm�1, due to the effects of conjugationwith the aromatic ring and intramolecular hydrogen bond-ing. Furthermore, an OH signal with intramolecularhydrogen bonding (dH 10.55) suggested that an hydroxylgroup was located on C-11, which was confirmed by itsHMBC correlations with C-9, C-11 and C-12. Thus, com-pound 4 was established as palmitoyl arucadiol.

Compound 5 was obtained as a yellow oil with a molec-ular formula of C9H10O3 as determined by HREIMS. TheEIMS of 5 gave a molecular ion peak at m/z 166 and majorfragment peaks at m/z 135, suggesting [M�CH3O]+. The1H NMR spectrum exhibited two trans-olefinic protonsat d 7.19 and 6.56, two aromatic signals at d 6.59 and6.35, one oxygenated methylene group as a singlet at d4.61, and one methyl singlet at d 2.28 (Table 1). The 13CNMR and DEPT spectra of 5 showed nine carbon signals(Table 2), which were assigned to one methyl, one oxygen-ated methylene, four sp2 methine, and two oxygenated qua-ternary sp2 carbons. The connectivities of H and C weredetermined by analysis of an HMQC experiment. TheHMBC data revealed the correlations of H-4 with C-2, -3, -5, and -6 and H-9 with C-7 and -8, indicating that 5 isa 2,5 disubstituted furan. The structure of 5 was therefore

O

O

O

O

2

OOH

1

O

O

Fig. 1. Selected HMBC correlations of 1 and 2.

Table 213C NMR spectroscopic data of 1–8 in CDCl3

Position 1 2 3 4 5 6 7 8

1 122.5 120.8 42.8 205.0 28.1 125.6 125.4 24.92 125.7 127.1 68.7 36.5 198.4 130.1 130.3 22.53 130.9 130.8 46.2 35.4 124.3 129.0 129.1 128.64 134.0 134.7 34.6 36.2 129.7 134.8 135.1a 131.05 136.4 135.0 48.9 157.6 150.9 135.6 135.0a 139.16 114.7 122.2 35.6 122.9 117.0 131.5 132.3 128.27 123.9 119.5 198.4 137.5 110.6 121.7 122.5 120.78 112.8 119.6 123.5 132.2 157.3 133.6 133.8 127.39 163.0 153.6 155.0 121.4 57.7 126.3 124.0 126.2

10 125.5 123.8 39.3 127.7 130.7 130.3 144.511 205.1 175.9 109.5 145.1 181.2 184.1 184.412 42.2 131.1 159.5 138.1 160.9 153.0 176.313 29.7 26.3 133.7 141.7 124.9 125.5 120.214 68.6 73.1 126.9 117.3 182.7 185.2 161.715 171.0 158.6 26.7 28.0 35.6 24.5 121.216 20.8 144.2 22.1a 22.8 80.4 20.0 141.317 17.1 16.5 22.4a 22.8 18.9 20.0 8.818 19.5 19.4 32.4 29.6 20.0 19.9 19.819 22.0 29.620 23.921 171.122 21.4

Palmitate

1 171.92 34.23 25.1

16 14.1

a Interchangeable.

M.-J. Don et al. / Phytochemistry 67 (2006) 497–503 499

assigned as (E)-4-[5-(hydroxymethyl)furan-2-yl]but-3-en-2-one.

Thirteen known compounds were also identified as dihy-droisotanshinone I (6) (Kong and Liu, 1984), dans-henxinkun B (7) (Luo et al., 1994), 1,2-dihydrotanshinoneI (8) (Feng and Li, 1980), tanshinone I (Ryu et al.,1997a), tanshinone IIA (Ryu et al., 1997a), methylene-tanshinquinone (Luo et al., 1994), 15,16-dihydrotanshi-none I (Ikeshiro et al., 1991), cryptotanshinone (Anet al., 2002), ferruginol (Harrison and Asakawa, 1987),sugiol (Gao and Han, 1997), norsavioxide (Li et al.,1991), and a mixture of danshenspiroketallactone and itsepi-isomer (Asari et al., 1990) by analysis of their MS,1D and 2D NMR spectroscopic data and by comparisonwith those in the literature; however, 13C NMR spectro-scopic data of compounds 6–8 have not been previouslyreported and are reported herein.

Compounds 1, 2, and 6–8 might be biosynthesized fromabietic acid (9) (Scheme 1). Abietic acid may firstly lead to7 and further to danshenxinkun A (10) via several steps ofoxidation. Dehydration of 10 potentially affords 6 anddihydrotanshinone I (11), of which the latter might leadto 8 by hydrogenation and dehydrogenation. After Bae-yer–Villiger oxidation, compound 10 is envisaged to pro-duce a seven-membered lactone intermediate 12, which isthen hydrolyzed, followed by esterification and dehydra-tion with ring closure to result in compound 2. Alterna-

tively, the lactone intermediate 12 can be followed in turnby hydrogenation, dehydration, retro-aldol reaction,hydration, hydrolysis and acetylation to afford compound1.

Compounds 1–4 and 7 were evaluated for their cytotox-icity against selected cancer cell lines by using MTTmethod and cisplatin as positive control. Among the testedcompounds, 4 was the most potent compound with CD50

values of 3.2 and 4.1 lg/ml against the HeLa andOVCAR-3 cells, respectively, which had slightly lowerCD50 values than cisplatin (Table 3). The cytotoxicities ofcompounds 1–4 and 7 against the above cancer cell lineshave not been reported so far. In addition, compounds1–4 were subjected to evaluation of antibacterial activityagainst Gram-positive Staphylococcus aureus and Entero-

coccus faecalis, and Gram-negative Escherichia coli, usingpaper disk methods. However, all tested compounds wereconsidered inactive (inhibition zone <10 mm/100 lg/disk).

3. Conclusions

So far about 50 abietanoids and diterpenoid tanshinoneshave been identified from the root of S. miltiorrhiza. Theoccurrence of tanshinones, however, can be used as a tax-onomic characteristic for the genus Salvia (Patudin et al.,1974). Some Salvia species such as Salvia yunnanensis (Qian

O

O

O

O

2

OOH O

O

1

OH

O

O

H

3

HOO

O

4

OHO

O

5

O

O

O

6

O

O

7

OH

O

OO

8

CH3(CH2)14

O

111

1

36

7

9 11

13

14

16

1213

16

1818

1717

15

15

11

14

15

16

18

20

3

9

5 7

13

4

7

9

11

15

16

18

3 7

O

2212

76

14

6

6

3

19


et al., 2002; Yang et al., 1996), Salvia przewalskii (Li et al.,1991), and Salvia paramiltiorrhiza f. purpureorubra (Wang,1981), which have been used as substitutes in Chinese folk

medicine for Danshen, were also reported to contain thepharmacologically active tanshinones. We report here thatfive new natural products, one furan derivative and four

O

OO

6

OO

O

8

O

O

OO

2

COOH

9

COOH

Dehydroabietic acid

O

OOH

7

O

OOH

10

OH

O

O

11

O

O

O

1

OH OO

O

O

OHOH

O

O

OOH

OH OOH

OOH

O

OHOH

O

HOOH

O

OO

OH

O

O

OOH

[O]

[O]

H2O[O][H]

H2O

[O] [O]

H2OH2O

H2O

H2O

12

Scheme 1. Proposed biosynthetic pathways for 1, 2, 6–8 of S. miltiorrhiza.


abietanoids, and 13 known diterpenoid tanshinones wereisolated from an ethyl acetate-soluble portion of ethanolextract of S. miltiorrhiza. These new components couldprovide a support of the chemotaxonomic significance forthe species of S. miltiorrhiza.

4. Experimental


Melting points were measured using a Yanaco MP-S9micro-melting point apparatus and uncorrected. UV spec-tra were performed on a Hitachi U-3310 spectrophoto-meter. IR spectra were recorded on a Nicolet Avatar 320FT-IR spectrometer. The 1H and 13C NMR spectra wererecorded on a Varian Unity Inova 500 spectrometer inCDCl3 with tetramethylsilane (TMS) as an internal stan-dard. COSY, HMQC, HMBC, and DEPT spectra wereobtained using standard Varian pulse sequences. EIMSspectra were measured with a direct insertion probe on aFinnigan GCQ spectrometer at 30 eV. HREIMS data weretaken on a Finnigan MAT 95XL mass spectrometer. Silicagel (Kieselgel 60, 70–230 mesh, Macherey-Nagel) was usedfor column chromatography. TLC was carried out on alu-minum sheets precoated with silica gel 60 F254 (layer thick-ness 0.2 mm, Merck Art. 5554). The chromatograms werevisualized under UV light (254 or 365 nm) or by sprayingwith 5% phosphomolybdic acid in 5% H2SO4 containinga trace of ceric sulfate, followed by heating on a hot plate(120 �C).

4.2. Plant material

The dried and sliced roots of S. miltiorrhiza (5 kg) werepurchased from the Cherng-Chi Chinese herbal shop inTaipei in April 2004. A voucher specimen (NRICM04024) was deposited in the herbarium of NationalResearch Institute of Chinese Medicine, Taipei.


The root of S. miltiorrhiza was extracted with EtOH(30 l) three times at 60 �C for 24 h. The EtOH extracts werecombined and concentrated in vacuo to 1 l. The concen-trated extract was suspended in H2O (4 l) and partitioned

successively with EtOAc. After concentration of the EtOAcextract, the concentrate was mixed with 700 g of silica gel(230–400 mesh). The air-dried mixture was subjected to sil-ica gel column chromatography (cc) using a mixture of hex-ane-EtOAc of increasing polarity as eluents. Fractionswere collected, with similar fractions (monitored by TLC)combined to give 5 fractions (F-1 to F-5). F-1 was furtherapplied to silica gel cc to give danshenxinkun B (7) (12 mg)and ferruginol (153 mg). F-2 was resubjected to silica gel ccto give tanshinone IIA (1.2 g), 1,2-dihydrodanshinone I (8)(35 mg), palmitoyl arucadiol (4) (10 mg), methylenetansh-inquinone (63 mg), and tanshinone I (480 mg), respectively.F-3 was separated by further silica gel cc to yield dihydro-isotanshinone I (6) (12 mg), sugiol (75 mg), norsalvioxide(18 mg), and a mixture of danshenspiroketallactone andepi-danshenspiroketallactone (30 mg). F-4 was reappliedto silica gel cc to afford salvianonol (1) (28 mg), cryptotan-shinone (700 mg), 2a-acetoxysugiol (3) (41 mg), and 15,16-dihydrotanshinone I (175 mg). F-5 was also subjected tosilica gel cc to give salviamone (2) (15 mg) and (E)-4-[5-(hydroxymethyl)furan-2-yl]but-3-en-2-one (5) (43 mg).

4.4. Salvianonol, 4-(1-hydroxy-5-methylnaphthalen-2-yl)-2-

methyl-4-oxobutyl acetate (1)

Yellow oil; ½a�25D þ 30� (CHCl3, c 0.1); UV (CH3OH)

kmax (log e) 215.0 (5.06), 256.8 (5.00), 371.4 (4.17) nm; IR(film) mmax 2963, 2925, 2853, 1739, 1623, 1576, 1471,1384, 1238, 1081, 1038, 796 cm�1; for 1H and 13C NMRspectra, see Tables 1 and 2, respectively; HREIMS m/z300.1363 (calcd. for C18H20O4 300.1356); EIMS m/z (rel.int.): 300 [M]+ (80), 240 (67), 225 (100), 197 (8), 185 (33),128 (10).

4.5. Salviamone, 4,8-dimethyl-8,9-dihydro-10,12-dioxa-

benzo[a]anthracene-7,11-dione (2)

Orange crystals, m.p. 203–204 �C, ½a�25D � 35� (CHCl3, c

0.2); UV (CH3OH) kmax (log e) 228.8 (4.83), 275.0 (4.36),360.4 (3.87) nm; IR (film) mmax 2967, 2925, 1745, 1644,1510, 1466, 1400, 1266, 1211, 1169, 1120, 772 cm�1; For1H and 13C NMR spectra, see Tables 1 and 2, respectively;HREIMS m/z 294.0887 (calcd. for C18H14O4 294.0887);EIMS m/z (rel. int.): 294 [M]+ (100), 249 (72), 235 (12).

4.6. 2a-Acetoxysugiol, (3S,4aS,10aS)-1,2,3,4,4a,9,10,10a-octahydro-6-hydroxy-7-isopropyl-1,1,4a-trimethyl-9-

oxophenanthren-3-yl acetate (3)

Orange oil; ½a�25D � 16� (CHCl3, c 1.0); UV (CH3OH)

kmax (log e) 205.2 (4.43), 232.2 (4.36), 282.2 (4.25) nm; IR(neat) mmax 3286, 2963, 2925, 2870, 1733, 1652, 1596,1464, 1366, 1268, 1179, 1129, 757 cm�1; for 1H and 13CNMR spectra, see Tables 1 and 2, respectively; HREIMSm/z 358.2146 (calcd. for C22H30O4 358.2144); EIMS m/z(rel. int.): 358 [M]+ (68), 298 (38), 283 (100), 241 (53).

Table 3Cytotoxicity of 1–4 and 7 against human cancer cell lines

Compound CD50 (lg/ml)

HeLa HepG2 OVCAR-3

1 17.4 37.5 >1002 >100 >100 >1003 25.5 37.5 30.24 3.2 25.1 4.17 40.5 34.5 33.5Cisplatin 7.2 7.1 9.0


4.7. Palmitoyl arucadiol, 1,2,3,4-tetrahydro-5-hydroxy-7-

isopropyl-1,1-dimethyl-4-oxophenanthren-6-yl palmitate (4)

Yellow oil; UV (CH3OH) kmax (log e) 216.8 (4.66), 271.6(4.30) nm; IR (neat) mmax 2959, 2925, 2854, 1762, 1642,1594, 1465, 1412, 1282, 1136 cm�1; for 1H and 13C NMRspectra, see Tables 1 and 2, respectively, 13C NMR of pal-mitoyl moiety d 14.11, 22.69, 25.11, 29.27, 29.34, 29.36,29.52, 29.70, 31.92, 34.25, 171.90; HREIMS m/z 536.3870(calcd. for C35H52O4 536.3866); EIMS m/z (rel. int.): 536[M]+ (3), 298 (100).

4.8. (E)-4-[5-(Hydroxymethyl)furan-2-yl]but-3-en-2-one

(5)

Yellow oil; for 1H and 13C NMR spectra, see Tables 1and 2, respectively; HREIMS m/z 166.0633 (calcd. forC9H10O3 166.0624); EIMS m/z (rel. int.): 166 [M]+ (31),151 (4), 135 (100), 67 (3).

Acknowledgements

We gratefully acknowledge the financial supportthrough the NRICM (94-DMC-03) for this work. Theauthors also thank Ms. Shu-Chi Lin of the InstrumentationCenter, National Tsing Hua University for HREIMS data.

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Oligomeric secoiridoid glucosides from Jasminum abyssinicum q

Francesca Romana Gallo a,*, Giovanna Palazzino a, Elena Federici a, Raffaella Iurilli a,Franco Delle Monache b, Kusamba Chifundera c, Corrado Galeffi a

a Dipartimento del Farmaco, Istituto Superiore di Sanita, V. le Regina Elena 299, 00161 Rome, Italyb CNR, Centro Chimica dei Recettori, Universita Cattolica S. Cuore, L.F. Vito 1, 00168 Rome, Italy

c Institut Superieur d’Ecologie pour la Conservation de la Nature, Lwiro, P.O. Box 293 Cyangugu, Rwanda

Received 29 March 2005; received in revised form 28 September 2005Available online 27 December 2005

Abstract

From the root bark of Jasminum abyssinicum (Oleaceae) collected in Congo was isolated tree oligomeric secoiridoid glucosides namedcraigosides A–C. The three compounds are esters of a cyclopentanoid monoterpene with an iridane skeleton, esterified with three, twoand two, respectively, units of oleoside 11-methyl ester. The structures were elucidated by spectroscopic methods and chemicalcorrelations.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Jasminum abyssinicum; Oleaceae; Root bark; Oligomeric secoiridoid glucosides; Craigoside A; Craigoside B; Craigoside C

1. Introduction

Genus Jasminum (Oleaceae) includes beyond 200 spe-cies, some of which are used in folk medicine or cultivatedto obtain essential oil from the fragrant flowers. The termJasminum (Oleaceae) was first mentioned in the ‘‘MateriaMedica’’ of Dioscoride (I A.D.). The phytochemical stud-ies of the aerial parts of some species, J. sambac [Soland.](Tanahashi et al., 1988), J. mesnyi Hance (Tanahashiet al., 1989), J. urophyllum Hemsl. (Shen and Hsieh,1997) and J. nudiflorum Lindl. (Tanahashi et al., 2000),resulted in the isolation of some secoiridoid glucosides, inparticular of oligomeric consisting of oleoside units linkedto a cyclopentanoid monoterpene named iridane.

This study deals with the structure elucidation of threeoligomeric secoiridoid glucosides, two trimer and one tetra-mer, isolated from the root bark of Jasminum abyssinicum

R. Br. (= Hochst. ex DC.) and named craigosides A, 1, B,

2, and C, 3, as a tribute to L.C. Craig (Craig and Post,1949) and his apparatus of counter-current distribution(CCD) utilised for our separations. The aerial parts of thisplant are used in Traditional Medicine in the South KivuProvince, Congo against endoparasitic worms and fortreatment of mumps (Chifundera, 2001).


Craigoside A, 1, is an amorphous powder, C61H86O34

(ICR-FTMS, m/z 1385.48602 [M + Na]+), ½a�20D ¼ �185

(MeOH), kmax 233 nm (log e 4.54). Its 1H and 13C NMRspectra (Tables 1 and 2) showed inter alia a pattern of sig-nals corresponding to oleoside methyl ester, viz., an acetalicmethine and an anomeric methine, a vinylic oxymethine, anethylidene and a carbomethoxy. In agreement with themolecular formula of 1, the signals of some carbons (6, 8,9, 1 0, 4 0 and 6 0) of this iridoid moiety appeared in triplicateand in particular the methoxy group, d 52.05, 52.03 and52.00. This accounted for the presence of three oleosidemethyl ester units and thus the presence of cyclic estersengaging two carboxylic groups of the same oleoside could


doi:10.1016/j.phytochem.2005.11.007

q Presented at FITOMED 2004, 1� Congresso Intersocieta sulle PianteMedicinali, Trieste, Italy, 16–19 September, 2004.

* Corresponding author. Tel.: +39 06 49903055; fax: +39 06 49903060.E-mail address: [email protected] (F.R. Gallo).



PHYTOCHEMISTRY


be ruled out. The LR HETCOR observed in 1 between thea-b unsaturated carboxyl group, d 168.6, and the methoxygroup, d 3.72, accounted for the methyl ester in position 11of the iridoid and therefore the other carboxyl group, (C-7,d 173.1), was engaged in the linkage with the non-iridoidicmoiety. The 10 minor 13C signals of craigoside A due tothis last part of the molecule corresponded to a cyclopenta-noid monoterpene, endowed with three oxymethylenegroups, d 68.4, 65.1 and 61.2, an oxymethine, d 80.0, anda methyl group, d 19.7.

By alkaline hydrolysis of 1 and subsequent methylationwith diazomethane, dimethyl oleoside, 4, and tetraol iridane5, C10H20O4 (ICR-FTMS, m/z 227.12552 [M + Na]+),½a�26

D ¼ þ2:4 (MeOH), were obtained. This last substanceappeared different from the two known tetraols 6 and 7

obtained by saponification of sambacosides A, E and F ofJ. sambac (Tanahashi et al., 1988) and of jasurosides Cand D of J. urophyllum (Shen and Hsieh, 1997), respec-tively. In particular, the cyclic methylene of 5, instead ofat d 37.7 and 37.0, as in 6 and 7, respectively, resonated athigher chemical shift (d 43.2), as in the known triol 8 (d43.7) obtained from nudiflosides A–C of J. nudiflorum

(Tanahashi et al., 2000) and having an hydroxy group inposition 400 instead of 500.

O10

COOMe

O

HMeOOC

OH

OHO

OH

OH

1'

2'

3'4'

5' 6'

3

1

4

56

7

89

11

Dimethyl oleoside (4)

The HETCOR and selective 1H-1H decoupling of thetetra-acetyl derivative of 5, 9, gave full account of its struc-ture and relative stereochemistry. Thus the irradiation ofH-400 of the acetoxymethine (d 5.12, quintet, J = 6.0, 3.0and 3.0) made the two signals of the adjacent methylene,d 1.52, dq, J = 13.5, 10.5 and 6.0, Ha-500, and d 1.83, m,J = 13.5, 6.8 and 3.0, Hb-500, into two dd with the loss ofthe couplings of J = 6.0 and 3.0, respectively. Moreover,

CH2H2C

H2C

CH3

OH

O

O

O

O10

COOMe

OGl

HO=C

O

MeOOC

OGl

H C=O

O

COOMeHO=C

OGl

1" 5"

4"3"

2"

3

1

4

56

7

89

11

8"

9" 10"

7"

6"

CH2H2C

H2C

CH3

OH

O

HO

O

O

COOMe

OGl

HO=C

O

MeOOC

OGl

H C=O

Craigoside B (2)Craigoside A (1)

CH2H2C

H2C

CH3

O

OH

HO

O

O

MeOOC

OGl

H C=O

O

COOMe

OGl

HO=C

Craigoside C (3)

F.R. Gallo et al. / Phytochemistry 67 (2006) 504–510 505

the irradiation in the range of d 4.0–4.2 corresponding tothe three acetoxymethylenic groups made the signal of H-800 (d 2.11, m) into a doublet with J = 6.3 and the signalof H-200 (d 1.63, m) into a perfect triplet, J = 9.0. The dihe-dral angle of H-200 both with H-100 and H-300 consistent withthis last coupling was about 140� (trans relationship withboth) and it corresponded to a position of C-200 out ofthe plane of the other four carbons of the cyclopentanoidring, thus allowing a quasi equatorial allocation of the sub-stituents in 200 and 300. This ring conformation in 9 was inagreement with the coupling constants J = 3.0 Hz betweenH-300 and H-400 and between H-400 and Hb-500 correspondingto the dihedral angle of about 115� (trans relationship forboth).

In tetraol 5, the signal d 4.08 of H-400 was a quartet, dueto the identical coupling constant, J = 5.1, with H-300, Ha-500 and Hb-500. The cis relationship between HO-400 andHb-500 was further confirmed by the downfield shift of thelatter, d 1.70, respect to Ha-500, d 1.48, owing to the aniso-tropic effect of the former.

In order to establish the absolute configuration of tet-raol 5, according the Mosher�s method through esterifica-tion of the secondary alcoholic function with (S)-MTPAand (R)-MTPA (Ohtani et al., 1991), tetraacyl derivatives10 and 11 were prepared, respectively. The results of Dd

(1H NMR) (dS � dR) showed, in line with the models ofFig. 1, the b configuration of the hydroxyl group in 400.The structure 5 was thus unambiguously established forthe tetraol iridane of craigoside A.

CH2H2C

H2C

R1

R6

R2

R3

R5

R4O

1"2"

3"4"

5"

9" 10"

CH

7" 8"

6"

B= (S)-MTPA radical C= (R)-MTPA radical

R1 R2 R3 R4 R5 R6

5 -- Me – OH H HH

OH6 -- Me H – OH OH

OH

OHOH

7 – Me H -- OH H OHOH8 --Me – OH H H H

9 --Me – OAc H Ac OAc OAc10 --Me – OB H B OB OB11 --Me – OC H C OC OC

The three 11-methyl oleoside units on tetraol 5 in craigo-side A, 1, were assigned in positions 400, 700 and 900 on the

Table 11H NMR spectroscopic data of compounds 1, 2, 3 and 5 in CD3OD

Position 1 2 3 5

1 5.97, bs 5.96, s 5.95, s

5.94, bs 5.94, s

3 7.54, s 7.54, s 7.55, s

7.53, s 7.55, s

5 4.10, dd (11.0; 4.8) 4.16, dd (11.0; 4.8) 4.29, dd (11.0; 4.8)4.19, dd (11.0; 4.8)

6 a 2.72, dd (13.0; 4.8) 2.76, dd (13.0; 4.8) 2.76, dd (13.0; 4.8)2.70, dd (13.0; 4.8)

6 b 2.49, dd (13.0; 11.0) 2.51, dd (13.0; 11.0) 2.52, dd (13.0; 11.0)2.47, dd (13.0; 11.0) 2.48, dd (13.0;11.0)

8 6.10, bq (6.8) 6.12, bq (6.8) 6.11, bq (6.8)6.11, bq (6.8) 6.10, bq (6.8)

10 1.77, d (6.8) 1.76, d (6.9) 1.77, d (6.8)1.75, d (6.8) 1.75, d (6.8)1.73, d (6.8)

MeO 3.72, s 3.74, s 3.73, s

10 4.82, d (7.6) 4.83, d (7.6) 4.83, d (7.6)20-50 A A A

60 a 3.66, m 3.66, m 3.66 m

60 b 3.94, m 3.99, m 3.99 m

100 1.93, m 1.94, m 1.93, m 1.97, m (6.9; 6.9)200 1.69, m 1.68, m 1.69, m 1.51, m

300 1.91, m 1.90, m 1.90, m 1.75, m (5.1)400 5.11, m 5.11, m 4.10, m 4.08, q (5.1; 5.1; 5.1)500 a 1.52, ddd (12.6; 6.7; 3.6) 1.53, ddd (13.0; 6.7; 2.5) 1.52, ddd (12.5; 6.7; 3.5) 1.48, m (11.9; 6.9; 5.1)500 b 1.95, m 1.86, m 1.88, m 1.70, m (11.9; 5.1)600 1.09, d (6.5) 1.10, d (6.6) 1.07, d (6.6) 1.03, d (6.9)700 4.05 m 4.06, m 4.05, m 3.70, m

800 1.89 m 1.83, m 1.86, m 1.70, m

900 4.12 m 3.66 m 3.63 m 3.63, m

3.61, m

1000 3.59 m 3.68 m 4.11 m 3.59, m

A In the range 3.3–3.5.

506 F.R. Gallo et al. / Phytochemistry 67 (2006) 504–510

basis of the a downfield effects (and the b upfield effects) onthe 13C resonances respect to the corresponding ones of thetetraol. Thus the chemical shifts of C-400, C-700 and C-900 in5, d 75.5, 65.8 and 62.7, respectively, moved to d 80.0, 68.4and 65.1 in craigoside A.

Craigoside B, 2, is an amorphous powder, C44H64O24

(ICR-FTMS, m/z 999.36655 [M + Na]+), ½a�20D ¼ �170

(MeOH), kmax 236 nm (log e 4.29). Its 1H and 13C NMRspectra (Tables 1 and 2) showed the signals typical of 11-methyl oleoside, the most of them in duplicate. By alkalinehydrolysis and subsequent methylation, the aforemen-tioned tetraol 5, and dimethyl oleoside, 4, were obtained.The downfield shifts observed in 2 only for C-400, d 80.3,

and C-700, d 68.7, respect to the corresponding values ofthe tetraol, d 75.5 and 65.8, respectively, showed unambig-uously the positions of the two iridoid moieties in the mol-ecule of craigoside B. Respect to craigoside A, 1, theabsence of an iridoid unit at C-900 in craigoside B, 2,resulted in the upfield shift of C-900 itself from d 65.1 to62.7 and the downfield shift of C-800 from d 43.5 to 46.8.

Craigoside C, 3, is an amorphous powder, C44H64O24

(ICR-FTMS, m/z 999.37081 [M + Na]+), ½a�20D ¼ �160

(MeOH), kmax 236 nm (log e 4.26 ). Its 1H and 13C NMRdata are reported in Tables 1 and 2, respectively. The alka-line hydrolysis of this trimer, isomer of 2, and the subse-quent methylation with diazomethane likewise gavetetraol 5 and dimethyl oleoside, 4. The downfield shiftsobserved in 3 only for C-700, d 67.7, and C-1000, d 64.2,respect to the corresponding values of 5, d 65.8 and 61.9,besides the b upfield shifts for C-200 and C-800 in the former,gave account of the positions 700 and 1000 for the two oleo-side 11-methyl ester units.

The CD curve of craigoside A, having the same config-uration at C-800 as molihuaside E from J. sambac (Zhanget al., 1995), showed an additional band at 247 nm besidesthe band at 228 nm.

In summary, the oligomeric secoiridoid glucosides, newrespect to the previously described ones occurring in the aer-ial parts of Jasminum genus plants, have been isolated fromthe root bark of J. abyssinicum from Congo. The three olig-omeric, craigoside A, tetramer, and craigosides B and C, tri-mer, have the same cyclopentanoid monoterpene, which is atetraol iridane, 5, esterified by oleoside 11-methyl ester units.

3. Experimental

3.1. General

A Craig-Post apparatus, 200 stages, 10:10 ml, upper andlower phase, for the CCD. 1H NMR, 300 MHz, 13C NMR,75 MHz, TMS as internal standard, chemical shifts (d) inppm, coupling constants (J) in Hz, Varian Gemini 300.ICR-FTMS, high resolution, APEX II Bruker; ESI-MS,Thermo Finnigan, and FAB-MS, VG 7070 EQ-HF. CD,Jasco 710.

3.2. Plant material

Root barks of J. abyssinicum R. Br. were collected inMarch 1999 near Bukavu (South Kivu Province, Congo).The plant material was identified in the Institut Superieurd�Ecologie pour la Conservation de la Nature, Lwiro(Cyangugu, Rwanda), where a voucher specimen (Mubeza,B 346) is deposited.


Air-dried root barks (310 g) were extracted three timeswith MeOH. The residue from the evaporation of the sol-

Table 213C NMR spectroscopic data of compounds 1, 2,3 and 5 in CD3OD

Position 1 2 3 5

1 95.2 95.3; 95.2 95.2; 95.13 155.1 155.5 155.34 109.4; 109.3 109.8; 109.7 109.55 31.8; 31.7 31.9; 31.8 32.06 41.4; 41.3; 41.2 41.6; 41.4 41.4; 41.37 173.1 173.0; 172.9 173.4; 173.38 125.0; 124.9; 124.8 125.1; 125.0 125.09 130.9; 130.7;130.6 130.6; 130.5 130.8; 130.710 13.8 13.9;13.8 13.911 168.6 168.8; 168.7 168.8; 168.7MeO 52.05; 52.03; 52.00 52.1; 52.0 52.2; 52.11 0 100.9; 100.8; 100.7 100.9; 100.8 100.9; 100.82 0 74.7 74.9; 74.8 74.9; 74.83 0 78.4; 78.3 78.6; 78.5 78.54 0 71.5; 71.4; 71.3 71.6 71.6; 71.55 0 77.9 78.0 78.06 0 62.9; 62.8; 62.7 62.8; 62.7 62.6100 37.0 37.3 35.7 35.1200 49.5 49.8 49.6 51.9300 49.9 49.7 52.6 52.6400 80.0 80.3 74.4 75.5500 41.3 41.8 43.6 43.2600 19.7 20.0 20.7 20.9700 68.4 68.7 67.7 65.8800 43.5 46.8 42.8 46.3900 65.1 62.7 61.6 62.71000 61.2 62.0 64.2 61.9

CF3

MeO Ph

(R)

C

CH

H

CF3

Ph OMe

(S)

C

CH

O

OH

H2C

HC

CF3

MeO Ph

(R)

O

OH

H2C

HC

CF3

Ph OMe

(S)

H

(5'')

(4'')

(3'')

downfield upfield

(4'')

(5'') (3'')

upfield downfield

4''

(5'')

(3'')

(5'')

(3'')

4''

2HC 2HC

Fig. 1. Configurational correlation models for the (R)-MTPA derivativesand the (S)-MTPA derivatives proposed by Mosher.


vent (32.6 g) was dissolved in water (350 ml) and extractedwith EtOAc (2 · 300 ml). The aqueous phase evaporated todryness under vacuum gave as residue 26.5 g.

Six grams of this was submitted to CCD with thebiphase system H2O:EtOAc:n-PrOH discontinuouslychanging the ratio from 10:9:1 to 10:7:3. The separationswere monitored by TLC, silica gel F254, solvent n-BuOH:-H2O:HOAc = 4:5:1 (upper phase); detection by fluores-cence quenching and/or by spray reagent anisaldehyde:H2SO4:HOAc:EtOH = 0.5:0.5:0.1:9. Three of the nine col-lected fractions, J6, 514 mg, J7, 441 mg, and J6/7, 138 mg,were submitted to CCD on recycling with the solvent sys-tem H2O:EtOAc:n-PrOH = 10:7:3 and three pure com-pounds, craigoside C, 3, 203 mg, craigoside B, 2, 170 mg,and craigoside A, 1, 366 mg, were obtained.

3.4. Craigoside A (1)

Amorphous powder, ½a�20D ¼ �185 (MeOH, c 0.5), UV

(MeOH), kmax nm (log e): 233 (4.54); CD (MeOH), k nm([H]): 211 (�8.1 · 107), 228 (�12.7 · 107), 247 (�8.8 · 107).Molecular formula C61H86O34, ICR-FTMS m/z:1385.48602 [M + Na]+, calcd 1385.48927; ESI-MS m/z:1386.6, 16 [M + Na]+, 1224.3, 100 [M + Na � 162]+,1062.4, 7 [M + Na � 162 · 2]+, 982.4, 67 [M + Na � aniridoid � H2O]+, 819.3, 32 [M + Na � an iridoid �H2O � 162]+. 1H and 13C NMR data in Tables 1 and 2,respectively.

3.5. Craigoside B (2)


(MeOH), kmax nm (log e): 236 (4.29); CD (MeOH), k nm([H]): 233 (�7.0 · 107). Molecular formula C44H64O24,ICR-FTMS m/z: 999.36655 [M + Na]+, calcd 999.36797;ESI-MS m/z: 1000.3, 41 [M + Na]+, 837.3, 55 [M +Na � 162]+, 595.3, 100 [M + Na � an iridoid � H2O]+.1H and 13C NMR data in Tables 1 and 2, respectively.

3.6. Craigoside C (3)


(MeOH), kmax nm (log e): 236 (4.26); CD (MeOH), k nm([H]): 232 (�5.7 · 107). Molecular formula C44H64O24,ICR-FTMS m/z: 999.37081 [M + Na]+, calcd 999.36797;ESI-MS m/z: 999.5, 34 [M + Na]+, 837.3, 100 [M + Na �162]+, 595.3, 15 [M + Na � an iridoid � H2O]+. 1H and13C NMR data in Tables 1 and 2, respectively.

3.7. Acetylation of craigosides A–C

Each substance (50 mg) was acetylated with pyridineand Ac2O (each, 0.5 ml). After evaporation of the reagentsunder vacuum, the compound was purified by CC (silicagel, solvents cyclohexane:EtOAc = 2:8) to give the corre-sponding pure peracetate.

3.7.1. Craigoside A tredeca-acetate

Crystals from cyclohexane, mp 87–89 �C, ½a�24D ¼ �153

(CHCl3, c 0.4). 1H NMR (CDCl3) d: 7.46 (3H, s, H-3 · 3);6.01, 5.99 (3H, bq, J = 6.7, H-8 · 3); 5.72 (3H, bs, H-1 · 3);5.28 (3H, t, J = 9.3, H-3 0 · 3); 5.13 (3H, t, J = 9.3, H-4 0 · 3); 5.12 (3H, dd, 9.3, 7.8, H-2 0 · 3); 5.11 (1H, m, H-400);5.06 (3H, d, J = 7.8, H-1 0 · 3); 4.33 (3H, dd, J = 12.3, 3.2,Ha-6 0 · 3); 4.10 (3H, dd, J = 12.3, 1.5, Hb-6 0 · 3); 3.96 (3H,dd, J = 11.0, 4.8, H-5 · 3); 3.78 (3H, m, J = 9.3, 3.2, 1.5,H-5 0 · 3); 3.71 (9H, s, MeO · 3); 2.66 (3H, dd, J = 13.0,4.8, Ha-6 · 3); 2.42 (3H, dd, J = 13.0, 11.0, Hb-6 · 3); 2.11(1H, m, H-800); 2.08, 2.04 (39H, s, CH3CO · 13); 1.94 (1H,m, Hb-500); 1.86 (1H, m, H-300); 1.75 (9H, d, J = 6.9, H3-10 · 3); 1.65 (1H, m, H-200); 1.51 (1H, m, Ha-500); 1.05 (3H,d, J = 6.3, H3-600). 13C NMR d: 170.9, 170.4, 170.0 (C-7 · 3); 169.3, 169.2 (CH3CO · 13); 166.6 (C-11 · 3); 152.9(C-3 · 3); 128.5, 128.2 (C-9 · 3); 124.7, 124.5 (C-8 · 3);108.5, 108.4 (C-4 · 3); 97.0 (C-100 · 3); 93.7 (C-1 · 3); 77.9(C-400); 72.4 (C-5 0 · 3); 72.1 (C-3 0 · 3); 70.6 (C-2 0 · 3); 68.0(C-4 0 · 3); 67.0 (C-700); 63.2 (C-900); 62.4 (C-1000); 61.5 (C-6 0 · 3); 51.4 (MeO · 3); 48.5 (C-300); 47.7 (C-200); 40.2 (C-500); 39.8 (C-6 · 3); 38.9 (C-800); 35.8 (C-100); 29.9 (C-5 · 3);21.0, 20.6 (CH3CO · 13); 19.4 (C-600); 13.5 (C-10 · 3).

3.7.2. Craigoside B deca-acetate


(CHCl3, c 0.4). 1H NMR (CDCl3) d: 7.46, 7.45 (2H, s,H-3 · 2); 6.01 (2H, bq, J = 6.6, H-8 · 2); 5.73, 5.72 (2H,s, H-1 · 2); 5.28 (2H, t, J = 9.4, H-3 0 · 2); 5.15 (2H, t,J = 9.4, H-4 0 · 2); 5.13 (1H, m, H-400); 5.12 (2H, dd,J = 9.4, 8.1, H-2 0 · 2); 5.06, 5.04 (2H, d, J = 8.1, H-1 0 · 2); 4.33 (2H, dd, J = 12.3, 4.5, Ha-6 0 · 2); 4.11 (2H,dd, J = 12.3, 2.1, Hb-6 0 · 2); 4.00, 3.96 (2H, dd, J = 11.0,4.8, H-5 · 2); 3.80 (2H, m, J = 9.4, 4.6, 2.1, H-5 0 · 2);3.71 (6H, s, MeO · 2); 2.71, 2.62 (2H, dd, J = 13.0, 4.8,Ha-6 · 2); 2.45, 2.40 (2H, dd, J = 13.0, 11.0, Hb-6 · 2);2.11 (1H, m, H-800); 2.09, 2.04, 2.03 (30H, s, CH3CO · 10);1.96 (1H, m, Hb-500); 1.84 (1H, m, H-300);1.76 (6H, d,J = 6.9, H3-10 · 2); 1.65 (1H, m, H- 200); 1.52 (1H, m, Ha-500); 1.05 (3H, d, J = 6.3, H3-600). 13C NMR d: 171.3,170.9 (C-7 · 2); 170.8, 170.7, 170.3, 169.5, 169.0(CH3CO · 10); 166.9 (C-11 · 2); 153.2 (C-3 · 2); 128.8,128.7 (C-9 · 2); 125.0, 124.8 (C-8 · 2); 108.8 (C-4 · 2);97.4, 97.3 (C-1 0 · 2); 94.2, 94.0 (C-1 · 2); 77.8 (C-400); 72.8(C-5 0 · 2); 72.4 (C-3 0 · 2); 71.0 (C-2 0 · 2); 68.5 (C-4 0 · 2);67.3 (C-700); 63.6 (C-900); 62.8 (C-1000); 61.9 (C-6 0 · 2); 51.6(MeO · 2); 49.1 (C-300); 48.1 (C-200); 40.7 (C-500); 40.2, 40.1(C-6 · 2); 39.2 (C-800); 36.0 (C-100); 30.4, 30.3 (C-5 · 2);21.0, 20.8 (CH3CO · 10); 19.6 (C-600); 13.8, 13.7 (C-10 · 2).

3.7.3. Craigoside C deca-acetate


(CHCl3, c 0.4). 1H NMR (CDCl3) d: 7.47 (2H, s, H-3 · 2);6.01 (2H, bq, J = 7.2, H-8 · 2); 5.71 (2H, bs, H-1 · 2); 5.28(2H, t, J = 9.6, H-3 0 · 2); 5.14 (2H, t, J = 9.6, H-4 0 · 2);5.13 (1H, m, H-400); 5.12 (2H, dd, J = 9.6, 8.1, H-2 0 · 2);5.04 (2H, d, J = 8.1, H-1 0 · 2); 4.33 (2H, dd, J = 12.3, 4.2,


Ha-6 0 · 2); 4.07 (2H, dd, J = 12.3, 1.8, Hb-6 0 · 2); 4.02 (2H,dd, J = 11.0, 4.8, H-5 · 2); 3.78 (2H, m, J = 9.6, 4.2, 1.8,H-5 0 · 2); 3.72 (6H, s, MeO · 2); 2.70, 2.69 (2H, dd,J = 13.0, 4.8, Ha-6 · 2); 2.44 (2H, dd, J = 13.0, 11.0, Hb-6 · 2); 2.11 (1H, m, H-800); 2.08, 2.03, 2.02, 2.00 (30H, s,CH3CO · 10); 1.96 (1H, m, Hb-500); 1.84 (1H, m, H-300);1.76 (6H, d, J = 6.9, H3-10 · 2); 1.63 (1H, m, H-200); 1.54(1H, m, Ha-500); 1.05 (3H, d, J = 6.6, H3-600). 13C NMR d:171.0, 170.8 (C-7 · 2); 170.5, 170.4, 170.2, 170.0, 169.2,169.1 (CH3CO · 10); 166.5 (C-11 · 2); 152.9 (C-3 · 2);128.4 (C-9 · 2); 124.7 (C-8 · 2); 108.5 (C-4 · 2); 97.0, 96.9(C-1 0 · 2); 93.7, 93.6 (C-1 · 2); 76.9 (C-400); 72.4 (C-5 0 · 2);72.1 (C-3 0 · 2); 70.7 (C-2 0 · 2); 68.1 (C-4 0 · 2); 66.1 (C-700);63.2 (C-900); 62.5 (C-1000); 61.6 (C-6 0 · 2); 51.3 (MeO · 2);48.4 (C-300); 48.0 (C-200); 40.5 (C-500); 39.8, 39.7 (C-6 · 2);38.9 (C-800); 35.2 (C-100); 30.0, 29.9 (C-5 · 2); 21.0, 20.4(CH3CO · 10); 19.1 (C-600); 13.4 (C-10 · 2).

3.8. Alkaline hydrolysis of craigosides A–C. Methylation

with diazomethane

Each compound (300 mg) was treated with 0.5 MNaOH (5 ml). After 20 h the solution was neutralized withweakly acid cation-exchanger (H+ form) and concentratedin vacuum to dryness. The residue was dissolved in MeOHand methylated with an ethereal solution of diazomethane.After 2 days the residue obtained by evaporation of the sol-vents was submitted to CCD with solvent system H2O:n-BuOH:EtOAc = 10:7.5:2.5 and dimethyl oleoside, 4, andtetraol iridane, 5, were separated. The former was identi-fied by NMR and rotatory power (Tanahashi et al., 1988).

3.8.1. Tetraol iridane (5)

Syrop, ½a�26D ¼ þ2:4 (MeOH, c 0.4). Molecular formula

C10H20O4, ICR-FTMS m/z: 227.12552 [M + Na]+, calcd227.12538; FAB-MS m/z: 205, 100 [M + 1]+, 187, 66[M � 17]+. 1H and 13C NMR data in Tables 1 and 2,respectively.

3.9. Acetylation of 5: iridane tetra-acetate (9)

Tetraol iridane 5 (28 mg) was acetylated with pyridineand Ac2O (each, 1 ml). After evaporation of the reagentsunder vacuum, the product was purified by CC (silica gel,solvents cyclohexane:EtOAc = 3:7) to give the correspond-ing tetra-acetate. Oily, ½a�23

D ¼ þ14:2 (CHCl3, c 0.3). Molec-ular formula C18H28O8, FAB-MS m/z: 372 (1, M), 329 (3,M-Ac), 313 (3, M-AcO), 269 (45, M-Ac-AcOH), 150 (100).1H NMR (CDCl3) d: 5.12 (1H, quintet, J = 6.0, 3.0, 3.0, H-400); 4.17, 4.05 (2H, m, H2-900); 4.13, 3.99 (2H, m, H2-1000);4.11, 4.00 (2H, m, H2-700); 2.11 (1H, m, J = 6.3, H-800); 2.08,2.06, 2.05, 2.02 (12H, s, CH3CO · 4); 1.96 (1H, m, J = 9.0,6.3, 3.0, H-300); 1.94 (1H, m, J = 10.5, 9.0, 6.8, 6.6, H-100);1.83 (1H, m, J = 13.5, 6.8, 3.0, Hb-500); 1.63 (1H, m, J =9.0, 9.0, H-200); 1.52 (1H, dq, J = 13.5, 10.5, 6.0, Ha -500);1.07 (3H, d, J = 6.6, H3-600). 13C NMR d: 169.9 (CH3CO ·4); 77.1 (C-400); 66.1 (C-700); 63.4 (C-900); 62.4 (C-1000); 48.6

(C-300); 48.1 (C-200); 40.5 (C-500); 39.0 (C-800); 35.2 (C-100);21.1, 20.7 (CH3CO · 4); 19.1 (C-600).

3.10. (S)-MTPA tetra-ester of 5 (10)

A suspension of 5 (40 mg) in anhydrous CH2Cl2 (15 ml)was added with (S)-MTPA (188 mg), DMAP (26 mg) andthen with DCC (176 mg). After 2 days of stirring, more(S)- MTPA (45 mg) and DCC (46 mg) were added. After 2days the mixture was diluted with water and extracted withadditional CH2Cl2. The residue of the evaporation of theorganic phase was submitted to CC (silica gel, solvents cyclo-hexane:EtOAc=8:2) and the tetra acyl derivative 10 wasobtained. Molecular formula C50H48O12F12, ICR-FTMSm/z: 1091.28111 [M + Na]+, calcd 1091.28464. 1H NMR(CDCl3) d: 7.47-7.37 (20H, m, ArH5 · 4); 5.15 (1H, m, H-400); 4.26 (1H, dd, J = 11.4, 4.9, Hb-1000); 4.22 (1H, dd,J = 12.0, 4.4, Ha-1000); 4.13, 4.11 (2H, m, H2-700); 4.08 (1H,m, Hb-900); 3.93 (1H, dd, J = 11.4, 6.8, Ha-900); 3.50, 3.47,3.45 (12H, s, MeO · 4); 2.05 (1H, m, H-800); 1.81 (1H, m,H-300); 1.69 (1H, m, Hb-500); 1.65 (1H, m, H-100); 1.46 (1H,m, H-200); 1.16 (1H, m, Ha-500); 0.83 (3H, d, J = 6.2, H3-600).13C NMR d: 166.2 (CO · 4); 132.2 (C Ar1 · 4); 129.8,128.7, 127.4, 127.3 (C Ar2-6 · 4); 122.3 (CF3 · 4); 84.3 (C-1 · 4); 79.6 (C-400); 68.0 (C-700); 64.3 (C-900); 63.3 (C-1000);55.6, 55.3 (MeO · 4); 48.9 (C-300); 48.1 (C-200); 40.0 (C-500);38.8 (C-800); 35.2 (C-100); 18.7 (C-600).

3.10.1. (R)-MTPA tetra-ester of 5 (11)

Tetraol 5 was likewise treated with (R)-MTPA and tetraacyl derivative 11 was obtained. Molecular formulaC50H48O12F12, ICR-FTMS m/z: 1091.27944 [M + Na]+,calcd 1091.28464. 1H NMR (CDCl3) d: 7.47–7.36 (20H, m,ArH5 · 4); 5.09 (1H, m, H-400); 4.36 (1H, dd, J = 11.7, 5.1,Hb-1000); 4.10 (1H, m, Ha-1000); 4.08, 4.06 (2H, m, H2-700);3.98, 3.92 (2H, m, H2-900); 3.49, 3.47 (12H, s, MeO · 4);2.04 (1H, m, H-800); 1.81 (1H, m, Hb-500); 1.74 (1H, m, H-100); 1.73 (1H, m, H-300); 1.54 (1H, m, H-200); 1.37 (1H, m,Ha-500); 0.86 (3H, d, J = 6.0, H3-600). 13C NMR d: 165.9(CO · 4); 132.0 (C Ar1 · 4); 129.7, 128.5, 127.2 (C Ar2-6 · 4);121.4 (CF3 · 4); 84.4 (C-1 · 4); 79.3 (C-400); 67.9 (C-700); 63.9 (C-900); 63.5 (C-1000); 55.3 (MeO · 4); 48.7 (C-300);47.8 (C-200); 39.8 (C-500); 38.9 (C-800); 35.4 (C-100); 18.7 (C-600).

Acknowledgements

The authors thank Professor Marcello Nicoletti (Univer-sita La Sapienza Rome, Italy) for his NMR data support.

References

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Ohtani, I., Kosumi, T., Kashman, Y., Kakisawa, H., 1991. High-field FT-NMR application of Mosher�s method. The absolute configuration ofmarine terpenoids. J. Am. Chem. Soc. 13, 4092–4096.

Shen, Y.C., Hsieh, P.W., 1997. Four new secoiridoid glucosides fromJasminum urophyllum. J. Nat. Prod. 60, 453–457.

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sambac. Tetrahedron Lett. 29, 1793–1796.

Tanahashi, T., Nagakura, N., Kuwajima, H., Takaiski, K., Inoue, K.,Inouye, H., 1989. Secoiridoid glucosides from Jasminum mesnyi.Phytochemistry 28, 1413–1415.

Tanahashi, T., Takenaka, Y., Nagakura, N., Nishi, T., 2000. Fivesecoiridoid glucosides esterified with a cyclopentanoid monoterpeneunit from Jasminum nudiflorum. Chem. Pharm. Bull. 48, 1200–1204.

Zhang, Y.J., Liu, Y.Q., Pu, X.Y., Yang, C.R., 1995. Iridoidal glycosidesfrom Jasminum sambac. Phytochemistry 38, 899–903.


Acetylated flavonol diglucosides from Meconopsis quintuplinervia

Xiao-Ya Shang a, Ying-Hong Wang a, Chong Li b, Cheng-Zhong Zhang b,Yong-Chun Yang a, Jian-Gong Shi a,*

a Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, Chinab Lanzhou Medical College, Lanzhou University, Lanzhou 730000, China

Received 11 March 2005; received in revised form 17 August 2005Available online 18 January 2006

Abstract

Four acetylated flavonol diglucosides, quercetin 3-O-[2000-O-acetyl-b-D-glucopyranosyl-(1! 6)-b-D-glucopyranoside] (1), quercetin 3-O-[2000,6000-O-diacetyl-b-D-glucopyranosyl-(1! 6)-b-D-glucopyranoside] (2), isorhamnetin 3-O-[2000-O-acetyl-b-D-glucopyranosyl-(1! 6)-b-D-glucopyranoside] (3), and quercetin 3-O-[2000-O-acetyl-a-L-arabinopyranosyl-(1! 6)-b-D-glucopyranoside] (4), together with fiveknown flavonol glycosides quercetin 3-O-b-D-glucopyranoside, kaempferol 3-O-b-D-glucopyranoside, quercetin 3-O-[b-D-galactopyrano-syl-(1! 6)-glucopyranoside], isorhamnetin 3-O-[b-D-galactopyranosyl-(1! 6)-b-D-glucopyranoside], and kaempferol 3-O-[b-D-gluco-pyranosyl-(1! 2)-b-D-glucopyranoside] have been isolated from Meconopsis quintuplinervia. Their structures were determined usingchemical and spectroscopic methods including HRFABMS, 1H–1H COSY, HSQC and HMBC experiments.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Meconopsis quintuplinervia Regel; Papaveraceae; Acetylated flavonol glycosides

1. Introduction

Meconopsis quintuplinervia Regel, a plant belonging tothe Papaveraceae family and widely distributed in theQingzang plateau of the northwest of China (Luoet al., 1984), is used as a traditional Tibetan medicinefor treatments of various diseases, such as inflammation,pain, hepatitis and tuberculosis (Luo et al., 1984). Thereare, however, very few reports (Wang et al., 1991; Wangand Chen, 1995) concerning secondary metabolites of M.

quintuplinervia. As part of our program to assess system-atically the chemical and biological diversity of medicinalplants distributed at higher altitude, we carried out achemical investigation of this plant. In previous papers(Shang et al., 2002, 2003a,b), we described the isolationand structural identification of three alkaloids, norsan-

guinarine, O-methylflavinantine and meconoquintupline,and seven flavonoids, quercetin, dihydroquercetin,luteolin, chrysoeriol, apigenin, huazhongilexone andhydnocarpin from the less polar fraction of the ethanolicextract of the plant. In continuation of this work, fournew acetylated flavonol diglucosides (1–4), together withfive known flavonol glycosides, have been isolated fromthe polar fraction of the same material. By comparisonof the spectroscopic data with those reported in theliterature, the known compounds were characterizedas quercetin 3-O-b-D-glucopyranoside (Veit et al.,1990), kaempferol 3-O-b-D-glucopyranoside (Chaurasiaand Wichtl, 1987), quercetin 3-O-[b-D-galactopyranosyl-(1! 6)-glucopyranoside] (Waage and Hedin, 1985),isorhamnetin 3-O-[b-D-galactopyranosyl-(1! 6)-b-D-glucopyranoside] (Degot et al., 1971) and kaempferol 3-O-[b-D-glucopyranosyl-(1 ! 2)-b-D-glucopyranoside](Song, 1990). The present paper deals with isolation andstructural elucidation of compounds 1–4.


doi:10.1016/j.phytochem.2005.12.002

* Corresponding author. Tel.: +86 10 83154789; fax: +86 10 63017757.E-mail address: [email protected] (J.-G. Shi).



PHYTOCHEMISTRY



The water soluble portion of the ethanolic extract of M.

quintuplinervia Regel was subjected successively to columnchromatography on macroporous adsorbent resin, normalphase and reversed phase silica gels and Sephadex LH-20,to afford two mixtures which were further purified by pre-parative reversed phase HPLC to yield compounds 1–4 andthe known compounds.

Compound 1 was isolated as a yellow amorphous pow-der. Its IR spectrum showed the presence of hydroxyl(3400 cm�1), conjugated carbonyl (1734 and 1655 cm�1)and aromatic ring (1506 and 1604 cm�1) functional groups.Its UV spectrum exhibited absorption bands characteristicfor flavonols at 207, 257, 270, 296, and 362 nm. The posi-

tive FABMS exhibited a quasi-molecular ion peak at m/z669 [M + H]+, with the molecular formula established asC29H32O18 by the positive HRFABMS at m/z669.1615[M + H]+ (calcd. for C29H33O18 669.1666). The1H NMR spectrum of 1 showed two anomeric proton dou-blets at d 5.06 (1H, d, J = 7.8 Hz, H-100) and 4.15 (1H, d, J= 8.1 Hz, H-1000) and an acetyl singlet at d 1.61 (3H, s), inaddition to resonances characteristic for a quercetin agly-cone moiety (Table 1), as well as signals attributed toremaining protons of two glycosyl units between d 2.84and 4.40. These data indicated that compound 1 was anacetylated quercetin diglycoside, which was confirmed byanalysis of the 13C NMR spectroscopic data of 1 (Table2). Acid hydrolysis of 1 produced glucose as the sole sugaras identified by TLC and PC comparison with authenticsugar samples. The 1H–1H COSY and HSQC experimentsof 1 led to unambiguous assignments of signals of the pro-tons and protonated carbons in the NMR spectra of 1,while the resolvable axial-axial couplings between vicinalprotons of the glycosyl units, excluding H-500, H2-600, H-5000 and H2-6000 (Table 1), confirmed that both glycosyl unitswere b-glucopyranosyls. The chemical shift of C-3 sug-gested that the quercetin aglycone was glycosylated at C-3, which was confirmed by a long range correlation fromH-100 to C-3 (d 136.0) in the HMBC spectrum of 1. Mean-while, HMBC correlations from H-1000 to C-600(d 69.2) andH2-600 to C-1000 (d 102.1) unequivocally revealed an 1! 6connectivity between the two b-glucopyranosyls. In addi-tion, the carbonyl (d 169.1) of the acetoxyl group corre-lated to H-2000 of the outer b-glucopyranosyl unit at d

Table 11H NMR spectroscopic data for compounds 1–4

No. 1 2 3 4

6 6.15 d (1.8) 6.16 d (2.1) 6.15 d (2.0) 6.15 d (2.1)8 6.38 d (1.8) 6.39 d (2.1) 6.38 d (2.0) 6.38 d (2.1)20 8.01 d (2.4) 8.07 d (2.1) 8.16 d (1.8) 8.09 d (2.1)50 6.84 d (9.0) 6.85 d (8.5) 6.87 d (8.7) 6.84 d (9.0)60 7.65 dd (2.4, 9.0) 7.68 dd (2.1, 8.5) 7.63 dd (1.8, 8.7) 7.69 dd (2.1, 9.0)100 5.06 d (7.8) 5.08 d (7.8) 5.25 d (7.8) 5.01 d (7.8)200 3.80 dd (7.8, 8.4) 3.82 dd (7.8, 7.8) 3.80 dd (7.8, 8.1) 3.83 dd (7.8, 7.8)300 3.54 dd (8.4, 7.5) 3.55 dd (7.8, 7.5) 3.54 dd (8.1, 9.4) 3.55 dd (7.8, 7.5)400 3.72 dd (7.5, 7.2) 3.70 dd (7.5, 7.8) 3.70 dd (9.4, 7.2) 3.69 dd (7.5, 7.2)500 3.51 m 3.52 m 3.50 m 3.53 m

600 3.68 m 3.64 m 3.68 m 3.64 m

1000 4.15 d (8.1) 4.16 d (8.1) 4.22 d (7.8) 4.01 d (7.8)2000 4.40 dd (8.1, 9.0) 4.37 dd (8.1, 8.4) 4.40 dd (7.8, 9.3) 4.63 dd (7.8, 9.3)3000 3.04 dd (9.0, 9.3) 2.98 dd (8.4, 9.3) 3.06 dd (9.3, 9.3) 2.94 dd (9.3, 9.3)4000 3.20 dd (9.3, 9.6) 3.15 dd (9.3, 9.3) 3.19 dd (9.3, 9.6) 3.52 m

5000 2.84 m 2.84 m 2.67 m (a) 3.00 brd (12.3)(b) 3.70 m

6000 (a) 3.69 m (a) 4.20 dd (2.1, 12.3) (a) 3.64 dd (2.4, 11.7)(b) 3.57 m (b) 4.03 dd (5.5,12.3) (b) 3.48 m

Ac 1.61 s 1.59 s 1.64 s 1.56 s

Ac 1.98 s

OMe 3.92 s

1H NMR data were measured in methanol-d4 at 300 MHz. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based onDEPT,1H–1H COSY, HSQC and HMBC experiments.

O

OH

OR1

OH

HO

OO

O

HOHO

OHOR3

O

HOOR2

R5

1 R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OH2 R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OAc3 R1 = Me, R2 = Ac, R3 = OH, R4 = H, R5 = CH2OH4 R1 = R3 = H, R2 = Ac, R4 = OH, R5 = H

R41"

1'"

512 X.-Y. Shang et al. / Phytochemistry 67 (2006) 511–515

4.40 (1H, dd, J = 8.1 and 9.0 Hz), demonstrating that theacetoxyl group was located at C-2000 of the glucopyranosyl.Thus, 1 was quercetin 3-O-[2000-O-acetyl-b-D-glucopyrano-syl-(1! 6)-b-D-glucopyranoside].

Compound 2 was obtained as a yellow powder with amolecular formula C31H34O19 as determined by the posi-tive FABMS at m/z 711.1810 [M + H]+ (calcd. forC31H35O19, 711.1772). The UV, IR and NMR spectra of2 were similar to those of 1, except for the appearance ofsignals due to one more acetoxyl unit at dH 1.98 (3H, s)and dC 19.6 (q) and 171.8 (s) in the NMR spectra of 2, indi-cating that it was an acetylated cognate of 1. This was sup-ported by acid hydrolysis and 2D NMR spectroscopicexperiments of 2. In the HMBC spectrum of 2 correlationsfrom H-2000 to one acetoxyl carbonyl and from H2-6000 to theother unequivocally established that the two acetyls wereesterified at C-2000 and C-6000 of the outer glucosyl moiety,respectively. Therefore, 2 was quercetin 3-O-[2000,6000-O-dia-cetyl-b-D-glucopyranosyl-(1! 6)-b-D-glucopyranoside].

Compound 3 was obtained as a yellow powder. Itsmolecular formula was determined as C30H34O18 by thepositive FABMS at m/z 683.1854 [M + H]+ (calcd. for

C30H35O18 683.1823). The UV, IR and NMR spectra of3 were similar to those of 1, except for the appearance ofsignals attributed to an aromatic methoxyl group at dH

3.92 (3H, s) and dC 57.0 in the NMR spectra of 3, indicat-ing that it was a methylated derivative of 1. A comparisonof the NMR spectroscopic data of 3, with those of theco-occurring isorhamnetin 3-O-[b-D-galactopyranosyl-(1! 6)-b-D-glucopyranoside] (Degot et al., 1971), demon-strated that the aglycone of 3 was isorhamnetin. Therefore,3 was isorhamnetin 3-O-[2000-O-acetyl-b-D-glucopyranosyl-(1! 6)-b-D-glucopyranoside].

Compound 4 was obtained as a yellow powder with amolecular formula C28H30O17 as established by the positiveHRFABMS at m/z 639.1546 [M + H]+. The UV and IRspectra of 4 were similar to those of 1. A comparison ofits NMR spectroscopic data with those of 1 (Tables 1and 2) indicated that the only difference between 1 and 4

was replacement of the outer glucopyranosyl of 1 by anarabinopyranosyl (Simon et al., 1993) in 4. This was sup-ported by acid hydrolysis of 4 yielding glucose and arabi-nose as the sugars. The location of the acetyl linkagebetween the glycosyls in 4 was further confirmed by 2DNMR experiments (1H–1H COSY, HSQC and HMBC).Consequently, 4 was quercetin 3-O-[2000-O-acetyl-a-L-arabi-nopyranosyl-(1! 6)-b-D-glucopyranoside].

Previous studies indicated that plants of the genusMeconopsis contained alkaloids (Hemingway et al., 1981;Allais et al., 1983; Liu and Wang, 1986; Wang and Chen,1995), triterpenoids (Zhang et al., 1997) and flavonoids(Tanaka et al., 2001), although the emphasis of the chemi-cal investigations thus far was focused on the alkaloids inspecies of this genus. However, our systematical chemicalinvestigation of M. quintuplinervia has revealed that diverseflavonoids represent the main metabolites in this specieswhile two morphinane alkaloids, O-methylflavinantineand meconoquintupline, and a benzophenanthrindine alka-loid norsanguinarine, were obtained (Shang et al., 2002,2003a,b). The structures of the acetylated flavonol diglyco-sides and meconoquintupline from M. quintuplinervia weredistinctive by the number and/or substitution position ofacetyl in the acetylated flavonol diglycosides and the8,14-dihydrogenation of the morphinane skeleton inmeconoquintupline, i.e., even though flavonoids fromMeconopsis grandis (Tanaka et al., 2001) and the morphi-nane/benzophenanthrindine alkaloids from several Mecon-

opsis species (Hemingway et al., 1981) have been reported,respectively. Both alkaloids and flavonoids may, therefore,have chemotaxonomically important roles in the genusMeconopsis though flavonoids from this genus havereceived relatively little attention.

In the cytotoxic and antioxidant assays compounds 1–4

and the known flavonoids showed neither cytotoxicityagainst human colon cancer (HCT-8), hepatoma (Bel-7402), stomach cancer (BGC-823), and lung adenocarci-noma (A549) cell lines (IC50 > 10 lg/mL) nor significantantioxidant activity inhibiting rat liver microsomal lipidperoxidation (IC50 > 5 lg/mL).

Table 21C NMR spectroscopic data for compounds 1–4

No. 1 2 3 4

2 158.2 156.9 158.1 157.83 136.0 134.8 135.5 136.24 179.2 178.1 179.4 179.25 162.9 161.8 163.0 162.96 100.0 98.8 100.0 100.17 166.2 165.1 166.2 166.78 94.9 93.8 95.0 95.09 158.3 157.2 158.4 158.310 105.6 104.4 105.8 105.41 0 122.6 121.4 122.8 122.62 0 117.9 116.7 114.5 117.93 0 145.9 144.8 148.5 146.04 0 150.2 149.2 151.1 150.35 0 116.5 115.4 116.2 116.36 0 122.9 121.6 123.6 122.8100 105.7 104.4 104.5 106.0200 73.2 72.0 73.1 73.2300 74.8 73.5 74.7 74.8400 70.5 69.6 70.4 70.7500 77.3 76.6 77.3 77.8600 69.2 68.0 68.8 69.11000 102.1 100.8 101.8 102.72000 75.3 74.0 75.3 73.73000 75.7 74.3 75.8 72.04000 71.3 70.2 71.3 69.85000 77.2 73.6 77.3 67.06000 62.2 63.1 62.1Ac 20.3 19.3 20.6 20.6

169.1 170.5 171.8 172.0Ac 19.6

171.8OMe 57.0

13C NMR data were measured in methanol-d4 at 75 MHz. The assign-ments were based on DEPT, 1H–1H COSY, HSQC and HMBCexperiments.

X.-Y. Shang et al. / Phytochemistry 67 (2006) 511–515 513

3. Experimental

3.1. General

Melting points were determined on an XT-4 micro melt-ing point apparatus and are uncorrected. IR spectra wererecorded as KBr disks on a Nicolet Impact 400 FT-IRSpectrophotometer. 1D- and 2D NMR spectra wereobtained at 300 and 75 MHz for 1H and 13C, respectively,on Inova 300 or 500 MHz spectrometers in methanol-d4 orDMSO-d6 with solvent peaks as references. FABMS andHRFABMS data were measured with a Micromass Auto-spec-Ultima ETOF spectrometer. Column chromatogra-phy was performed with silica gel (200–300 mesh), RP-18reversed phase silica gel (43–60 lm) and Sephadex LH-20. HPLC separation was performed on an instrument con-sisting of a Waters 600 controller, a Waters 600 pump, anda Waters 2487 dual k absorbance detector with an Alltima(250 · 22 mm) preparative column packed with C18

(10 lm). TLC was carried out with glass precoated silicagel GF254 plates. Spots were visualized under UV light orby spraying with 3% FeCl3 in EtOH or 7% H2SO4 in95% EtOH followed by heating.

3.2. Plant material

M. quintuplinervia Regel (4 kg) was collected at Dabanmountain at an altitude of 3400–3600 m, Qinghai province,China, in August of 1999. The plant was identified by Prof.Guo-liang Zhang (Department of Biology, Lanzhou Uni-versity, Lanzhou 730000, China). A voucher specimen(No. 200025) was deposited at the Herbarium of theDepartment of Medicinal Plants, Institute of Materia Med-ica, Beijing, China.


Air dried aerial parts of M. quintuplinervia (4 kg) wereextracted with 11.0 L of 90% EtOH at room temperaturefor 3 · 48 h. The ethanolic extract was evaporated toalmost dryness in vacuo to yield a dark brown viscidresidue (470 g). The residue was suspended in H2O(1100 mL) and then partitioned successively with petro-leum ether (4 · 800 mL), and EtOAc (4 · 650 mL). Theaq. phase resulting from the partition was applied to amacroporous adsorbent resin (RA, Seventh Factory of Bei-jing Chemical Industry, China) (650 g, dried weight) col-umn using H2O and EtOH–H2O (6:4) as eluents. Aftersolvent removal, the fraction (7.8 g) eluted by EtOH–H2O (6:4) was subjected to normal phase silica gel CC elut-ing with a gradient of increasing MeOH in CHC13. TheCHCl3–MeOH (4:1) eluent gave a mixture that was sepa-rated into three subfractions by gel chromatography overSephadex LH-20 eluted with CHCl3–MeOH (1:1). Thethird subtraction was purified by reversed-phase HPLCusing MeOH–H2O (45:55) as mobile phase to give querce-tin 3-O-[b-D-galactopyranosyl-(1! 6)-glucopyranoside]

(35 mg) and quercetin 3-O-b-D-glucopyranoside (21 mg).The CHCl3–MeOH (2:1) eluent was separated into foursubfractions by gel chromatography over Sephadex LH-20 eluted with CHCl3–MeOH (1:1). The third and fourthsubfractions were further purified, respectively, by prepara-tive reversed phase HPLC using MeOH–H2O (40:60) as themobile phase to afford 1 (18 mg), 2 (21 mg), 3 (17 mg), 4

(15 mg), kaempferol 3-O-b-D-glucopyranoside (27 mg),isorhamnetin 3-O-[b-D-galactopyranosyl-(1! 6)-b-D-glucopyranoside] (31 mg) and kaempferol 3-O-[b-D-gluco-pyranosyl-(1! 2)-b-D-glucopyranoside] (13 mg).

3.4. Quercetin 3-O-[2000-O-acetyl-b-D-glucopyranosyl-

(1! 6)-b-D-glucopyranoside] (1)

Amorphous yellow powder; ½a�20D +20.6 (MeOH c 0.16);

UV kMeOHmax nm (log e): 207 (4.37), 257 (4.14), 270 (4.03), 296

(3.73), 362 (4.08); IR mKBrmax cm�1: 3400, 2910, 1734, 1655,

1604, 1506, 1444, 1360, 1304, 1200, 1169, 1074, 1022. For1H and 13C NMR spectroscopic data, see Tables 1 and 2;FABMS (m/z): 669 [M + H]+. HRFABMS (m/z):669.1615 [M + H]+, C29H33O18 requires 669.1666.

3.5. Quercetin 3-O-[2000,6000-O-diacetyl-b-D-glucopyranosyl-


Amorphous yellow powder; ½a�20D +30.8 (MeOH; c 0.25);


(3.77), 363 (4.13); IR mKBrmax cm�1: 3419, 2908, 1732, 1653,

1604, 1506, 1444, 1361, 1244, 1078. For 1H and 13CNMR spectroscopic data, see Tables 1 and 2; FABMS(m/z): 711[M + H]+, HRFABMS (m/z): 711.1810[M + H]+, C31H35O19 requires 711.1773.

3.6. Isorhamnetin 3-O-[2000-O-acetyl-b-D-glucopyranosyl-



UV kMeOHmax nm (log e): 206 (4.39), 255(4.08), 269(3.99),

298(3.75), 357(4.03); IR mKBrmax cm�1: 3415, 2908, 1732,

1653, 1604, 1514, 1431, 1356, 1290, 1203, 1074, 1028. For1H and 13C NMR spectroscopic data, see Tables 1 and 2;FAB MS m/z: 683 [M + H]+; HRFABMS m/z: 683.1854[M + H]+, C30H35O18 requires 683.1823.

3.7. Quercetin 3-O-[2000-O-acetyl-a-L-arabinopyranosyl-(1! 6)-b-D-glucopyranoside] (4)



(3.94), 363 (4.26); IR mKBrmax cm�1: 3400, 2918, 1732, 1653,

1604, 1498, 1446, 1360, 1304, 1201, 1171, 1072, 1020. For1H and 13C NMR spectroscopic data, see Tables 1 and 2;FABMS m/z: 639 [M + H]+; HRFABMS m/z: 639.1546[M + H]+, C28H31O17 requires 639.1561.

514 X.-Y. Shang et al. / Phytochemistry 67 (2006) 511–515

3.8. Acid hydrolysis of 1–4

A solution of each compound (5 mg) in 2 N HCl (2 mL)was individually refluxed for 16 h at 94 �C. The reactionmixture was partitioned with EtOAc, with the aqueousphase neutralized with 1 N NaOH and dried using a streamof N2. The resulting residue was dissolved in EtOH(0.5 mL) and analyzed by TLC and PC together withauthentic sugar samples, using as developing solvent sys-tems CHCl3–MeOH (2.5:1) for TLC and the upper layerof n-BuOH–AcOH–H2O (4:1:5) for PC; products werevisualized by spraying aniline hydrogen phthalate followedby heating at 105 �C.

Acknowledgements

The authors are grateful to A. Zeper for mass spectrameasurements. Financial support is from the NSFC (GrantNo. 20432030).

References

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Chaurasia, N., Wichtl, M., 1987. Flavonol glycosides from Urtia dioica.Planta Med. 53, 432–434.

Degot, A.V., Litvinenko, V.I., Kurinnaya, N.V., 1971. Flavonoids ofOrthantha lutea. Khim. Prir. Soedin. 7, 117–119.

Hemingway, S.R., Phillipson, J.D., Verpoorte, R., 1981. Meconopsis

cambrica alkaloids. J. Nat. Prod. 44, 67–74.Liu, S.Y., Wang, X.K., 1986. Studies on chemical constituents of

Meconopsis punicea. Zhong Yao Tong Bao 11, 360–362.Luo, D.S., Sun, A.L., Xia, G.C., 1984. Tibetan drug in Qingzang plateau,

a preliminary investigation of resources Meconopsis. Zhong Cao Yao15, 359–360.

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Shang, X.Y., Shi, J.G., Yang, Y.C., Liu, X., Li, C., Zhang, C.Z., 2003a.Alkaloids from a Tibetan medicine Meconopsis quintuplinervia Regel.Acta Pharmaceut. Sin. 38, 276–278.

Shang,X.Y.,Jiao,H.S.,Yang,Y.C.,Shi,J.G.,2003b.Amorphinanealkaloidfrom Meconopsis quintuplinervia. Chin. Chem. Lett. 14, 597–598.

Simon, A., Chulia, A.J., Kaouadji, M., Allais, D.P., Delage, C., 1993.Further flavonoid glycosides from Calluna vulgaris. Phytochemistry 32,1045–1049.

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Waage, S.K., Hedin, P.A., 1985. Quercetin 3-O-galactopyranosyl-(1! 6)-glucopyranoside, a compound from narrowleaf vetch with antibacte-rial activity. Phytochemistry 24, 243–245.

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quintuplinervia Regel. Nat. Prod. Res. Develop. 7, 32–34.Wang, M.A., Chen, S.N., Zhang, H.D., Chen, Y.Z., 1991. Studies on the

chemical constituents of Meconopsis quintuplinervia Regel, a Tibetanmedicinal herb. J. Lanzhou Univ. (Nat. Sci.) 27, 80–92.

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X.-Y. Shang et al. / Phytochemistry 67 (2006) 511–515 515

Lignan, phenolic and iridoid glycosides fromStereospermum cylindricum

Tripetch Kanchanapoom a,*, Pawadee Noiarsa a, Hideaki Otsuka b, Somsak Ruchirawat c

a Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailandb Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan

c Chulabhorn Research Institute, Vipavadee Rangsit Highway, Bangkok 10210, Thailand

Received 21 February 2005; received in revised form 25 August 2005Available online 28 November 2005

Abstract

A lignan glycoside [(+)-cycloolivil 4 0-O-b-D-glucopyranoside], a phenolic glycoside [3,4-dimethoxyphenyl 1-O-b-D-xylopyranosyl-(1! 6)-b-D-glucopyranoside] and a iridoid glycoside (stereospermoside) were isolated from the leaves and branches of Stereospermum

cylindricum, together with (+)-cycloolivil, (+)-cycloolivil 6-O-b-D-glucopyranoside, (�)-olivil, (�)-olivil 4-O-b-D-glucopyranoside, (�)-olivil 4 0-O-b-D-glucopyranoside, vanilloloside, decaffeoyl-verbascoside, isoverbascoside, 3,4,5-trimethoxyphenyl 1-O-b-D-xylopyrano-syl-(1! 6)-b-D-glucopyranoside, ajugol, verminoside, and specioside. The structure elucidations were based on spectroscopic evidence.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Stereospermum cylindricum; Bignoniaceae; Lignan glucoside; Phenolic glycoside; Iridoid glucoside; Stereospermoside

1. Introduction

As part of our systematic investigation on Thai Bigno-niaceous plants, especially in tribe Tecomeae, we reportedthe constituents of Fernandoa adneophylla (Kanchanapoomet al., 2001), Markhamia stipulata (Kanchanapoom et al.,2002a) and Barnettia kerrii (Kanchanapoom et al.,2002b). To further study plants in the same tribe, we inves-tigated the constituents of Stereospermum cylindricumPierre ex P. Dop. (Thai name: Khae-Khao), collected fromthe Botanical garden, Faculty of Pharmaceutical Sciences,Khon Kaen University, Thailand. S. cylindricum is a treeup to 25 m high, distributed in South-east Asia. The barkof this plant is used in Thai traditional medicine for anti-fever purposes, as well as an anti-inflammatory agent. Nophytochemical study has been carried out on this species.Preliminary studies on plants in this genus have led toisolation of several compounds such as lignans (Ghogomu

et al., 1985), and quinones (Onegi et al., 2002; Kumar et al.,2003). The present paper deals with the isolation of 15compounds, including a new lignan glycoside, a new phe-nolic diglycoside and a new iridoid glycoside, as well as12 known compounds.


The methanolic extract was suspended in H2O and defat-ted with Et2O. The aqueous layer was subjected to DiaionHP-20 column chromatography, using H2O, MeOH andMe2CO as eluants, successively. The portion eluted withMeOH was repeatedly subjected to silica gel, RP-18, andpreparative HPLC-ODS chromatography to afford 15 com-pounds. Twelve were identified as (+)-cycloolivil (1) (Abeet al., 1988), (+)-cycloolivil 6-O-b-D-glucopyranoside (2)(Sugiyama et al., 1993), (�)-olivil (4) (Abe et al., 1988),(�)-olivil 4-O-b-D-glucopyranoside (5) (Abe et al.,1988), (�)-olivil 4 0-O-p-glucopyranoside (6) (Abe et al.,1988), vanilloloside (7) (Ida et al., 1993), decaffeoyl-verbas-coside (8) (Karasawa et al., 1986), isoverbascoside (9)


doi:10.1016/j.phytochem.2005.10.009

* Corresponding author. Tel.: +66 43 202378; fax: +66 43 202379.E-mail address: [email protected] (T. Kanchanapoom).



PHYTOCHEMISTRY


(Kanchanapoom et al., 2002a), 3,4,5-trimethoxyphenyl 1-O-b-D-xylopyranosyl-(1! 6)-b-D-glucopyranoside (10)(Kosuge et al., 1994), ajugol (12) (Nishimura et al.,1989), verminoside (13) (Sticher and Afifi-Yazar, 1979),specioside (14) (Compadre et al., 1982) by comparison ofphysical data with literature values and from spectroscopicevidence.

The molecular formula of compound 3 was determinedas C26H34O12 by negative ion HR-FAB mass spectrometricanalysis. The 1H and 13C NMR spectroscopic data showedthe presence of a b-glucopyranosyl unit from the anomericproton signal at dH 4.83 (d, J = 7.3 Hz) and from the car-bon signals at dC 102.9, 78.1, 77.8, 74,9, 71.3 and 62.5, inaddition to the signals of the aglycone moiety. The chemi-cal shifts of the aglycone moiety were similar to those of(+)-cycloolivil (1), suggesting that compound 3 is (+)-cycloolivil glucoside. This was supported by enzymatichydrolysis of 3 with crude hesperidinase, providing com-pound 1. The location of the sugar unit was assigned byan NOESY experiment, in which NOESY correlationwas found between dH 4.83 (d, J = 7.3 Hz, H-1 0 0) and dH

7.08 (d, J = 8.3 Hz, H-5 0), indicating that the sugar partwas attached at C-4 0. Moreover, HMBC spectrum pro-vided the further confirmation of the structure from thethree-bond correlation between H-1 0 0 (dH 4.83) and C-4 0

(dC 146.6) as illustrated in Fig. 1. Therefore, structure ofcompound 3 was elucidated as (+)-cycloolivil 4 0-O-b-D-glucopyranoside.

The molecular formula of compound 11 was deter-mined as C19H28O12 by negative ion HR-FAB mass spec-trometric analysis. The 1H NMR spectrum indicated thepresence of a tri-substituted aromatic ring (ABX system)from the signals at dH 6.83 (d, J = 8.8 Hz), 6.64 (d,J = 2.1 Hz) and 6.59 (dd, J = 8.8, 2.7 Hz), two singletmethoxyl signals at dH 3.72 and 3.68, and two anomericproton signals at dH 4.69 (d, J = 7.6 Hz) and 4.16 (d,J = 7.3 Hz). The 13C NMR spectrum displayed 19 carbonsignals, of which three were assignable to three oxy-aryl

carbons at dC 151.9, 149.3 and 144.0, three aryl-methinesat dC 112.8, 107.3 and 102.3, and two methoxyl groups atdC 56.1 and 55.5 for the aglycone moiety. The remainingcarbon signals belonged to the sugar moiety, and could beidentified as b-D-xylopyranosyl-(1! 6)-b-D-glucopyrano-syl unit by comparing chemical shifts with those of3,4,5-trimethoxyphenyl 1-O-b-D-xylopyranosyl-(1! 6)-b-D-glucopyranoside (10). All protonated carbons wereassigned by the result from HSQC spectrum. From thesespectral data, compound 11 is a glycoside of dimethoxyl-phenol. The locations of two methoxyl groups and thesugar moiety were assigned by NOESY experiment Thesignificant correlations observed between the signals wereat: (i) dH 4.69 (H-1 0) and dH 6.64 (H-2), (ii) dH 4.69 (H-1 0)and dH 6.59 (H-6), (iii) dH 6.64 (H-2) and dH 3.72 (MeO-3), and (iv) dH 6.83 (H-5) and dH 3.68 (MeO-4); indicatingthat two methoxyl groups were attached at C-3 and C-4,as well as the sugar moiety was substituted at C-1. Con-sequently, the structure of compound 11 was identifiedto be 3,4-dimethoxyphenyl 1-O-b-D-xylopyranosyl-(1! 6)-b-D-glucopyranoside.

The molecular formula of compound 15 was deter-mined as C24H30O13 negative ion HR-FAB mass spectro-metric analysis. The 13C NMR spectroscopic datashowed the presence of one b-glucopyranosyl unit, onecoumaroyl moiety in addition to nine carbons signalfor the aglycone moiety. DEPT experiments indicatedthat compound 15 contained one quarternary carbinoliccarbon (dC 80.7), seven methines (dC 140.8, 106.0, 93.0,85.8, 84.3, 48.3 and 36.5) and one methylene (dC 64.3)for the aglycone part, consistent with cyclopentanopyranring of an iridoid skeleton. The chemical shift at dC 93.0was characteristic of an acetal group of C-l. The methinesignals at dC 140.8 and 106.0 were assigned to a disubsti-tuted olefin group at C-3 and C-4, respectively. The cou-maroyl moiety was assigned as trans by the couplingconstant of the proton signals at dH 7.60 and 6.32 (eachd, J = 15.9 Hz). The remaining signals in the 1H NMRspectrum at dH 5.50 (d, J = 4.4 Hz), 6.22 (dd, J = 6.1,1.8 Hz), 5.19 (dd, J = 6.1, 3.4 Hz), 2.71 (m), 4.71 (dd,J = 5.6, 5.4 Hz), 4.09 (d, J = 5.6 Hz), and 2.45 (dd,J = 9.8, 4.4 Hz) were assignable to H-1, H-3, H-4, H-5,H-6, H-7 and H-9, respectively. Also, it showed the ABtype of methylene protons at dH 3.98 and 3.78 (each d,J = 12.0 Hz), ascribable to H-10. The 1H and 13CNMR spectroscopic data were closely related to thoseof decinnamoyl-globularimin (16) (Chaudhuri and Sti-cher, 1981), except for a set of additional signals arisingfrom the coumaroyl moiety. This ester moiety wasassigned to be located at C-6 since the chemical shiftsof C-6, C-5 and C-7 were significantly changed by+2.7, �0.8, and �2.1 ppm, respectively. Moreover, theHMBC spectrum (Fig. 2.) provided further confirmationfrom the three-bond correlation from H-6 (dH 4.71) toC-9 0 0 (dC 169.1). Therefore, the structure of compound 15

was determined as 6-O-trans-p-coumaroyl-decinnamoyl-globularimin, namely stereospermoside.Fig. 1. Significant HMBC correlations of compound 3.

T. Kanchanapoom et al. / Phytochemistry 67 (2006) 516–520 517

Among the compounds isolated, the present studyyielded two phenylethanoid glycosides (8–9) and four irid-oid glucosides (12–15). The phenylethanoid glycosides,decaffeoyl-verbascoside (8) and isoverbascoside (9), arewidely distributed in Bignoniaceous plants, such as inThailand (Kanchanapoom et al., 2001, 2002a,b). Iridoidsare also widespread in the family Bignoniaceae (von Poseret al., 2000), especially those lacking a carboxylic acidfunctionality at C-4. These appears to be the most com-mon for tribe Tecomeae such as ajugol (12), verminoside(13), specioside (14), and stereospermoside (15). Thus,these two classes of compounds may serve as usefulchemotaxonomical methods in tribe Tecomeae of the fam-ily Bignoniaceae.

3. Experimental

3.1. General

1H, 13C and 2D NMR spectra were recorded using aJEOL JNM a-400 spectrometer (400 MHz for 1H NMRand 100 MHz for 13C NMR). The NMR spectroscopicdata were measured in CD3OD and DMSO-d6 with tetra-methylsilane (TMS) as internal standard. The negative-ionmode FAB-MS spectra were recorded on a JEOL JMS-SX102 spectrometer. IR spectra were measured with a PerkinElmer Spectrum one FT-IR spectrometer. Optical rota-tions were determined on a Union PM-1 digital polarime-ter. For column chromatography, silica gel G (Merck no.7734), YMC-gel ODS (50 lm, YMC) and highly porouscopolymer resin of styrene and divinylbenzene (MitsubishiChem. Ind. Co. Ltd.) were used. HPLC (Jasco PU-980pump) were carried on columns of ODS (150 · 20 mmi.d., YMC) and Polyamine II (250 · 4.6 mm i.d.) with aToyo Soda refractive index (RI-8000) detector. The flowrate was 6 ml/min. The solvent systems were (I) EtOAc–MeOH (9:1), (II) EtOAc–MeOH–H2O (40:10:1), (III)EtOAc–MeOH–H2O (70:30:3), (IV) 10–50% aqueousMeOH, (V) 10% aqueous MeCN, (VI) 12% aqueousMeCN, (VII) 15% aqueous MeCN, (VIII) 20% aque-ous MeCN, (IX) 20% aqueous MeOH and (X) 80%aqueous MeCN, respectively.

3.2. Plant material

The leaves and branches of S. cylindricum Pierre ex. P.Dop were collected from Botanical Garden, Faculty ofPharmaceutical Sciences, Khon Kaen University in July,2004, and identified by Mr. Bamrung Tavinchiua of theDepartment of Pharmaceutical Botany and Pharmacog-nosy, Faculty of Pharmaceutical Sciences, Khon Kaen Uni-versity, Thailand. A voucher sample (PSKKU-0050) is keptin the Herbarium of the Faculty of Pharmaceutical Sci-ences, Khon Kaen University, Thailand.

Fig. 2. Significant HMBC correlations of compound 15.

518 T. Kanchanapoom et al. / Phytochemistry 67 (2006) 516–520


Dried leaves and branches (2.5 kg) of S. Cylindricum

were extracted with hot MeOH three times/(under reflux,5 l, for each extraction). After removal of the solvent byevaporation, with Et2O. The aqueous layer was appliedto a column of Diaion HP-20 and eluted successivelywith H2O, MeOH and Me2O. The fraction eluted withMeOH (49.3 g) was subjected to a silica gel cc using sol-vent systems I (2.0 l), II (5.0 l) and III (4.0 l). Five frac-tions were collected. Fraction 2 (5.1 g) was located ontoa column of RP-18 using solvent system IV to providecompounds 1 (1.23 g) and 4 (519.9 mg). Fraction 3(18.9 g) was applied to a RP-18 column using solventsystem IV, affording 12 fractions. Fraction 3–1 was puri-fied by preparative HPLC-ODS with solvent system V togive compounds 7 (195.6 mg), 8 (154.8 mg) and 12

(36.4 mg). Fraction 3–3 was purified by preparativeHPLC-ODS with solvent system VII to obtain com-pounds 5 (353.8 mg) and 6 (92.3 mg). Fraction 3–5 wasfurther purified by preparative HPLC-ODS with solventsystem VIII to afford compounds 9 (68.8 mg), 13(627.5 mg) and 15 (49.2 mg). Compound 14 (430.0 mg)was crystallized from fractions 3–7 and 3–8. Fraction 4(15.4 mg) was similarly separated on a column of RP-18 using solvent system IV to give eight fractions. Frac-tion 4–3 was purified by preparative HPLC-ODS withsolvent system VI to give compound 10 (135.4 mg) andfraction 4-3-2. This fraction was further purified by pre-parative HPLC-ODS with solvent system IX to givecompound 2 (54.2 mg) and fraction 4-3-2-2, which wasfinally purified by analytical HPLC-Polyamine II withsolvent system X to provide compounds 3 (26.3 mg)and 11 (13.7 mg).

3.4. (+)-Cycloolivil-4 0-O-b-D-glucopyranoside (3)

Amorphous powder, ½a�24D +17.6 (MeOH, c 1.59); IR

mKBRmax cm�1: 3434, 2920, 1617, 1514, 764; for 1H and 13C

NMR (CD3OD) spectra, see Table 1; negative HR-FAB-MS, m/z: 537.1967 [M � H]� (calcd for C26H33O12,537.1972).

3.5. Enzymatic hydrolysis of compound 3

Compound 3 (13.0 mg) was hydrolyzed with crude hes-peridinase (25 mg) in 2 ml of H2O. After stirring at 37 �Cfor 24 h, the reaction mixture was extracted with EtOAc,and then evaporated to dryness to provide (+)-cycloolivil(5.2 mg), ½a�24

D +85.4), whose structure was identified usingphysical and NMR spectral analyses.

3.6. 3,4-Dimethoxyphenyl 1-O-b-D-xylopyranosyl-(1! 6)-

b-D-glucopyranoside (11)

Amorphous powder, ½a�24D �100.7 (DMSO, c 0.45); IR

mKBrmax cm�1: 3533, 3380, 3275, 2921, 1602, 1522, 833; for

1H and 13C NMR (DMSO-d6) spectra, see Table 2; nega-tive HR-FAB-MS, m/z: 447.1477 [M � H]� (calcd forC19H27O12 447.1503).

Table 1NMR Spectroscopic data of compound 3

No. dC dH

1 39.9 3.17 (1H, d, J = 16.8 Hz)2.57 (1H, d, J = 16.8 Hz)

2 74.92a 69.3 3.75 (1H)a

3.55 (1H, d, J = 11.2 Hz)3 47.5 2.00 (1H, br d, J = 11.5 Hz)3a 60.8 3.75 (1H)a

3.49 (1H, dd, J = 11.2, 4.2 Hz)4 45.0 4.03 (1H, br d, J = 11.5 Hz)5 117.3 6.11 (1H, s)6 145.47 147.68 113.0 6.59 (1H, s)9 133.110 126.5MeO-7 56.7 3.75 (3H, s)MeO-30 56.4 3.74 (3H, s)10 142.020 114.9 6.75 (1H, d, J = 1.9 Hz)30 150.840 146.650 117.9 7.08 (1H, d, J = 8.3 Hz)60 123.6 6.72 (1H, dd, J = 8.3, 1.9 Hz)100 102.9 4.83(1H, d, J = 7.3 Hz)200 74.9 3.43 (1H, dd, J = 8.3, 7.3 Hz)300 77.8 3.42 (1H, dd, J = 9.0, 8.3 Hz)400 71.3 3.36 (1H, dd, J = 9.0, 8.3 Hz)500 78.1 3.26 (1H, m)600 62.5 3.84 (1H, br d, J = 12.0 Hz)

3.65 (1H, dd, J = 12.0, 5.6 Hz)

a Chemical shifts obtained approximately by HSQC.

Table 2NMR Spectroscopic data of compound 11 (DMSO-d6)

No. dC dH

1 151.92 102.3 6.64 (1H, d, J = 2.7 Hz)3 149.34 144.05 112.8 6.83 (1H, d, J = 8.8 Hz)6 107.3 6.59 (1H, d, J = 8.8, 2.7 Hz)MeO-3 55.5 3.72 (3H, s)MeO-4 56.1 3.68 (3H, s)10 101.2 4.69 (1H, d, J = 7.6 Hz)20 73.2 3.21 (1H)a

30 76.5 3.22 (1H)a

40 69.8 3.11 (1H, dd, J = 9.0, 8.1 Hz)50 75.9 3.45 (1H, m)60 68.5 3.93 (1H, br d, J = 11.0 Hz)

3.56 (1H, dd, J = 11.0, 6.6 Hz)100 103.9 4.16 (1H, d, J = 7.3 Hz)200 73.4 2.94 (1H, dd, J = 8.8, 7.3 Hz)300 76.6 3.05 (1H, d, J = 8.8, 8.5 Hz)400 69.6 3.25 (1H, m)500 65.7 3.65 (1H, dd, J = 11.2, 5.1 Hz)

2.95 (1H, dd, J = 11.2, 3.9 Hz)

a Chemical shifts obtained approximately by HSQC.

T. Kanchanapoom et al. / Phytochemistry 67 (2006) 516–520 519

3.7. Stereospermoside (15)

Amorphous powder, ½a�24D �90.8 (MeOH, c 1.74); IR

mKBrmax cm�1: 3427, 2927, 1692, 1605, 832; for 1H and 13C

NMR (CD3OD) spectra, see Table 3; negative HR-FAB-MS, m/z: 525.1599 [M � H]� (calcd for C24H29O13,525.1608).

Acknowledgements

The authors are grateful to Khon Kaen University, andNRCT-JSPS Core University Program for financial sup-port of this work. We also thank Mr. Savian Jantavisetof the Faculty of Pharmaceutical Sciences, Khon KaenUniversity, for help in obtaining the plant material.

References

Abe, F., Yamauchi, T., Wan, A.S.C., 1988. Lignans related to olivil fromgenus Cerbera (Cercera.) VI). Chemical and Pharmaceutical Bulletin36, 795–799.

Chaudhuri, R.K., Sticher, O., 1981. New iridoid glucosides and a lignandiglucoside from Globularia alypum L. Helvetica Chimica Acta 64, 3–15.

Compadre, C.M., Jauregui, J.F., Nathan, P.J., Enrıquez, R.G., 1982.Isolation of 6-O-(p-coumaroyl)-catapol from Tabebuia rosea. PlantaMedica 46, 42–44.

Ghogomu, R.T., Bodo, B., Nyasse, B., Sondengam, B.L., 1985. Isolationand identification of (�)-olivil and (+)-cycloolivil from Stereospermum

kunthianum. Planta Medica 45, 464.Ida, Y., Satoh, Y., Ohtsuka, M., Nagasao, M., Shoji, J., 1993. Phenolic

constituents of Phellodendron amurense bark. Phytochemistry 35, 209–215.

Kanchanapoom, T., Kasai, R., Yamasaki, K., 2001. Lignan and phenyl-propanoid glycosides from Fernandoa adenophylla. Phytochemistry 57,1245–1248.

Kanchanapoom, T., Kasai, R., Yamasaki, K., 2002a. Phenolic glycosidesfrom Markhamia stipulata. Phytochemistry 59, 557–563.

Kanchanapoom, T., Kasai, R., Yamasaki, K., 2002b. Phenolic glycosidesfrom Barnettia kerrii. Phytochemistry 59, 565–570.

Karasawa, H., Kobayashi, H., Takizawa, N., Miyase, T., Fukushima, S.,1986. Studies on the constituents of Cistanchis herba. VII. Isolationand structures of cistanosides H and I. Yakugaku Zasshi 106, 562–566.

Kosuge, K., Mitsunaga, K., Koiki, K., Ohmoto, T., 1994. Studies on theconstituents of Ailanthus integrifolia. Chemical and PharmaceuticalBulletin 42, 1669–1671.

Kumar, U.S., Aparna, P., Rao, R.J., Rao, T.P., Rao, J.M., 2003. 1-Methyl anthraquinones and their biogenetic precursors from Stereo-

spermum personatum. Phytochemistry 63, 925–929.Nishimura, H., Sasaki, H., Morota, T., Chin, M., Mitsuhashi, H.,

1989. Six iridoids from Rehmannia glutinosa. Phytochemistry 28,2705–2709.

Onegi, B., Kraft, C., Kohler, L., Freund, M., Jenett-Siems, K., Siems, K.,Beyer, G., Melzig, M.F., Bienzle, U., Eich, E., 2002. Antiplasmodialactivity of naphthoquinones and one anthraquinone from Stereosper-

mum kunthianum. Phytochemistry 60, 39–44.Sticher, O., Afifi-Yazar, F.U., 1979. Minecoside and verminoside, two new

iridoid glucosides from Veronica officinale L. (Scrophulariaceae).Helvetica Chimica Acta 62, 535–539.

Sugiyama, M., Nagayama, E., Kikuchi, M., 1993. Lignan and phenyl-propanoid glycosides from Osmanthus asiaticus. Phytochemistry 33,1215–1219.

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Table 3NMR Spectroscopic data of compound 15

No. dC dH

1 93.0 5.50 (1H, d, J = 4.4 Hz)3 140.8 6.22 (1H, dd, J = 6.1, 1.8 Hz)4 106.0 5.19 (1H, dd, J = 6.1, 3.4 Hz)5 36.5 2.71 (1H, m)6 85.8 4.71 (1H, dd, J = 5.6, 5.4 Hz)7 84.3 4.09 (1H, d, J = 5.6 Hz)8 80.79 48.3 2.45 (1H, dd, J = 9.8, 4.4 Hz)10 64.3 3.98 (1H, d, J = 12.0 Hz)

3.78 (1H, d, J = 12.0 Hz)10 99.5 4.62 (1H, d, J = 7.8 Hz)20 74.7 3.16 (1H, dd, J = 8.8, 7.8 Hz)30 77.9 3.22 (1H, dd, J = 9.0, 8.8 Hz)40 71.6 3.26 (1H, dd, J = 9.0, 8.0 Hz)50 78.1 3.27 (1H, m)60 62.8 3.83(1H, br d, J = 11.7 Hz)

3.62 (1H, dd, J = 11.7, 3.9 Hz)100 127.1200, 600 131.2 7.41 (2H, d, J = 8.8 Hz)300, 500 116.8 6.76 (2H, d, J = 8.8 Hz)400 161.3700 146.9 7.60 (1H, d, J = 15.9 Hz)800 115.0 6.32 (1H, d, J = 15.9 Hz)900 169.1

520 T. Kanchanapoom et al. / Phytochemistry 67 (2006) 516–520

Corrigendum

Corrigendum to ‘‘Flavones and flavone synthases’’[Phytochemistry 66 (2005) 2399–2407]

Stefan Martens a, Axel Mithofer b,*

a Institut fur Pharmazeutische Biologie, PhilippsUniversitat Marburg, Deutschhausstr. 17A, D-35037 Marburg/Lahn, Germanyb Bioorganische Chemie, Max-Planck-Institut fur Chemische Okologie, Hans-Knoll Str. 8, D-07745 Jena, Germany

Available online 30 January 2006

The authors regret that Fig. 2 (p. 2400) was published incorrectly. The correct figure is given below.


doi:10.1016/j.phytochem.2006.01.004

DOI of original article: 10.1016/j.phytochem.2005.07.013.* Corresponding author. Tel.: +49 0 3641 571263; fax: 49 0 3641 571256.

E-mail address: [email protected] (A. Mithofer).

5-Deoxy-flavonoids

Chalcones

3x Malonyl-CoA

CHS

CHSCHKR

O

FlavanonesDihydro-flavonols

FHT

O

O

Leuco-anthocyanins

Flavones Flavonols

p-Coumaroyl-CoA

CHI

FNS I orFNS II

O

O

DFR ANS FGT

FLS

Isoflavones

trans Flavan-3-ols

IFS +IFD

Antho-cyanidins

Antho-cyanins

LAR

cis Flavan-3-ols

ANR

Flavan-4-ols

6‘-Deoxy-chalcones

5-Deoxy-flavanones

CHI

IFS +IFD

3-Deoxy-flavonoids

O

OH

O

O

OH

OH

O

OH

+O

O-Glc

+

O

O O

OH

O

OHO

O

OH

Fig. 2.


Phytochemistry 67 (2006) 521

PHYTOCHEMISTRY

http://dx.doi.org/10.1016/j.phytochem.2005.07.013


Announcement

The Phytochemical Society ofEurope–Pierre-Fabre 2006Award for Phytochemistry

The Phytochemical Society of Europe and Pierre-Fabre

Laboratories are pleased to publish this call for candidatesfor the 2006 Award, to be presented to a young Europeanscientist who has made an outstanding contribution tophytochemistry or plant biochemistry. Candidatures areinvited from all disciplines within this general field. TheAward aims to reward an exceptional contribution to theresearch field in which the candidate is working.

The Award consists of a cheque for 2000 euros, togetherwith a parchment certificate. Recipients will be invited topresent their research in the form of an award lecture to bedelivered during one of the Society’s meetings in 2007.Expenses for attending the meeting are paid by theAwarding Committee.

Previous PSE–PF Award winners are:

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All members of the PSE are strongly encouraged tomake a nomination. This should consist of: a CV, a short

summary of the significant points of the candidate’s re-search career (1–2 pages); a list of publications; the ad-dresses of 2 or 3 independent referees; a letter of support.In particular, Professors and Directors of Institutes arestrongly encouraged to nominate colleagues or promisingformer PhD students or post-doctoral researchers.

Nominations for the 2006 Award should be sent before15 April 2006—preferably in electronic format—to the PSEVice-Chairman:

Dr Richard Robins,LAIEM, CNRS UMR6006,Faculte des Sciences et des Techniques,Universite de Nantes,B.P. 92208,F-44322 Nantes 03, France.E-mail: [email protected]

Further information about the PSE and its Awards canbe found on the website http://www.phytochemicalsociety.org

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Phytochemistry 67 (2006) 522

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doi: 10.1016/j.phytochem.2006.01.029

Phytochemistry Vol. 67, No. 5, 2006

Author Index

Abdelgaleil, S.A.M., 452Abdel-Kader, M.S., 429

Ahmad, M.S., 429

Ahmed, A.A., 424

Al-Rehaily, A.J., 429

Asakawa, Y., 424

Atta-ur-Rahman, 439

Chifundera, K., 504Choudhary, M.I., 439

Coombes, P.H., 459

Cutillo, F., 481

D�Abrosca, B., 481

Deachathai, S., 464

DellaGreca, M., 481

Devkota, K.P., 439Di, Y., 486

Ding, Y.-H., 497

Doe, M., 452

Don, M.-J., 497

Duplex, W.J., 475

Emerenciano, V.P., 492

Federici, E., 504

Ferreira, M.J.P., 492

Fiorentino, A., 481

Florent, L., 444

Fomum, Z.T., 459, 475

Fotso, S., 475

Frappier, F., 444

Frederich, M., 433

Galeffi, C., 504

Gallo, F.R., 504

Grellier, P., 444

Hao, X., 486Harakat, D., 433

Harmut, L., 475

Iurilli, R., 504

Jacquier, M.-J., 433

Joyeau, R., 444

Kamdem Waffo, A.F., 459, 475

Kanchanapoom, T., 516

Kaplan, M.A.C., 492

Karchesy, J., 424

Kenmogne, M., 433

Laatsch, H., 475

Li, C., 511Li, S., 486

Mahabusarakam, W., 464, 470

Mambu, L., 444

Martens, S., 521

Maskey, R.P., 475

Mithofer, A., 521

Mohamed, A.E.H., 424Monache, F.D., 504

Moreira, D.L., 492

Morimoto, Y., 452

Mossa, J.S., 429

Mulholland, D.A., 459

Musharraf, S.G., 439

Nakatani, M., 452Ndom, J.C., 475

Nguefa, H.E., 475

Njamen, D., 475

Nkengfack, A.E., 459

Noiarsa, P., 516

Nuangnaowarat, W., 470

Otsuka, H., 516

Palazzino, G., 504

Phongpaichit, S., 464

Prost, E., 433

Ramanitrahasimbola, D., 444

Ranjit, R., 439

Rasoanaivo, P., 444Ruchirawat, S., 516

Santos, M.I.S., 492

Shang, X.-Y., 511

Shen, C.-C., 497

Shi, J.-G., 511

Shrestha, T.M., 439

Sondengam, L.B., 433Sun, C.-M., 497

Syu, W.-J., 497

Taylor, W.C., 464, 470

Velozo, L.S.M., 492

Waffo-Teguo, P., 433Wandjii, J., 475

Wang, J., 486

Wang, Y., 486

Wang, Y.-H., 511

Wansi, J.D., 475

Yang, C.-R., 464

Yang, X., 486Yang, Y.-C., 511

Zarrelli, A., 481

Zeches, M., 433

Zhang, C.-Z., 511

Zhang, Y.-J., 464

doi:10.1016/S0031-9422(06)00050-1

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