Bioactive indole alkaloids isolated from Alstonia angustifolia

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Phytochemistry Letters xxx (2014) xxx–xxx

G Model

PHYTOL 731 1–6

Bioactive indole alkaloids isolated from Alstonia angustifolia

Li Pan a, Cesar Terrazas b, Ulyana Munoz Acuna c, Tran Ngoc Ninh d, Heebyung Chai a,Esperanza J. Carcache de Blanco a,c, Djaja D. Soejarto e,f, Abhay R. Satoskar b,A. Douglas Kinghorn a,*a Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, OH 43210, United Statesb Department of Pathology, College of Medicine, The Ohio State University, Columbus, OH 43210, United Statesc Division of Pharmacy Practice and Administration, College of Pharmacy, The Ohio State University, Columbus, OH 43210, United Statesd Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hoang Quoc Viet, Cau Giay, Hanoi, Vietname Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, United Statesf Division of Collections, Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, IL 60605, United States

A R T I C L E I N F O

Article history:

Received 2 May 2014

Received in revised form 11 June 2014

Accepted 23 June 2014

Available online xxx

This paper forms part of a special issue of

Phytochemistry Letters dedicated to the

memory of Andrew Marston (1953–2013),

outstanding phytochemist who is much

missed by his friends.

Keywords:

Alstonia angustifolia (Apocynaceae)

Stem bark

Sarpagine-type alkaloid

Macroline-type alkaloid

Macroline-pleiocarpamine bisindole

alkaloid

Cytotoxicity

NF-kB (p65) inhibitory activity

Antileishmanial activity

A B S T R A C T

Bioassay-guided fractionation was conducted on a CHCl3-soluble extract of the stem bark of

Alstonia angustifolia ( Apocynaceae) collected in Vietnam using the HT-29 human colon cancer cell

line, and led to the isolation of a new sarpagine-type indole alkaloid ( 1), together with nine known

alkaloids, including four macroline-derived alkaloids ( 2–5), a sarpagine-type alkaloid ( 6), and four

macroline-pleiocarpamine bisindole alkaloids ( 7–10). The structure of the new compound ( 1) was

determined on the basis of spectroscopic data interpretation. Compounds 1–10 were evaluated in

vitro for their NF-kB (p65) inhibitory activity against the Hela cells in an ELISA assay. The new

sarpagine alkaloid, N(4)-methyltalpinine ( 1), was found to show significant NF-kB inhibitory activity

(ED50 = 1.2 mM). Furthermore, all the isolates ( 1–10) were evaluated in vitro for their

antileishmanial activity, and compounds ( 1–4, 6 and 8–10) exhibited leishmaniacidal activity

against promastigotes of Leishmania mexicana.

� 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.

Contents lists available at ScienceDirect

Phytochemistry Letters

jo u rn al h om ep ag e: ww w.els evier .c o m/lo c ate /p hyt ol

22232425262728293031

1. Introduction

Species of Alstonia (Apocynaceae), a genus of evergreen trees andshrubs, are mainly distributed in the tropical and subtropical areasof Africa, Central America, southeast Asia, Polynesia and Australia,with most of these in the Malesian region (Perry and Metzger,1980). Many Alstonia species are commercial timbers, and somespecies are also used as traditional medicines for the treatment ofcough, ulcer, dysentery, fever, malaria, sore throats, toothache,rheumatism, and snake bites (Perry and Metzger, 1980; Sidiyasa,

3233343536

* Corresponding author.

E-mail addresses: [email protected],

[email protected] (A.D. Kinghorn).

Please cite this article in press as: Pan, L., et al., Bioactive indole alkahttp://dx.doi.org/10.1016/j.phytol.2014.06.010

http://dx.doi.org/10.1016/j.phytol.2014.06.010

1874-3900/� 2014 Published by Elsevier B.V. on behalf of Phytochemical Society of Eu

1998; Gadekar et al., 2010). Alstonia angustifolia Wall. ex A.DC., asmall to medium-sized tree, with the common name ‘‘Red-leafedPulai’’, is native to Indonesia, Malaysia, the Philippines,and Vietnam. In Malaysia, the leaves of A. angustifolia are appliedexternally to the spleen area for the treatment of remittent fever,and the bark is used as an antimalarial remedy (Flora andSingapura, 2014; Slik JWF (2009 onwards), Plants of SoutheastAsia, 2014). According to previous phytochemical investigationson Alstonia species, the effects described in traditional medicinalusage are probably due to the presence of indole alkaloids, whichhave been reported to be the major secondary metabolites ofthese plants (Hamaker and Cook, 1995; Osorio et al., 2008). Thusfar, more than 60 indole alkaloids, with the majority beingmacroline-type derivatives, have been isolated and identified fromA. angustifolia species (Ghedira et al., 1988; Hu et al., 1989; Kam

loids isolated from Alstonia angustifolia. Phytochem. Lett. (2014),

rope.


mailto:[email protected]

mailto:[email protected]


http://www.sciencedirect.com/science/journal/18743900

www.elsevier.com/locate/phytol


37 an38 co39 tie40 in41

42 bi43 pa44 co45 HT46 m47 us48 id49 w50 de51 pi52 al53 m54 af55 pl56 1957 st58 et59

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R = CH2Cl

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N(4') po siti vel y charge d

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N N

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HOHH

H H

H H

Fig. 1. Structures of compounds 1–10.

L. Pan et al. / Phytochemistry Letters xxx (2014) xxx–xxx2

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PHYTOL 731 1–6

d Choo, 2002; Tan et al., 2011, 2013), and some of thesempounds have been reported to possess antiprotozoal proper-s (Wright et al., 1992) and activities against multidrug-resistant

KB cells (Tan et al., 2011).As part of our continuing efforts to discover naturally occurring

ologically active agents from plants, a chloroform-solublertition of a methanol extract from the stem bark of A. angustifolia

llected in Vietnam was found to show cytotoxicity against the-29 human colon cancer cell line, with an ED50 value of 12.0 mg/

L, and thus was fractionated by bioactivity-guided isolationing this assay. The present investigation led to the isolation andentification of a new sarpagine-type indole alkaloid (1), togetherith nine known alkaloids (Fig. 1), including four macrolinerived alkaloids, 21-deoxy-N(4)-methyl-21-oxo-N(4),21-secotal-nine [N(4)-methyl-N(4),21-secotalpinine, (2) (Kam et al., 2004)],stonerinal (3) (Kam et al., 1999), alstonerine (4) (Bi et al., 1994),acrocarpine B (5) (Kam et al., 2004), a sarpagine-type alkaloidfinisine (6) (Zocoler et al., 2005), as well as four macroline-eiocarpamine bisindole alkaloids, villalstonine (7) (Ghedira et al.,88), villalstonine N(4)-oxide (8) (Ghedira et al., 1988), villal-

onidine D (9) (Tan et al., 2013) and villalstonidine E (10) (Tan al., 2013).

Although the crude extracts of two other Alstonia species, macrophylla and A. congensis, have been reported to show

Please cite this article in press as: Pan, L., et al., Bioactive indole alkhttp://dx.doi.org/10.1016/j.phytol.2014.06.010

pronounced antileishmanial activities (Camacho et al., 2003;Lumpu et al., 2013), and certain alkaloids isolated fromA. angustifolia have been tested for their antiamoebic andantiplasmodial activities (Wright et al., 1992), to this date theleishmanicidal activities of alkaloids from Alstonia specieshave not been evaluated. Thus, besides the human cancer cellline and NF-kB inhibitory bioassays employed to evaluate thecytotoxic and NF-kB inhibitory activities of the principlesfrom this plant, all the isolates obtained in the present studywere also screened for their potential leishmanicidal activitiesagainst promastigotes of Leishmania mexicana.

2. Results and discussion

Compound 1 was obtained as a light brownish resin, and gave apositive test for Dragendorff’s reagent. In the UV spectrum,absorptions at 219, 269, 282 and 290 nm are corresponding toan indole Q2chromophore (Houlihan, 1972). The molecular formulaof compound 1 was determined as C21H27N2O2 based on the [M]+

ion peak at m/z 339.2059 (calcd 339.2073) in the HRESIMS. In the1H NMR spectrum (Table 1), proton signals at dH 7.53 (br d, J = 7.9,H-9), 7.12 (br t, J = 7.8, H-10), 7.25 (br t, J = 7.7, H-11) and 7.43 (br d,J = 8.2, H-12), were recognized belonging to the aromatic ring ofthe indole moiety. Also observed were two nitrogen-attached

aloids isolated from Alstonia angustifolia. Phytochem. Lett. (2014),


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Fig. 2. Key HMBC (!) correlations observed for compound 1.

Fig. 3. Preferred conformation and selected NOESY ($) correlations observed for

compound 1.

Table 11H and 13C NMR spectroscopic data of compound 1a

Position 2

dH, mult (J in Hz)b dCc

2 134.2

3 4.99, d (10.7) 53.1

5 3.91, t (5.4) 61.3

6b 3.09, d (16.6) 24.7

6a 3.36, dd (17.4, 5.3)

7 101.7

8 127.3

9 7.53, d (7.9) 119.5

10 7.12, t (7.8) 121.0

11 7.25, t (7.7) 123.7

12 7.43, d (8.2) 110.6

13 139.7

14b 1.98, ddd(13.2, 5.0, 1.8) 32.4

14a 2.47, brt (12.2)

15 2.36, brs 22.7

16 1.77, brs 38.2

17b 3.80, d (11.7) 63.1

17a 3.54, dd (11.9, 2.1)

18 1.38, d (6.8) 15.7

19 4.15, q (6.8) 72.7

20 2.04, brs 48.2

21 4.95, d (1.9) 98.4

N1-CH3 3.73, s 29.9

N4-CH3 3.07, s 43.5

a Obtained in CD3OD. Assignments are based on 1H–1H COSY, HSQC, and HMBC

spectroscopic data.b Measured at 400 MHz with residual signals of CD3OD at d 3.31 ppm used as

reference.c Measured at 100 MHz with residual signals of CD3OD at d 49.0 ppm used as

reference.

L. Pan et al. / Phytochemistry Letters xxx (2014) xxx–xxx 3

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PHYTOL 731 1–6

methyl groups at dH 3.73 and 3.07 (each 3H, s, N1–CH3 and N4–CH3), and an alkyl methyl group at dH 1.38 (3H, d, J = 6.8 Hz, H-18).In addition to the protons mentioned above, another 13 protonsignals remained to be assigned in the 1H NMR spectrum, and wereascribed to three methylene groups [dH 3.36 (dd, J = 17.4 and5.3 Hz) and 3.09 (d, J = 16.6 Hz), each 1 H, H-6a and H-6b 2.47 (brt,J = 12.2 Hz) and 1.98 (ddd, J = 13.2, 5.0 and 1.8 Hz), each 1 H, H-14aand H-14b 3.54 (dd, J = 11.9, 2.1 Hz) and 3.80 (d, J = 11.7 Hz), each1 H, H-17a and H-17b], and seven methine groups [dH 4.99 (d,J = 10.7 Hz, H-3), 4.15 (q, J = 6.8 Hz, H-19), 3.91 (t, J = 5.4 Hz, H-5),2.36 (brs, H-15), 1.77 (brs, H-16), 2.04 (brs, H-20), 4.95 (d,J = 1.9 Hz, H-21), each 1H]. The 13C NMR spectrum (Table 1)displayed a total of 21 carbon resonances, which were classifiedfrom the DEPT and HSQC spectra as three methyl carbon signals(including two nitrogen-attached methyls and one alkyl methyl),three methylene carbon signals (including one oxygenatedmethylene and two alkyl methylenes), seven methine carbonsignals (including two N-vicinal methines, a oxygenated methine,a hemiaminal methine, and three alkyl methines), as well as eightsp2 carbon signals (including four tertiary carbons and fourquaternary carbons) of the indole moiety. By studying the COSYand TOCSY spectra, a large spin system that conjugated withthe indole unit comprising three methylene (C-6, C-14 and C-17),seven methine (C-3, C-5, C-15, C-16, C-19, C-20, C-21) and onemethyl (C-18) groups was established. In the HMBC spectrum,the proton signal of the methyl group attached on N4 (dH 3.07, s,3H), exhibiting a strong correlation with a hemiaminal methinecarbon (dC 98.4, C-21), suggested that C-21 is connected with N-(4)in compound 1 to form a ring containing a carbinolaminefunctionality. Based on above NMR spectroscopic analysis,compound 1 was found to resemble closely to a knownsarpagine-type derived indole alkaloid, talpinine (Naranjo et al.,1972; Yu and Cook, 1998). Talpinine was oringinally isolatedfrom the stem bark of Pleiocarpa talbotii (Apocynaceae), with its


structure confirmed by enantiospecific synthesis. Although the 1Hand 13C NMR data of talpinine have been reported (Yu and Cook,1998), the full assigments are not available so far. Furthermore, theplanar structure of 1 was confirmed by HMBC correlations of H-5with C-3, C-7 and C-17, H-6 with C-2 and C-16, H-14 with C-2, C-16and C-20, H-15 with C-21, H-16 with C-6 and C-20, H-17 with C-5,C-15 and C-19, H-18 with C-20, H-19 with C-21, H-20 with C-15and C-21, as well as H-21 with C-3 and C-19. The relative stereoconfigurations at the stereogenic centers of 1 were establishedbased on the important NOE correlations, including H-16 with H-15 and H-6b, H-15 with H-18, H-20 and H-17b, H-5 with H-6a,H-17a, and N(4)-CH3, H-3 with N(1)-CH3 and N(4)-CH3, as well asH-21 with H-5, H-19 and N(4)-CH3, consistent with those oftalpinine (Fig. 2). In addition, an energy-minimized model wasgenerated by Chem3D Ultro 10.0 based on a presumed configura-tion, which matched well with all the observed key NOESYcorrelations (Fig. 3). Thus, the structure of compound 1 wasdeduced as N(4)-methyltalpinine, which is a new sarpagine-typealkaloid.

Compounds 1–10 obtained in the present study were evaluatedagainst the HT-29 human colon cancer cell line for theircytotoxicity, as summarized in Table 2. Among these compounds,a monomeric macroline indole alkaloid, alstonerial (3), togetherwith two macroline–pleiocarpamine type bisindole alkaloids,villalstonine (7) and villalstonidine E (10), were found to exhibitcytotoxicity against the HT-29 cells, with ED50 values of 8.6, 8.0and 6.5 mM, respectively.

In the present investigation, all the isolates were also evaluatedfor their NF-kB (p65) inhibitory activity against Hela cells in anenzyme-based Elisa assay. Only the new compound, N(4)-methyltalpinine (1), was found to show significant NF-kB (p65)



148 in149 th150 fo151 ob152 pi153 al154

155 A.

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169 qu170 ag171 in172 ev

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Table 2Bioactivity evaluation of compounds isolated from A. angustiforlia.

Compound Bioassay

HT-29a NF-kB (p65)a L. mexicana

promastigotesb

1 >20 1.2 183.3

2 >20 >20 57.8

3 8.6 >20 104.7

4 >20 >20 145.4

5 NT >20 >200

6 >20 >20 138.0

7 8.0 >20 >200

8 >20 >20 80.3

9 >20 >20 120.4

10 6.5 >20 78.0

Paclitaxelc 0.006 – –

Rocaglamided 0.005 0.08 –

Amphotericin Be 0.09

a Results are expressed as ED50 values (mM); compounds with ED50> 20 mM

were considered inactive.b Results are expressed as IC50 values (mM); compounds with IC50> 200 mM are

considered to be inactive.c Used as a positive control substance for HT-29 cell line.d Used as a positive control substance for NF-kB (p65) cell line.e Used as a positive control substance for L. mexicana promastigote assay.


G Model

PHYTOL 731 1–6

hibitory activity, with an ED50 value of 1.2 mM, which impliedat the four ring system containing a carbinolamine featurermed by the junction of N(4) and C-21 in 1 is important for theserved activity. To the best of our knowledge, N(4)-methyltal-

nine (1) is the first member of the sarpagine-type indolekaloids reported to have NF-kB inhibitory activity.

As mentioned before, several alkaloids isolated from angustifolia have been evaluated for their antiprotozoal activitiesainst Entamoeba histolytica and Plasmodium falciparum in vitroright et al., 1992). In this study, the bisindole alkaloids

aluated, were found to possess significant activity againstth protozoal species, but were less potent than the positiventrols, and the monomeric alkaloids were all considerablyss active than the dimers tested. Most of the compounds (1–4, 6,10) obtained in present study were found to possess leishma-

cidal activities against promastigotes of L. mexicana (Table 2).sed on the present results, the antileishmanial activitieshibited by the dimeric- and monomeric indole alkaloidsd not show obvious differences. Of the four bisindole alkaloids–10), villalstonine (7) was inactive against promastigotes of

mexicana, while its derivatives (8–10), all possessing aaternary ammonium cation at N(40), were found to be activeainst the same parasites. This is the first time that puredole alkaloids isolated from Alstonia species have beenaluated for their antileishmanial activities.

Experimental

1. General experimental procedures

Optical rotations were measured on a Perkin-Elmer 343tomatic polarimeter (Perkin-Elmer, Waltham, MA). UV spectra

ere obtained on a Hitachi U-2910 spectrophotometer (Hitachi,kyo, Japan). NMR spectroscopic data were recorded at roommperature on a Bruker Avance DRX-400 or a Bruker Avance-300ectrometer (Bruker, Billerica, MA), using standard Bruker pulsequences, and the data were processed using MestReNova 6.0ftware (Mestrelab Research SL, Santiago de Compostela, Spain).gh-resolution electrospray ionization mass spectra (HRESIMS)ere performed on a Micromass Q-TofTM II (Micromass, Wythen-awe, UK) mass spectrometer operated in the positive-ion mode,


with sodium iodide being used for mass calibration. Columnchromatography was carried out with silica gel (230–400 mesh;Sorbent Technologies, Atlanta, GA). Analytical TLC was conductedon precoated 250 mm thickness silica gel UV254 aluminum-backedplates (Sorbent Technologies). Waters Xbridge1 (4.6 � 150 mm),semi-preparative (10 � 150 mm), and preparative (19 � 150 mm)C18 (5 mm) columns were used for analytical, semi-preparative andpreparative HPLC, respectively, as conducted on a Waters systemcomprised of a 600 controller, a 717 Plus autosampler, and a 2487dual wavelength absorbance detector. 205 nm and 254 nm wereselected as UV detection wavelength.

3.2. Plant material

A stem bark sample of Alstonia angustifolia Wall. ex A. DC.(Apocynaceae) was collected in Nui Chua National Park, Ninh ThuanProvince, Vietnam by DDS, TNN, and Vuong Tan Tu, on January 11,2010, who also identified this plant. A voucher specimen (originalcollection Soejarto et al.14599; recollection on July 26, 2011Soejarto et al., 1861) has been deposited in the John G. SearleHerbarium of the Field Museum of Natural History (underaccession number FM 2294419 and 2300815, respectively),Chicago, Illinois.

3.3. Extraction and isolation

The dried and milled stem bark (340 g) of A. angustifolia wasmacerated overnight using MeOH at room temperature (3� 3 L).After evaporation under reduced pressure, the dried MeOHextract (50 g) was suspended in a mixture of methanol –water(9:1) solution and was then partitioned sequentially withhexane (3� 1 L) and chloroform (3� 1 L) to yield a CHCl3-soluble partition (20 g), which was found to be active against theHT-29 cell line (ED50 = 12.0 mg/mL). Accordingly, this fractionwas subjected to separation over a Si gel column (7 � 50 cm),eluted with a gradient mixture of CH2Cl2 –acetone of increasingpolarity (20:1 to 1:1, then pure acetone), to yield eight fractions(F 01–F08). Of the sub-fractions obtained, F01 and F03 werefound to be the most active against the HT-29 cells (ED50 = 6.3and 1.3 mg/mL, respectively). Fraction F01 (55 mg) was chro-matographed on a silica gel column and eluted in a gradientmanner with hexane-acetone (10:1 to pure acetone), to givethree subfractions (F 101–F103). Subfraction F102 which con-taining the major compounds was purified by HPLC (MeOH –H2O = 60:40, 0.05% triethylamine) to afford compounds 3(2.0 mg) and 4 (4.0 mg). Part of fraction F02 (1.0 g) wassubjected to passage over a Sephadex LH-20 column withelution by MeOH to give four subfractions (F 201–F204). F0201(eluted was further purified by preparative TLC (20 � 20 cm,500 mm), developed by CHCl3–MeOH (15:1, saturated withNH3 �H2O), to yield compounds 7 (Rf = 0.8; 11.0 mg), and 8(Rf = 0.5; 2.0 mg), and the same method was applied to F202 toyield 10 (Rf = 0.4; 12.0 mg). F203 and F204 were combined asF20(34) and subjected to an open C18 column to give fivesubfractions. Fraction F20(34)3 was purified by HPLC withMeOH–H2O (55:45, 0.05% triethylamine) to yield compounds 6(4.0 mg), 5 (2.0 mg) and 2 (2.0 mg). Compound 1 (Rf = 0.65;4.0 mg) and 9 (Rf = 0.6; 3.0 mg) were purified from subfractionsF20(34)4 and F20(34)8, respectively, by preparative TLC(20 � 20 cm, 500 mm), using CHCl3–MeOH (10:1, saturatedwith NH3 �H2O) as developing solvent system.

3.4. New compound information

N(4)-methyltalpinine (1): light brownish resin. [a]D23–10

(c = 0.1, MeOH); UV (MeOH) lmax (log e) 219 (4.60), 269 (3.95),



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L. Pan et al. / Phytochemistry Letters xxx (2014) xxx–xxx 5

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PHYTOL 731 1–6

282 (3.90) and 290 (3.86) nm; 1H and 13C NMR data shown inTable 1; HRESIMS obsd m/z 339.2059 [M]+ (calcd for C21H27O2N2,339.2073).

3.5. Biological assay

3.5.1. Cytotoxicity assay

Human colon cancer cells (HT-29) purchased from AmericanType Collection were cultured in MEME medium supplementedwith streptomycin (100 mg/mL), penicillin (100 units/mL),amphotericin B (Fungizone, 0.25 mg/mL) and 10% fetal bovineserum (FBS), and incubated with an atmosphere with 5% CO 2 at37 8C. Cells were trypsinized and split for subculture five days orlater when they reached near-confluent state. Upon reachingabout 60–70% confluence, the medium was changed and thecells were used for test procedures one day later. Afterappropriate dilutions, the harvest cells were seeded in 96-well(9500 cells/190 mL) plates using complete medium, and treatedwith the test compounds (10 mL/well in triplicate) at variousconcentrations. Test samples were initially dissolved in DMSOand then diluted 10-fold with H 2O2. Serial dilutions were madeusing 10% DMSO as the solvent. For the control groups, 10 mL of10% DMSO were also added to each well. After two day’sincubation, the cells were fixed to the plates by 100 mL of cold20% trichloroacetic acid (TCA) and incubated at 4 8C for 30 min.The plates were washed three times with tap water and driedovernight. The fixed cells were dyed with sulforhodamine Bsolution at 0.4% (w/v) in 1% acetic acid, and incubated at roomtemperature for 30 min. After washed three times with 1% aceticacid and allowed to air dry, the bound SRB stain was thensolubilized with 10 mM unbuffered Tris base. The absorbancewas read at 515 nm using a Bio-Tek mQuant microplate reader.The ED 50 values of test samples with serial dilutions werecalculated using non-liner regression analysis (Table curve2Dv4;AISN Software, Inc., Mapleton, OR). The cytotoxic activity ofextracts, chromatographic fractions of extracts, and all purecompounds were evaluated against the HT-29 (human coloncancer) cell line, according to an established protocol (Pan et al.,2010).

3.5.2. Enzyme-based ELISA NF-kB assay

The NF-kB assay was carried out according to a publishedprotocol (Renard et al., 2001; Deng et al., 2009). In summary,a nuclear extract was prepared from HeLa cells obtainedfrom the American Type Culture Collection. An EZ-DetectTM

Transcription Factor Assay System ELISA kit (Pierce Biotechnol-ogy, Rockford, IL) was used to assess the specific bindingability of activated NF-kB to the biotinylated-consensussequence under the presence of tested compounds. Compoundswere tested at four concentration levels to obtain theinhibitory concentration. The binding activity of the p65 subunitof NF-kB was measured by detecting the chemiluminescentsignal in a Fluostar Optima plate reader (BMG Labtech Inc.,Durham, NC).

3.5.3. Parasite strain and promastigote assay

Leishmania mexicana ((MNYC/BZ/62/M379) amastigotesexpressing DSRed fluorescent protein was recovered fromfootpads of Balb/c infected mice. Promastigotes were obtainedby in vitro culture of amastigotes in Schneider’s mediumsupplemented with 10% fetal calf serum (FCS, Sigma, St. Louis,MO) and incubated at 28 8C. After transformation in promastigotesparasites were cultured in M199, serial passages were carriedout until reach log growth phase. All work including experimentalanimals was approved by the Institutional Animal Care andUse Committee (IACUC) of The Ohio State University.


Promastigotes were seeded in 1 � 106/mL of complete M199medium, after 48 h of treatment, live parasites were identified byflow cytometry based in their DSRED fluorescence and deathparasites were identified by their lost of fluorescence. Theproportion of death parasites was used to calculate IC50 values.Untreated parasites were used as a live control and Quillaja

saponaria (Quillajaceae) saponin-treated parasites resulted in 100%dead parasites, as reported in the literature (Lezama-Davila et al.,2012). Amphotericin B is used in this assay as a positive control.

Acknowledgement

Support from grant P01 CA125066 (to ADK) from the QNationalCancer Institute, NIH, Bethesda, MD, and from RC4 AI092624 (toARS and ADK) from National Institute of Allergy and InfectiousDiseases, NIH, Bethesda, MD, are acknowledged.

We thank Dr. Craig McElroy, College of Pharmacy, andDr. Chun-hua Yuan, Campus Chemical Instrumentation Center(CCIC), The Ohio State University, for facilitating the acquisitionof NMR spectra. The plant material was collected under theterms and conditions of a Memorandum of Agreement betweenthe University of Illinois at Chicago and the Institute of Ecologyand Biological Resources (IEBR) of the Vietnam Academy ofScience and Technology, Hanoi, Vietnam. Thanks are expressedto the Director of Nui Chua National Park for permission, and tothe Director of IEBR for overseeing the field operation in thecollection of the plant.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at http://dx.doi.org/10.1016/j.phytol.2014.06.010.

References

Bi, Y., Zhang, L.H., Hamaker, L.K., Cook, J.M., 1994. Enantiospecific synthesis of (�)-alstonerine and (+)-macroline as well as a partial synthesis of (+)-villalstonine. J.Am. Chem. Soc. 116, 9027–9041.

Deng, Y., Balunas, M.J., Kim, J.-A., Lantvit, D.D., Chin, Y.-W., Chai, H.-B., Sugiarso, S.,Kardono, L.B.S., Fong, H.H.S., Pezzuto, J.M., Swanson, S.M., Carcache-Blanco, E.J.,Kinghorn, A.D., 2009. Bioactive 5,6-dihydro-alpha-pyrone derivatives fromHyptis brevipes. J. Nat. Prod. 72, 1165–1169.

Camacho, M.d.R., Phillipson, J.D., Croft, S.L., Solis, P.N., Marshall, S.J., Ghazanfar, S.A.,2003. Screening of plant extracts for antiprotozoal and cytotoxic activities. J.Ethnopharmacol. 89, 185–191.

Flora Singapura, 2014. Further Information available at http://florasingapura.com/Alstonia-Angustifolia.php (accessed April 2014).

Ghedira, K., Zeches-Hanrot, M., Richard, B., Massiot, G., Le Men-Olivier, L., Sevenet,T., Goh, S.H., 1988. Alkaloids of Alstonia angustifolia. Phytochemistry 27,3955–3962.

Gadekar, R., Singour, P.K., Chaurasiya, P.K., Pawar, R.S., Patil, U.K., 2010. A potentialof some medicinal plants as an antiulcer agent. Pharmacogn. Rev. 4, 136–146.

Hamaker, L.K., Cook, J.M., 1995. In: Pelletier, S.W. (Ed.), The synthesis of macrolinerelated sarpagine alkaloids. Alkaloids:chemical and biological perspectives, vol.9. Elsevier Science, New York, NY, pp. 23–84.

Hu, W., Zhu, Lan., Hesse, J.P.M, 1989. Indole alkaloids from Alstonia angustifolia.Planta Med. 55, 463–466.

Kam, T.S., Choo, Y.M., 2002. Alkaloids from Alstonia angustifolia. Phytochemistry 65,603–608.

Kam, T.S., Iek, I.H., Choo, Y.M., 1999. Alkaloids from the stem-bark of Alstoniamacrophylla. Phytochemistry 51, 839–844.

Kam, T.S., Choo, Y.M., Komiyama, K., 2004. Unusual spirocyclic macroline alkaloids,nitrogenous derivatives, and a cytotoxic bisindole from Alstonia. Tetrahedron60, 3957–3966.

Lezama-Davila, C.M., Isaac-Marquez, A.P., Kapadia, G., Owens, K., Oghumu, S.,Beverley, S., Satoskar, A.R., 2012. Leishmanicidal activity of two naphthoqui-nones against Leishmania donovani. Biol. Pharm. Bull. 35, 1761–1764.

Lumpu, S.L., Kikueta, C.M., Tshodi, M.E., Mbenza, A.P., Kambu, O.K., Mbamu, B.M.,Cos, P., Maes, L., Apers, S., Pieters, L., Cimanga, R.K., 2013. Antiprotozoalscreening and cytotoxicity of extracts and fractions from the leaves, stem barkand root bark of Alstonia congensis. J. Ethnopharmacol. 148, 724–727.

Osorio, E.J., Robledo, S.M., Bastida, J., 2008. In: Cordell, G.A. (Ed.), The Alkaloids:Chemistry and Biology, vol. 66. Academic Press, Oxford, UK, pp. 113–190.

Pan, L., Kardono, L.B.S., Riswan, S., Chai, H., Carcache de Blanco, E.J., Pannell, C.M.,Soejarto, D.D., McCloud, T.G., Newman, D.J., Kinghorn, A.D., 2010. Isolation and



http://florasingapura.com/Alstonia-Angustifolia.php

http://florasingapura.com/Alstonia-Angustifolia.php


377

378

379 Pe380

381 Re382

383

384 Sid385

386

387 Sli388

389

390391392393394395396397398399400401402


G Model

PHYTOL 731 1–6

characterization of minor analogues of silvestrol and other constituents from alarge-scale recollection of Aglaia foveolata. J. Nat. Prod. 73, 1873–1878.

rry, L.M., Metzger, J., 1980. In Medicinal Plants of East and Southeast Asia:Attributed Properties and Uses. MIT Press, Cambridge, London, MA, pp. 1–620.

nard, P., Ernest, I., Houbion, A., Art, M., Le Calvez, H., Raes, M., Remacle, J., 2001.Development of a sensitive multi-well colorimetric assay for active NFkB.Nucleic Acids Res. 29, e21.

iyasa, K., 1998. Taxonomy, phylogeny, and wood anatomy of Alstonia (Apoc-ynaceae). Blumea, Suppl. 11. In: Rijksherbarium/Hortus Botanicus. Leiden Uni-versity, Leiden, The Netherlands, pp. 1–230.

k JWF (2009 onwards) Plants of Southeast Asia. url., 2014. Further Informationavailable at http://www.asianplant.net/Apocynaceae/Alstonia_angustifolia.htm(accessed 04.14).


Tan, S.-J., Robinson, W.T., Komiyama, K., Kam, T.-S., 2011. Macrodasine A-G, macro-line indole alkaloids incorporating fused spirocyclic tetrahydrofuran-tetrahy-drofuran and tetrahydrofuran-tetrahydropyran rings. Tetrahedron 67,3830–3838.

Tan, S.-J., Lim, K.-H., Subramaniam, G., Kam, T.-S., 2013. Macroline-sarpagine andmacroline-pleiocarpamine bisindole alkaloids from Alstonia angustifolia. Phy-tochemistry 85, 194–202.

Wright, C.W., Allen, D., Cai, Y., Phillipson, J.D., Said, I.M., Kirby, G.C., Warhurst, D.C.,1992. In vitro antiamoebic and antiplasmodial activities of alkaloids isolatedfrom Alstonia angustifolia roots. Phytother. Res. 6, 121–124.

Zocoler, M.A., de Oliveira, A.J.B., Sarragiotto, M.H., Grzesiuk, V.L., Vidotti, G.J., 2005.Qualitative determination of indole alkaloids of Tabernaemontana fuchsiaefolia(Apocynaceae). J. Braz. Chem. Soc. 16, 1372–1377.


http://www.asianplant.net/Apocynaceae/Alstonia_angustifolia.htm


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Bioactive indole alkaloids isolated from Alstonia angustifolia