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Isolation, characterization and quantification of bioactive molecules…… Chapter 1
1
Isolation, characterization and quantification of bioactive molecules from Cedrus
deodara, Albizzia chinensis and Podophyllum hexandrum
1.1 Isolation, characterization and quantification of bioactive molecules from
Cedrus deodara (Roxb.) Loud.
1.1.1 Introduction
The genus of true cedars, Cedrus consists of
four closely related species with
geographically separated distributions in
Mediterranean and western Himalayas
[Farjo´n (1990, 2001)], i.e. C. deodara
(Roxb.) Loud. in the Hindu Kush, Karakoram
and Indian Himalayas, C. libani A. Rich. in
Turkey, Lebanon and Syria, C. brevifolia
(Hook. f.) Henry in Cyprus, and C. atlantica
(Endl.) Manetti ex Carrie´re in North Africa (Algeria, Morocco) (Table 1.1.1).
The Himalayan cedarwood, Cedrus deodara (Roxb.) Loud., grows extensively on the
slopes of the Himalayas in northern India, Pakistan and Afghanistan and is often the most
important conifer at the elevations of 1650-2400 m. In India, deodar forests are naturally
distributed in an average estimated area of about 203263 hectares comprising of 69872,
20391, and 113000 hectares in Himachal Pradesh, Uttar Pradesh and Jammu & Kashmir.
respectively, yielding 0.75 million m3 annual production of wood [Anonymous (1950)].
Sizeable quantities of wood are employed for distillation of essential oils, used worldwide
in the soap industry as an inexpensive source of perfume. The oil is distilled from roots and
stumps of the plant left after cutting of trees for timber extraction. Himalayan cedarwood
oil is relatively a recent addition to the list of cedarwood oils produced commercially.
Production began in India in late 1950s and is estimated to be around 150 tonnes per
annum out of world’s production of cedarwood oil of 3000 tonnes per annum [Coppen
(1995)].
The plant leaves (called needles) are widely used for flavoring foods, beverages, (powders,
wine, and tea) [Kim and Chung (2000)] and for the treatment of rheumatism, diabetes,
obesity, gonorrhea, chronic bronchitis, cancer, stomach and cardiovascular diseases [Zhang
et al. (2011); Atwal et al. (1976)]. Their application lessen the inflammation in tuberculous
glands and have mild terebinthinate properties [Krishnappa and Dev (1978); Bhan et al.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
2
(1984); Mukherjee (2001)]. It is recorded in the dictionary of Chinese Crude Drugs as an
effective herbal drug for expelling wind, removing dampness, destroying parasites,
termites, moths, beetles and relieving itching. The wood has been used since ancient time
in Indian medical practice for the treatment of inflammations and rheumatoid arthritis
[Kirtikar and Basu (1933); Agarwal et al. (1980)]. The wood and bark extracts are
carminative, diaphoretic and useful in fever, piles, kidney stones, flatulence, pulmonary,
urinary, diarrhoea, dysentery problems [Parveen et al. (2010); Sharma et al. (1997);
Bhushan et al. (2006)]. The root oil is used as anti-ulcer drug by Hakims. Previous
chemical investigations indicated the presence of terpenes, lignans [Agarwal et al. (1982)],
and flavonoids [Agarwal et al. (1980)]. The lignans have been known for a number of
pharmacological activities such as antioxidant, antimitotic, antiviral, antitumor [Mercer and
Towers (1984)].
Table 1.1.1: List of Cedar trees with botanical/common names
Botanical Name Common Name
Family Pinaceae
Cedrus deodara Himalayan cedarwood, deodar
Cedrus libani Lebanon cedar or Cedar of Lebanon
Cedrus brevifolia Cyprus cedar
Cedrus atlantica Atlas cedar
Family Cupressaceae
Cupressus funebris Endl. Chinese cedarwood
Juniperus virginiana L. Virginia cedarwood, Eastern red cedar
J. mexicana Schiede Texas cedarwood
J. procera Hochst East African cedarwood
Widdringtonia whytei Rendle Mulanje cedarwood
1.1.2 Chemical constituents
Phytochemical research carried out on C. deodara has led to the isolation of
sesquiterpenes, flavonoids, alkaloids, tannins, saponins, lignans, organic acids and few
other classes of chemical constituents from its different parts. Sesquiterpenes are present in
almost all parts of C. deodara, in fact, they are mainly responsible for the pharmacological
activities of the plant.
1.1.2.1 Constituents of wood essential oil
The cedarwood oil is used as perfume fixative in cosmetic, soap and essence for household
or industrial use. The oil enriched with himachalenes is known as ‘Supper Rectified Oil’
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
3
and that in atlantones is called ‘Perfumery Grade’. The therapeutic actions of Cedrus
species have partially been ascribed to sesquiterpenes. Generally, the Cedrus oils were
characterized by high percentage of himachalenes [Bhushan et al. (2006)]. In the essential
oil of C. deodara wood, a number of sesquiterpenes including the himachalenes (�, � and
�) (1-3), isocentdarol (4), himachalol (5), allohimachalol (6) [Bisarya and Dev (1968)],
aryl himachalene (7), (E)-�-atlantone (8), (E)-γ-atlantone (9), (Z)-�-atlantone (10), (Z)-γ-
atlantone (11) [Pande et al. (1971)], deodarone (12), [Shankaranarayan et al. (1973);
Gopichand and Chakravarti (1974)], oxidohimachalene (13), �-himachalene monoepoxide
(14), atlantolone (15) [Shankaranarayan et al. (1977)], deodardione (16), diosphenol (17),
limonene carboxylic acid (18) were reported [Krishnappa and Dev (1978)]. Nigam et al.
(1990) described the presence of twenty three sesquiterpenic compounds in the essential
oil.
H
H
HO
OH OH
HO
H
(1) (2) (3) (4) (5) (6)
O
O
O O
(7) (8) (9) (10) (11)
O
O
O
O
O
HO
O
OH
O
(12) (13) (14) (15) (16)
OH
O
COOHH
(17) (18)
1.1.2.2 Constituents of stem bark and wood extract
Flavonoids [taxifolin (19), taxifolin-3'-glucoside (20), deodarin (21), cedeodarin (22),
cedrin (23), cedrinoside (24), quercetin (25)] [Raghunathan et al. (1971)], neolignans
[dihydrodehydroconiferyl alcohol (26), cedrusin (27), cedrusinin (28), triacetyl cedrusinin
(29)], lignans [lariciresinol (30), isolarciresinol (31), secoisolaricirosinol (32), meso-
secoisolariciresinol (33), (-)-wikstromal (34), (-)-matairesinol (35), dibenzylbutyrolactol
(36), (-)-nortrachelogenin (37)], sesquiterpenoids [himachalol (5), centdarol (38)], phenolic
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
4
sesquiterpene [himasecolone (39)] [Agarwal and Rastogi (1981)], diterpene [isopimaric
acid (40)], fatty acid ester [ethyl-23-methyl pentacosanoate (41)] [Khan and Naheed
(1988)] and acids [� 10-dehydroepitodomatuic acid (42), �7-dehydrotodomatuic acid (43) and
7-hydroxytodomatuic acid (44)] [Agarwal et al. (1982); Bhan et al. (1984)] were identified
from the extracts of wood and stem bark.
O
OH
HO
O
OR4
R3
OH
OH
R1
R2
O
OH
HO
O
OH
OH
OH
R
R1 R2 R3 R4 R
H H H H (19) H (25)
H H H glu (20) OH (45)
H Me H H (21)
Me H H H (22)
Me H OH H (23) Me H OH glu (24)
H H OH H (46)
OROH2C
OR1
OR2
OMe
R3
O
HO
H H
OMe
OH
MeO
HO
R R1 R2 R3 (30)
H H H OMe (26) H H H OH (27)
H H H H (28) Ac Ac Ac H (29)
MeO
HO
OH
OMe
CH2OH
CH2OH
HO
MeO
OH
OMe
CH2OH
CH2OH
HO
MeO
OH
OMe
CH2OH
CH2OH
(31) (32) (33)
1.1.2.3 Constituents of needles and roots
The compounds obtained from 95% ethanolic extract of C. deodara needles were
characterized as taxifolin (19), quercetin (25), myricetin (45), 2R,3R-dihydromyricetin
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
5
(46), cedrusone A (47), �-sitosterol (48), 1-[3-(4-hydroxyphenyl)-2-propenoate]-�-D-
glucopyranoside (49), 10-nonacosanol (50), dibutyl phthalate (51), phthalic acid bis-(2-
ethylhexyl)ester (52), protocatechuic acid (53), shikimic acid (54) and 5p-trans-
coumaroylquinic acid (55) [Zhang et al. (2011); Liu et al. (2011)]. A diterpene acid,
centdaroic acid (56) has been reported from roots [Srivastava et al. (2001)].
O
MeO
HO
O
OH
OMe
O
MeO
HO
O
OH
OMe
HO
H
O
MeO
HO
OH
OMe
OH
(34) (35) (36)
O
O
MeO
HO
OH
OMe
H
H
OH
OH
OH
O
Me
Me
HOOCH COOC2H520
(37) (38) (39) (40) (41)
HHO
HOOC
HO
HOOC
HO
HOOC
OH
(42) (43) (44)
O
OMe
OH
OMe
O
OOH
HO
O
O
O
HO
OH
OH
H
HH
HO
H
O
O
O
HOHO
OH
OH
OH
(47) (48) (49)
OH
18
O
O
O
O
O
O
O
O
(50) (51) (52)
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
6
O OH
OH
OH
O OH
OHHO
OH OH
OH
HOOC
HO
O
O
OH
Me
Me
Me
Me COOH
(53) (54) (55) (56)
1.1.3 Pharmacological and biological activities
The use of C. deodara (Roxb.) Loud. is recommended in the Ayurvedic system of
medicine for treatment of various ailments [Nayar and Chopra (1956)]. The alcoholic
extract of C. deodara is known to possess a variety of biological effects such as anticancer,
anti-inflammatory, diuretic and spasmolytic activities [Kulshreshtha and Rastogi (1976);
Agarwal et al. (1980)]. Major pharmacological activities included insecticidal,
molluscicidal, antifungal and anti-inflammatory activities [Zhang et al. (2009)].
1.1.3.1 Insecticidal activity
Cedarwood oil, being non-pollutant, has been used as an alternative for conventional
pesticides against different insect-pests. Chromatography fractions of the oil showed
insecticidal activity against Callosobruchus analis and Musca domestica; himachalol (5)
and �-himachalene (2) being active against C. analis [Singh and Agarwal (1988)]. The oil
showed Knock Down (KD) property against adult Anopheles stephensi, at low
concentrations (KD50 0.4452% in acetone) [Singh et al. (1984)]. The commercial products of C.
deodara such as Himax and Pestoban were found effective against skin diseases of goats,
sheep and dogs [Hazarika et al. (1995); Sharma et al. (1997); Dimri and Sharma (2004b)].
Oils obtained from A. indica, C. deodara and their combination (1:1) exhibited fumigant
potential against adults of Callosobruchus chinensis L. [Raguraman and Singh (1997)].
Essential oil of C. deodara, in combination with other oils showed low activity against the
adults of Aedes agypti (LC50 2.48%) [Makhaik (2005)].
1.1.3.2 Molluscicidal activity
Different combinations of the essential oils of cedar and neem tree in combination with
powder from bulbs of Allium sativum Linn., oleoresin extracted from rhizomes of Zingiber
officinale Rosc., custard apple seed powder, fruit powder of Embelia ribes showed toxicity
against the snail Lymnaea acuminata L. [Singh and Singh (1998); Singh and Singh (2001);
Rao and Singh (2001); Singh and Singh (2004)]. C. deodara along with other plant-derived
molluscicides exhibited dose and time dependent toxicity against Achatina fulica [Rao and
Singh (2000)]. Sublethal in vivo 24 h exposure of oil of Azadirachta indica, C. deodara,
bulb powder of Allium sativum and bark powder of Nerium indicum (LC50 40% and 80%)
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
7
significantly reduced the activity of A. fulica by altering acetylcholinesterase (AChE),
lactic dehydrogenase (LDH) and acid/alkaline phosphatase enzymes activity [Rao et al.
(2003)].
1.1.3.3 Antifungal activity
The oil proved to be effective against Absidia sp., Alternaria alternata, A. porri,
Aspergillus flavus, A. fumigatus, A. niger, A. ruber, A. versicolor, Cladosporium
cladosporioides, Curvularia lunata, Paecilomyces variotii, Fusarium oxysporum and
Rhizopus spp. [Dikshit et al. (1983); Pawar and Thaker (2007)]. Himachalol (5) exhibited
a significant protection and reduced colony forming units against A. fumigatus (minimum
inhibitory concentration, MIC, 46.4 �g/ml) [Khan and Jain (2000); Chowdhry et al. (1996);
Parveen et al. (2010)] and showed protection against invasive Aspergilli [Chowdhry et al.
(1997)]. The essential oil exhibited absolute toxicity, inhibiting the mycelial growth of A.
niger and Curvularia ovoidea, the two storage fungi found in blackgram, Vigna mungo L.
showing MIC of 1000 ppm [Singh and Tripathy (1999)]. The oil showed a broad
fungitoxic spectrum inhibiting the mycelial growth of fungi (Achaetomium strumarium
Guarro, Acremonium album Cattaneo, Alternaria alternata (Fr.) Keisaler, Aspergillus
aculeatus Iizuka, A. flavus Link ex Fries, A. japonicus Saito, A. niger van Teighem, A.
tamarri Kita, A. terreus Thom., Curvularia ovoidea (Hiroe & Watanabe) Muntanola,
Fusarium moniliforme Scheldon, F. oxysporum Schl., Penicillium chrysogenum Thom. and
P. funiculosum Thom., Rhizopus arrhizus Fischer [Singh et al. (1999)]. Eleven �-
himachalene derivatives displayed moderate to excellent activity against Botrytis cinerea
after 6 days; dihydroxyhimachalene derivative was found most promising [Daoubi et al.
(2005)]. Ethanolic extract of fresh plants of C. deodara caused complete inhibition of
mycelial growth of Sclerotium rolfsii Sacc. [Devi et al. (2007)].
1.1.3.4 Anti-inflammatory
The wood of C. deodara has been used since ancient days in Ayurvedic medical practice
for the treatment of inflammations and arthritis [Kirtikar and Basu (1933)]. The activity of
the oil could be attributed to its mast cell stabilizing activity and the inhibition of
leukotriene synthesis [Shinde et al. (1999a)]. The essential oil reduced the number of
neutrophils at the site of inflammation and the release of inflammatory mediators. It
produced significant inhibition of carrageenan-induced rat paw edema and of both
exudative-proliferative and chronic phases of inflammation in adjuvant arthritic rats at
doses of 50 and 100 mg/kg body weight [Shinde et al. (1999b); Tandan et al. (1998);
Baylac and Racine (2004)].
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
8
1.1.3.5 Other activities
The spasmolytic activity was shown by 50% ethanolic extract of the wood of C. deodora
and himachalol (5) [Dhar et al. (1968); Kar et al. (1975)]. Antibacterial action of the
ethanolic extract was found against Staphylococcus aureus, Enterococcus faecalis, Bacillus
cereus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli [Zeng et al.
(2011)]. A standardized lignan composition (AP9-cd) from C. deodara consisting of (-)-
wikstromal (34), (-)-matairesinol (35) and dibenzylbutyrolactol (36) showed cytotoxicity in
several human cancer cell lines [Bhushan et al. (2006)]. Higher doses (100 & 200 mg/kg)
of alcoholic extract of heart wood of the plant showed anxiolytic, anticonvulsant activities
and significant CNS depression by reducing locomotor activity in mice through modulation
of GABA levels in brain [Dhayabaran et al. (2010)]. The mortality was found against
larvae of Culex quinquefasciatus by hot water, acetone, and methanolic extracts of stem
bark with LC50 values of 133.85, 141.60, 95.19 ppm and LC90 values of 583.14, 624.19 and
639.99 ppm, respectively [Rahuman (2009)]. Study showed that petroleum ether extract of
the heart wood exhibited protection against sodium oxalate induced nephrolithiasis, thus
showing diuretic and anti-urolithiatic activities [Ramesh et al. (2010)].
There has been a tremendous interest in this plant as evidenced by the voluminous work
carried out by different researchers in the identification of chemical constituents of
essential oil. Yet very less work on the extracts from wood of this species has so far been
reported. With the efforts to search for the novel bioactive constituents from natural source,
we aimed to work on isolation and identification of bioactive constituents of C. deodara
and their quantification by different analytical techniques. In the following pages results
and discussion section followed by experimental sections are mentioned.
1.1.4 Results and discussion
1.1.4.1 Phytochemical studies
The sawdust of C. deodara stump was successively extracted with n-hexane (hexane),
followed by chloroform (CHCl3), methanol (MeOH) and water (H2O) using percolation
extraction. From hexane extract one known sesquiterpene, (E)-�-atlantone (8) and from
chloroform extract, two new sesquiterpenes, (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57), (E)-
(2S, 3S, 6S)-atlantone-2,3,6-triol (58) and one known sesquiterpene atlantolone (15) were
isolated by repeated column chromatography over silica gel.
The ethanolic extract of the needles was fractionated into petroleum ether, ethyl acetate
(EtOAc), n-butanol (n-BuOH) and water fractions. From petroleum ether fraction
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
9
protocatechuic acid (53) and from ethyl acetate fraction, taxifolin (19) and myricetin (45)
were isolated and characterized.
The chemical composition of the essential oil obtained by hydrodistillation of woodchips
of C. deodara by GC-MS was investigated for their secondary metabolites. A mixture of
three himachalenes: �-himachalene (1), �-himachalene (2), γ-himachalene (3) were isolated
and characterized from essential oil.
1.1.4.1.1 (E)-�-Atlantone (8)
Compound 8 was isolated as a yellowish gum. Its positive ESI-QTOF-MS showed
molecular ion peak at m/z 219.3411 [M+H]+ (calcd. 219.3425) corresponding to the
molecular formula C15H23O. HPLC chromatogram showed 88.1% purity (tR 8.03 min)
using water:methanol (20:80, v/v) as mobile phase (Figure 1.1.1). UV spectrum showed
absorption maxima at 268 nm. FT-IR spectrum indicated absorption maxima for �,�-
unsaturated ketone (1665, 1614 cm-1
) and olefinic groups (3139, 2979 cm-1
). 1H NMR
spectrum (Table 1.1.2) showed three methine signals at � 5.32 (1H, br s), 5.98 (2H, s)
assignable to H-2, H-8 and H-10 respectively. Six methylene protons resonated at � 1.87-
1.97 (4H, m) and 1.68-1.72 (2H, m) corresponding to carbons at C-1, C-4 and C-5
positions whereas four methyls were observed at � 2.08 (6H), 1.80 (3H) and 1.52 (3H).
OH
31
114
2
79
0.0 2.5 5.0 7.5 10.0 12.5 min
0
500
1000
1500
2000
2500
3000mAU
Figure 1.1.1: Chemical structure of 8 and its HPLC chromatogram
HMBC
31
114
2
79
OH
Figure 1.1.2: Selected HMBC correlations of 8
13C NMR spectrum displayed fifteen signals including one �,�-unsaturated carbonyl at �
191.9 (C-9) and three quaternary carbons at � 153.9 (C-11), 161.6 (C-7) and 133.5 (C-3).
Three tertiary carbon signals at � 120.1, 125.5 and 124.2 were assigned to C-2, C-8 and C-
10 respectively. The long-range interactions were observed in HMBC spectrum between
the protons at �H 5.98 with the carbons resonating at � 191.9 (C-9), 153.9 (C-11) and 161.6
(C-7) suggesting that these carbons are present in the same chain (Figure 1.1.2). The
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
10
methyl protons resonating at �H 1.52 exhibited long-range interactions with the carbons at �
133.5 (C-3), 30.4 (C-1) and 30.3 (C-4). The HMBC correlations displayed interaction of C7
methyl (�H 2.08) and C-8 methylene (�H 5.98) protons with C-6 carbon (� 44.8). Thus, on
the basis of above spectral data and comparison with earlier reported spectral values
[Shankaranarayan et al. (1977); Pande et al. (1971); Crawford et al. (1972)], the structure
of compound 8 was assigned as (E)-�-atlantone (Figure 1.1.1).
Table 1.1.2: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 8 in CDCl3 Position �C (ppm) �H (ppm) m (J Hz) Position �C �H (ppm)m (J Hz)
1 30.4 1.87-1.97 m 9 191.9 -
2 120.1 5.32 br s 10 124.2 5.98 s
3 133.5 - 11 153.9 -
4 30.3 1.87-1.97 m 11-trans-CH3 27.4 1.80 s
5 27.3 1.68-1.72 m 3-CH3 23.4 1.52 s
6 44.5 2.25-2.28 m 7-CH3 17.4 2.08 s
7 161.6 - 11-cis-CH3 20.8 2.08 s 8 125.5 5.98 s
1.1.4.1.2 (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)
Compound 57 was obtained as a light brownish gum and displayed a molecular ion peak at
m/z 253.3562 [M+H]+ (calcd. 253.3572) in its HRESI-QTOF-MS, corresponding to the
formula C15H25O3 suggesting 4 degrees of unsaturation. FT-IR spectrum indicated
absorption maxima for hydroxyl (3421 cm-1) and �,�-unsaturated ketone (1660, 1617 cm-1)
functional groups. The UV spectrum showed absorption maxima at �max 296 nm. The
compound showed 92.3% purity (tR 4.83 min) determined by HPLC using
water:acetonitrile (30:70, v/v) as mobile phase (Figure 1.1.3). 1H,
13C and DEPT NMR
spectra revealed 15 carbon signals constituting four methyls, three methylenes, four
methines and four quaternary carbons. The four methyl singlets were found at � 1.24, 1.93,
2.14 and 2.15 corresponding to carbons at � 27.8 (C3-CH3), 27.8 (C11-trans-CH3), 18.1 (C7-
CH3) and 20.9 (C11-cis-CH3), respectively, two olefinic protons at � 6.16 (C-8, C-10) and
one hydroxymethine signal at � 3.59 (C-1) (Table 1.1.3). One carbonyl moiety at � 194.3
was assignable to C-9, the characteristic signal of an �,�-unsaturated ketone moiety. Four
olefinic signals at � 164.1, 125.4, 127.5 and 156.0 revealed the position of two double
bonds at C-7 and C-8; C-10 and C-11. Since the 13C NMR spectral data of 57 was similar
to (E)-�-atlantone, it was assumed to be an atlantone type of sesquiterpene.
Analysis of its COSY data disclosed two proton-proton networks corresponding to H-2, H-
1, H-5, H-6, H-7, H3-7, H-8 and H-10, H-11 and H3-11 (Figure 1.1.4). Long-range proton-
carbon correlations observed in the HMBC spectrum (Figure 1.1.4) provided corroborative
evidence to support these subunits deduced from COSY data. The HMBC spectrum
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
11
showed correlations of H2-1 (�H 1.99-2.08, 1.61-1.69)/C-2, H3-3 (�H 1.24)/C-2, H2-4 (�
1.73-1.78, 1.46-1.55)/C-2, H1-2 (�H 3.59)/C-3, CH3-3 (�H 1.24)/C-3, and H2-4 (�H 1.73-
1.78, 1.46-1.55)/C-3. The NOESY correlations H-2 (�H 3.59)/H-1 (�H 1.99-2.08); H3-3 (�H
1.24)/H-1 (�H 1.99-2.08); H-5 (�H 1.73-1.78)/H-6 (�H 2.39-2.46); H-1 (�H 1.61-1.69)/H-6
(�H 2.39-2.46) supported a trans configuration between C-2 and C-3 hydroxyl groups
[Werf et al. (1999); Demyttenaere et al. (2001)]. MS2 spectra generated the fragments at
m/z 275 [M+Na]+ due to the sodiated molecular ion peak and the fragments at m/z 235 [M-
H2O]+, 217 [M-2H2O]+ were due to the sequential loss of two 18 (H2O) mass units
confirming presence of two free hydroxyl groups. Thus, on the basis of above evidences
compound 57 was unambiguously characterized as (E)-(2S, 3S, 6R)-atlantone-2,3-diol
(Figure 1.1.3).
O
OHOH
31
8
11 65
4
279
10
0.0 2.5 5.0 7.5 10.0 12.5 min
0
25
50
75
100
mAU
Figure 1.1.3: Structure of compound 57 and its HPLC chromatogram
H-H COSY
HMBC
O
OHOH
Figure 1.1.4: Selected HMBC and COSY correlations of 57
Table 1.1.3: 1H NMR (300 MHz) and 13C NMR (75.4 MHz) data of 57 in CD3OD Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 34.8 1.99-2.08 m; 1.61-1.69 m 9 194.3 -
2 74.2 3.59 br s 10 127.5 6.16 s
3 71.5 - 11 156.0 -
4 34.2 1.73-1.78 m; 1.46-1.55 m 11-trans-CH3 27.8 1.93 s
5 27.0 1.73-1.78 m; 1.46-1.55 m 3-CH3 27.8 1.24 s 6 42.4 2.39-2.46 m 7-CH3 18.1 2.14 s
7 164.1 - 11-cis-CH3 20.9 2.15 s
8 125.4 6.16 s
1.1.4.1.3 (E)-(2S, 3S, 6S)-atlantone-2,3,6-triol (58)
Compound 58 was obtained as a brownish gum. Its positive HR-ESI-QTOF-MS showed a
molecular ion peak at m/z 269.3581 [M+H]+ (calcd. 269.3566) correspond to the molecular
formula C15H25O4. FT-IR spectrum indicated absorption maxima for a hydroxyl (3404 cm-
1) and �,�-unsaturated ketone (1650, 1615 cm
-1). UV spectrum showed absorption maxima
at �max 293 nm. The purity (95.6%) of the compound (tR 3.84 min) was determined by
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
12
HPLC using water:ACN (30:70, v/v) as mobile phase (Figure 1.1.5). It possessed
spectroscopic data closely comparable to those of 57 except that proton (� 2.39) at C-6
position in 57 replaced with a hydroxyl group as evident from its 1H NMR data (Table
1.1.4). The presence of OH functional group at C-6 (� 77.0) was further confirmed by the
HMBC correlations H3-7 (�H 2.10)/C-6 and H-8 (�H 6.50)/C-6 (Figure 1.1.6). The NOESY
showed correlations of H-1 (�H 1.55-1.60)/H-5 (�H 1.41-1.49); H3-3 (�H 1.28)/H-1 (�H 1.55-
1.60) and H-5 (�H 2.17-2.18)/H-4 (�H 1.98-2.02).
31
8
11
6
54
279
10
O
OHOH
OH
0.0 2.5 5.0 7.5 10.0 12.5 min
0
250
500
750mAU
Figure 1.1.5: Structure of compound 58 and its HPLC chromatogram
MS2 spectra generated the fragments at m/z 291 [M+Na]+ due to the sodiated molecular
ion peak and the fragments at m/z 251, 233, 215 were due to the sequential loss of three 18
(H2O) mass units confirming presence of three free hydroxyl groups. On the basis of 1H-1H
COSY, HMBC and NOESY correlations the structure of 58 was deduced as (E)-(2S, 3S,
6S)-atlantone-2,3,6-triol (Figure 1.1.5) [Thappa et al. (1976); Kozma et al. (2004)].
H-H COSY
HMBC
O
HOOH
OH
Figure 1.1.6: Selected HMBC and COSY correlations of 58
Table 1.1.4: 1H NMR (300 MHz) and 13C NMR (75.4 MHz) data of 58 in CD3OD Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 36.4 2.25-2.31 m; 1.55-1.60 m 9 195.0 -
2 75.2 3.56 br s 10 127.5 6.20 s
3 71.6 - 11 156.7 -
4 30.1 1.98-2.02 m; 1.41-1.49 m 11-trans-CH3 27.8 1.93 s
5 31.6 2.17-2.18 m; 1.41-1.49 m 3-CH3 27.3 1.28 s 6 77.0 - 7-CH3 15.6 2.10 s
7 162.3 - 11-cis-CH3 20.9 2.15 s
8 124.7 6.50 s
1.1.4.1.4 Atlantolone (15)
Compound 15 was isolated as brownish oil. Its positive ESI-QTOF-MS showed molecular
ion peak at m/z 237.3563 [M+H]+ (calcd. 237.3578) corresponding to the molecular
formula C15H25O2. UV spectrum showed absorption maxima at 241 nm. FT-IR spectrum
indicated absorption maxima for hydroxyl (3435 cm-1
) and �,�-unsaturated ketone (1664,
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
13
1604 cm-1
) and olefinic (2914 cm-1
) functional groups. The purity (85.6%) of the
compound (tR 3.37 min) was determined by HPLC using water:ACN (20:80, v/v) as mobile
phase (Figure 1.1.7). 1H and 13C NMR spectra revealed fifteen carbon signals and
constituted four methyls, four methylenes, three methines and four quaternary carbons as
evident from DEPT spectra. The four methyl singlets were found to be at � 2.14, 1.64, 1.26
corresponding to carbons at � 18.1 (C7-CH3), 23.5 (C3-CH3), 29.5 (C11-trans-CH3, C11-cis-
CH3), respectively, two olefinic protons at � 5.39 (C-2, 1H, br s) and � 6.01 (C-8, 1H, s)
(Table 1.1.5).
OHO H
31
811 6
54
2
7910
0.0 2.5 5.0 7.5 10.0 12.5 min
0
25
50
75
mAU
Figure 1.1.7: Chemical structure of compound 15 and its HPLC chromatogram
HMBC
OHO
31
11 5
2
79
Figure 1.1.8: Selected HMBC correlations of 15
Table 1.1.5: 1H NMR (300 MHz) and
13C NMR (75.4 MHz) data of atlantolone in CDCl3
Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 30.4 1.98-2.04 m 9 202.9 -
2 120.0 5.39 br s 10 54.4 2.58-2.60 m
3 134.0 - 11 70.1 -
4 30.3 1.98-2.04 m 11-trans-CH3 29.5 1.26 s
5 27.5 1.75-1.84 m 3-CH3 23.5 1.64 s
6 44.7 2.19-2.23 m 7-CH3 18.1 2.14 s
7 164.8 - 11-cis-CH3 29.5 1.26 s
8 122.7 6.01 s
One carbonyl moiety at � 202.9 was assignable to C-9, the characteristic signal of an �,�-
unsaturated ketone moiety. Four olefinic signals at � 120.0, 134.0, 164.8 and 122.7
revealed the position of two double bonds at C-2 and C-3; C-7 and C-8. The presence of
OH functional group at C-11 (� 70.1) was confirmed by the HMBC correlations H3-3 (�H
1.26)/C-11, H2-10 (�H 2.58-2.60)/C-11 (Figure 1.1.8). The HMBC spectrum showed
correlations of H3-3 (�H 1.64) to C-1, C-2, C-3; H3-7 (�H 2.14) to C-6, C-8 and H2-5 (�H
1.75-1.84) to C-1, C-4, C-7. MS2 spectra generated the fragments at m/z 259 [M+Na]
+ due
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
14
to the sodiated molecular ion peak and the fragment at m/z 219 was due to the loss of one
18 (H2O) mass units confirming presence of one free hydroxyl groups. Thus, on the basis
of above spectral data and comparison with previously known spectral values
[Shankaranarayan et al. (1977)], the structure of compound 15 was assigned as atlantolone
(Figure 1.1.7).
1.1.4.1.5 Protocatechuic acid (53)
COOH
OH
OH
126
4
O
OH
HO
OH
O
OH
OH
O
OH
HO
OH
OH
O
OH5
8
4a
4'
OH
2
53 19 45
Figure 1.1.9: Chemical structures of compounds 53, 19 and 45
Compound 53 was isolated as a white amorphous powder. Its positive ESI-QTOF-MS
showed molecular ion peak at m/z 319.2411 [M+H]+ (calcd. 319.2430) corresponding to
the molecular formula C7H7O4. The FT-IR spectrum showed the absorption bands at 3456,
1680, 1544, 1167 cm-1
suggesting the existence of hydroxyl, carbonyl, C=C and ether
functionalities, respectively.
Table 1.1.6: 1H NMR (300 MHz) and 13C NMR (75.4 MHz) data of 53 in CD3OD Position �C (ppm) �H (ppm) m (J Hz)
1 122.0 -
2 116.7 7.46 s
3 145.0 -
4 150.5 -
5 114.7 6.81 d (8.6)
6 122.9 7.43 d (8.1)
COOH 169.2 -
1H NMR spectrum of this compound (Table 1.1.6) showed signals at � 7.46 (1H, s), 7.43
(1H, d, J = 8.1 Hz), and 6.81 (1H, d, J = 8.6 Hz) indicating the presence of a 1,3,4-
trisubstituted benzene ring. The 13C NMR spectrum showed the presence of seven carbons
with a signal at � 169.2 assignable to carboxyl carbon (COOH). The four signals at � 122.9,
122.0, 116.7, and 114.7 were assigned to C-6, C-1, C-2, and C-5 carbons respectively, by
HMQC and HMBC correlations. The signals at � 145.0 and 150.5 were assigned to C-3 and
C-4 aromatic carbons bearing hydroxyl groups. MS2 spectra generated the fragments at m/z
155 [M+H]+ due to the protonated molecular ion peak and the fragments at m/z 137
[M+H-H2O]+ and 111 [M+H-CO2]+, showed presence of hydroxyl and acidic groups. Thus,
compound 53 was identified as 3,4-dihydroxybenzoic acid (protocatechuic acid) (Figure
1.1.9) on the basis of spectral data that matched with the reported values [He et al. (2009)].
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
15
1.1.4.1.6 Taxifolin (19)
Compound 19 was isolated as a yellow amorphous powder. Its positive ESI-QTOF-MS
showed molecular ion at m/z 305.2579 [M+H]+ (calcd. 305.2595) corresponding to the
molecular formula C15H13O7. FT-IR spectrum showed the absorption bands at 3566, 1663,
1588, 1128 cm-1
suggesting the existence of hydroxyl, carbonyl, C=C and ether
functionalities, respectively. The 1H NMR spectrum gave signals (Table 1.1.7) due to C-2
and C-3 protons at � 4.94 (1H, m), 4.52 (1H, d, J = 11.2 Hz). The coupling constant and �
value indicated that C-2 and C-3 protons were of trans-type and of (2R, 3R) configuration
[Sakushima et al. (2002)]. The signals at � 6.98 (1H, br s), 6.83 (1H, m), and 6.80 (1H, m)
indicated the presence of a 1,3,4-trisubstituted benzene ring. 13
C NMR spectrum displayed
fifteen signals including one carbonyl at � 198.0 (C-4) and eight oxygen bearing quaternary
carbons at � 169.1 (C-5), 165.7 (C-7, C-8a), 147.5 (C-3'), 146.7 (C-4'), 85.5 (C-2), 74.1 (C-
3). Five methine signals at � 121.3, 116.5, 116.2, 97.7 and 96.7 were assigned to C-6', C-5'
and C-2', C-6 and C-8 carbons respectively. Thus, 19 was identified as (2R, 3R)-Taxifolin
(Figure 1.1.9) and spectral data of 19 matched with the previous literature [Han et al.
(2007)].
Table 1.1.7: 1H NMR (300 MHz) and
13C NMR (75.4 MHz) data of 19 in CD3OD
Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 - 8a 165.7 -
2 85.5 4.94 m 4a 102.3 -
3 74.1 4.52 d (11.2) 1' 130.3 -
4 198.0 - 2' 116.2 6.98 br s
5 169.1 - 3' 147.5
6 97.7 5.94 br s 4' 146.7
7 165.7 - 5' 116.5 6.80 m
8 96.7 5.90 br s 6' 121.3 6.83 m
1.1.4.1.7 Myricetin (45)
Compound 45 was isolated as a yellow amorphous powder. Its positive ESI-QTOF-MS
showed molecular ion at m/z 319.2411 [M+H]+ (calcd. 319.2430) corresponding to the
molecular formula C15H10O8. UV spectrum showed absorption maxima at 296 and 375 nm
characteristic of flavonols. FT-IR spectrum showed the absorption bands at 3556, 1675 cm-
1 suggesting the existence of hydroxyl and carbonyl groups, respectively. The IR spectrum
also exhibited absorptions at 1590 and 1025 cm-1 representing C=C and ether
functionalities, respectively. 1H NMR spectrum (Table 1.1.8) showed aromatic signals at �
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
16
7.35 (2H, br s), 6.39 (1H, br s), 6.19 (1H, br s) assignable to H-2', H-6', H-8, and H-6
respectively. 13C NMR spectrum displayed fifteen signals including one flavonol carbonyl
at � 177.4 (C-4) and eight oxygen bearing quaternary carbons at � 165.7 (C-7), 162.6 (C-5),
158.3 (C-8a), 148.1 (C-2), 146.8 (C-3', C-5'), 137.5 (C-4'), 137.1 (C-3). Four methine
signals at � 108.7, 99.3 and 94.5 were assigned to C-2', C-6', C-6 and C-8 respectively.
Thus, on the basis of above spectral data and comparison with previously known spectral
values [He et al. (2009)], the structure of compound 45 was assigned as myricetin (Figure
1.1.9).
Table 1.1.8: 1H NMR (300 MHz) and 13C NMR (75.4 MHz) data of 45 in CD3OD Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 - - 8a 158.3 -
2 148.1 - 4a 104.6 -
3 137.1 - 1' 123.2 -
4 177.4 - 2' 108.7 7.35 s
5 162.6 - 3' 146.8 -
6 99.3 6.19 br s 4' 137.5 -
7 165.7 - 5' 146.8 -
8 94.5 6.39 brs s 6' 108.7 7.35 s
1.1.4.1.8 �-, �- and �-Himachalene (1-3)
1 2 3
Figure 1.1.10: Chemical structures of mixture of three himachalenes (1-3)
Compound (1-3) was isolated as colorless oily liquid. The GC-MS showed three peaks at
m/z 204 corresponding to the molecular formula C15H24. 1H,
13C NMR, IR and GC-MS
showed a mixture of three compounds identified as �- (1), �- (2) and �-himachalene (3)
(Figure 1.1.10). 1H,
13C NMR and MS values are detailed in section 1.4.1.1.3.3 and were
compared with literature values [Bisarya and Dev (1968); Daoubi et al. (2005)].
1.1.4.2 Chemical composition of hydrodistilled and solvent volatiles extracted from
woodchips of C. deodara
The yields of wood essential oils and extracts from the hydrodistillation and percolation of
woodchips of C. deodara were 1.0% and 14.5% on dry weight basis, respectively. GC and
GC-MS analyses resulted in the identification of thirty four and twenty six constituents in
oil and extract of woodchips of C. deodara, respectively. Table 1.1.9 indicated the
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
17
constituents identified, their percentage composition and Kovats index (KI) values listed in
order of elution from the BP-20 capillary column. The identified constituents identified
were 26.7-79.5% sesquiterpene hydrocarbons and 18.9-67.9% oxygenated sesquiterpenes.
A total of twenty sesquiterpene hydrocarbons and nineteen oxygenated sesquiterpenes were
observed in the essential oil and extract; major constituents being himachalenes (23.5-
68.5%) and atlantones (15.0-61.6%). The other constituents identified were himachalene
oxide, himachalol, oxidohimachalene, dehydro-ar-himachalene and cis-�-bisabolene. C.
libani wood extract was reported to be rich in himachalenes (~42%) and needle oil in �-
pinene (~24%) and carophyllene (~7%) [Fleisher (2000)]. Himachalenes (68.5%),
atlantones (15.0%), himachalol (1.0%) present in the oil were reported in various
publications variation in their percentage composition [Nigam et al. (1990); Fleisher
(2000)]. A significantly higher percentage of atlantones (~67%) was observed in the C.
deodara extract as compared to other species whereas these constituted less than 10% of
the total oil. It may be pointed that the essential oil or extract content may differ greatly in
the same genus or different organs of same species [Salido et al. (2002); Cavaleiro et al.
(2002); Duquesnoy et al. (2006)]. Some earlier reported major constituents (cedrene and
cedrol) were not detected in this study [Nigam et al. (1990)].
Table 1.1.9: Chemical composition C. deodara woodchips essential oil and extract
Compounds KI Oil % Extract % Compounds KI Oil % Extract %
Longifolene 1517 0.6 - �-Dehydro-ar-himachalene 1849 0.7 0.3
Aromadendrene 1560 0.6 - trans-�-Bergamotene 1873 - 0.6
allo-Aromadendrene 1579 0.3 - Vestitenone 1883 0.4 1.6
� –Himachalene 1593 17.1 4.7 cis-�-Bergamotene 1890 - 0.6
� –Humulene 1633 0.5 - Oxidohimachalene 1931 0.3 0.2
Z-�-Farnesene 1637 0.4 - �-Himachaleneoxide 1965 0.3 -
�-Himachalene 1646 12.6 3.3 Caryophyllene oxide 1993 0.4 0.5
Cubinene 1649 2.3 0.6 �-Bisabolol 2036 - 0.2
�-Himachalene 1666 38.8 15.5 Longiborneol 2099 0.2 0.2
�-Cadinene 1672 0.4 - �-Atlantone 2127 0.4 0.8
8-Cedren-13-ol-acetate 1675 0.4 0.2 (Z)-�-Atlantone 2152 2.3 7.8
�-Cadinene 1677 0.7 0.2 Himachalol 2158 1.0 1.5
(E),(E)-Farnesol 1680 0.2 - (E)-�-Atlantone 2173 2.4 10.2
Albicanol 1685 0.2 - Deodarone 2181 0.3 0.8
4,5-Dehydroisolongifolene 1690 0.2 - Deodarone isomer 2185 0.3 0.9
�-Vativenene 1694 0.2 - (Z)-�-Atlantone 2200 1.4 4.3
cis-�-Bisabolene 1734 2.2 0.7 Aristolone 2265 - 0.1
ar-Curcumene 1773 0.9 - (E)-�-Atlantone 2278 8.6 38.5
�-Dehydro-ar-himachalene 1811 0.7 0.2 14-Oxy-�-muurolene 2292 - 0.2
9,10-Dehydroisolongifolene 1818 0.3 - Total identification 98.3 94.6
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
18
1.1.4.3 Chemical constituents of essential oil fractions
Total forty compounds were identified using GC and GC-MS analyses from n-pentane and
acetonitrile fractions. The identified constituents, percentage composition and their Kovats
index (KI) values are shown in Table 1.1.10. In the n-pentane fraction, twenty seven
compounds were identified representing 90.89% of the total constituents detected. A total
of eleven sesquiterpene hydrocarbons and sixteen oxygenated sesquiterpenes were
identified constituting 59.68 and 31.21%, respectively.
Table 1.1.10: Chemical constituents of fractions of C. deodara essential oil Compounds KI A5 (%) A6 (%) A4 (%) A3 (%)
4-Acetyl-1-methylcyclohexene 1499 - 2.10 - -
Longifolene 1503 0.91 - - -
Aromadendrene 1545 0.25 - - -
�-Himachalene 1579 13.29 2.56 20.33 -
�-Humulene 1617 0.48 - - - �-Himachalene 1627 11.28 2.29 17.95 -
�-Curcumene 1629 - 0.40 - -
�-Himachalene 1651 27.78 6.85 52.61 -
3-Cyclohexene-1-methanol 1660 - 0.33 - -
8-Cedren-13-ol-acetate 1677 0.21 - - -
�-Vativenene 1702 0.50 - - -
cis-�-Bisabolene 1715 1.58 0.36 - -
3-Methylacetophenone 1716 - 1.30 - -
4,5-Dehydroisolongifolene 1755 0.23 - - -
�-Dehydro-ar-himachalene 1791 1.94 0.96 - -
�-Dehydro-ar-himachalene 1826 1.44 1.02 - - Vestitenone 1860 0.55 3.00 - -
�-Himachalene oxide 1923 9.12 1.17 - -
Calarene epoxide 1969 0.83 1.74 - -
Nerolidol 1977 - 0.55 - -
Carophyllene oxide 2002 0.25 0.50 - -
(+)-8(15)-Cedren-9-ol 2006 - 0.47 - -
Aromadendrene oxide 2009 0.96 - - -
Longiborneol 2070 0.58 2.01 - -
�-Bisabolol 2077 0.22 0.48 - -
�-Atlantone 2097 0.94 2.71 - 1.95
(Z)-�-Atlantone 2122 0.79 8.63 - 11.38 Himachalol 2129 2.04 4.51 - -
m-Tolyldimethylactealdehyde 2137 - 0.45 - -
(E)-�-Atlantone 2143 0.87 8.83 - 15.70
Deodarone 2152 0.53 4.18 - -
Deodarone isomer 2156 - 3.70 - -
Humulane-1,6- dien-3-ol 2161 - 4.37 - -
(Z)-�-Atlantone 2172 3.40 5.23 - 4.99
(E)-�-Atlantone 2248 9.45 16.00 - 61.82
Longifolenaldehyde 2303 0.47 - - -
2-Butyl-1-methyl-1,2,3,4-tetrahydro-naphthalen-
1-ol
2345 - 1.09 - -
8-�-Acetoxyelemol 2349 - 0.47 - - 7�,3�-Dihydroxy-1�-2,6-cyclohimachalane 2401 - 0.36 - -
14-Hydroxy-9-epi-(E)-caryophyllene 2412 - 0.62 - -
Total identification 90.89 89.24 90.89 95.74 A5: n-pentane fraction; A4: himachalenes; A3: atlantones; A6: acetonitrile fraction; A2: crude oil
In the acetonitrile fraction, thirty one compounds were identified representing 89.24% of
the constituents of oxygenated monoterpene (3.73%), sesquiterpene hydrocarbon (14.44%),
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
19
and oxygenated sesquiterpene (71.07%) types. The major constituents in n-pentane fraction
were himachalenes (52.35%) while in acetonitrile fraction were atlantones (41.40%). The
other constituents identified in these fractions were himachalene oxide (1.17-9.12%),
himachalol (2.04-4.51%), �-dehydro-ar-himachalene (0.96-1.94%), cis-�-bisabolene (0.36-
1.58%) and �-dehydro-ar-himachalene (1.02-1.44%). Further chromatography of n-pentane
and acetonitrile fractions led to the increase in percentage of himachalenes and atlantones
to 90.89 and 95.74%, respectively. The results of the present study are indicative of the
utilization of oil, major constituents and extract of C. deodara for further modifications.
1.1.4.4 Determination of major flavonoids by UPLC-MS in C. deodara needles extract
A simple, sensitive, selective, precise and robust ultra-performance liquid
chromatography–tandem mass spectrometry (UPLC-MS) method was developed and
validated for determination of four flavonoids, taxifolin, quercitrin, myricetin, quercetin in
needles of C. deodara. All the four flavonoids were detected and determined in the needles
extracts. The chromatographic separation of four flavonoids was achieved in less than 8
min by UPLC® BEH C18 column (100 × 2.1 mm i.d., 1.7 �m) using linear gradient elution
of water (0.05 % formic acid) and acetonitrile with flow rate of 0.3 ml/min at � 254 nm.
The different extraction techniques such as soxhlet, maceration, ultrasound-assisted
extraction (UAE) and microwave-assisted extraction (MAE) were applied and compared to
bring out the best extraction procedure for flavonoids with methanol. In soxhlet extraction,
maximum yield of flavonoids was obtained. Soxhlet and microwave were found to show
comparable results, but taking into consideration the solvent and time consumption for
extraction, MAE was found to be the best approach for the rapid and efficient extraction of
flavonoids.
R1 R2 R3
OH OH H Myricetin
OH H rha Quercitrin
OH H H Quercetin
Taxifolin
Figure 1.2.14: Chemical structures of myricetin, quercitrin, quercetin and taxifolin
In UPLC analysis, various mobile phase compositions and chromatographic conditions
were tried to find the optimal chromatographic conditions. For optimization of
chromatographic conditions, good resolution was recorded with acetonitrile-water-formic
acid system for all active components than with methanol-water or acetonitrile-water
system. Water with formic acid (0.05%) (solvent A) and ACN (solvent B) was used as
O
OH
HO
OH
OR3
O
R1
R2 O
OH
HO
OH
O
OH
OH
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
20
mobile phase for the analysis with a linear gradient elution as follows: 0-2.5 min, 15% B;
2.5-4.0 min, 15-30% B; 4.0-5.5 min, 30% B, 5.5-6.0 min, 30-15% B, 6.0-8.0 min, 15% B.
The peak resolution was also recorded with variation in the column temperature. Column
temperature was optimized systematically from 25 to 40°C, and it was observed that all the
components achieved a baseline resolution at 35°C. All parameters like optimal separation,
high sensitivity and good peak shape without tailing of the analytes were standardized.
Optimal chromatographic conditions were obtained after running different mobile phases
with a reversed-phase C18 column (BEH C180, 100 × 2.1 mm i.d., 1.7 �m). Four
flavonoids, viz. taxifolin (RT: 1.36 min), quercitrin (RT: 4.79 min), myricetin (RT: 5.11
min), and quercetin (RT: 6.14 min) were well resolved. The representative chromatograms
of the standard mixture and sample of C. deodara have been shown in Figures 1.1.11 and
1.1.12. The chromatographic peaks were identified and confirmed by comparing their
retention times with reference compounds, overlaying of UV spectra with those of
reference compounds and spiking of samples with the reference compounds. The results
indicated that flavonoids were well resolved and their quantitative determination in C.
deodara was possible. Taxifolin (1.16-1.92%) and myricetin (0.54-1.25%) were detected in
major amount in the extract as compared to quercitrin (0.19-0.29%) and quercetin (0.17-
0.27%) (Table 1.1.14).
The calibration curves were linear in the range of 1.56-100 �g/ml for taxifolin, quercitrin,
myricetin, and quercetin. Regression equation and coefficient of correlation (r2 = 0.9952-
0.9962) revealed a good linearity response for developed method and are presented in
Table 10. The LODs for taxifolin, quercitrin, myricetin, and quercetin were 0.20, 0.10,
0.78, 0.39 and LOQs for same analytes were found to be 0.66, 0.32, 2.34, and 1.25 �g/ml,
respectively (Table 1.1.11). This indicated that the proposed method exhibited a good
sensitivity for the quantification of flavonoids. The intra- and inter-day precisions
(expressed in terms of %RSD) were observed in the range of 0.21-0.70% and 0.42-0.51%
respectively, demonstrating the good precision of the proposed method (Table 1.1.12).
Accuracy of the proposed method was expressed as the recovery of standard compounds
added to the pre-analyzed sample. Samples spiked with 150, 75, 37.5 �g/ml of taxifoiln,
50, 25, 12.5 �g/ml of quercitrin, quercetin and 100, 50, 25 �g/ml of myricetin, were used in
triplicate to assess accuracy. The amount of compounds was calculated from related linear
regression equation. The percentage recovery ranged from 95.99 to 99.86% with RSD
values in the range 0.11-1.53%, for the detected compounds (Table 1.1.13).
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
21
a
b
c
d
Figure 1.1.11: UPLC chromatogram of standard mixture of flavonoids; a = taxifoiln; b =
quercitrin; c = myricetin; d = quercetin
a
dbc
Figure 1.1.12: UPLC chromatogram of extract C. deodara needles; a = taxifoiln; b =
quercitrin; c = myricetin; d = quercetin
Table 1.1.11: Method validation data of four compounds in C. deodara needles
Analytes rt Regression
equation Linearity range
(�g/ml) r
2
LOD (�g/ml)
LOQ (�g/ml)
Taxifoiln 1.36 121.15x+181.84 1.56-100 0.9953 0.20 0.66
Quercitrin 4.79 177.47x+244.36 1.56-100 0.9952 0.10 0.32
Myricetin 5.11 119.23x+4.646 1.56-100 0.9957 0.78 2.34
Quercetin 6.14 138.82x+142.9 1.56-100 0.9962 0.39 1.25
The UV spectra suggested that the detected flavonoids present in the extracts were
flavanols and dihydroflavonols. The peaks showed characteristic absorption bands at 346-
371 nm (band A) and 251-265 nm (band B), indicating that the flvonols in extracts were
substituted in the 3-OH position. In dihydroflavonols, band A was reduced to little more
than a shoulder at 327 nm and band B, at 288 nm, detected as main peak.
Table 1.1.12: Inter-, intra-day precision of detected compounds in needles of C. deodara
Analytes Intra-day Precision (n=3) Inter-day Precision (n=9)
Day 1 Day 2 Day 3
RT* PA** RT* PA** RT* PA** RT* PA**
Taxifoiln 0.42 0.58 0.43 0.70 0.86 0.40 0.75 0.51
Quercitrin 0.12 0.50 0.12 0.55 0.12 0.44 0.13 0.49
Myricetin 0.11 0.59 0.11 0.40 0.11 0.45 0.13 0.42
Quercetin 0.09 0.50 0.09 0.21 0.09 0.21 0.09 0.43
RT*, %RSD of retention time. PA**, %RSD of peak area.
ESI-QTOF-MS/MS (in positive ion mode) of the constituents showed characteristic
distribution of fragment ions as illustrated in Figure 1.1.13. The fragmentation patterns
observed in the mass spectrum were useful in characterization of the compounds.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
22
Dihydroflavonols (taxifolin) exhibited an initial dehydration of [M+H]+
to [M+H-H2O]+
(m/z 287) and sequential losses of two carbonyl groups to form [M+H-H2O-CO]+ (m/z 259)
and [M+H-H2O-2CO]+ (m/z 231) (Table 1.1.15). Additional fragments were the 1,3A+ (m/z
153), [M+H-B ring]+
(m/z 159), 0,2
A+ (m/z 165) and
1,4B
+ (m/z 179) [Tsimogiannis et al.
(2007)]. Flavonol (quercetin) [M+H]+ product ions underwent dehydration, followed by
two sequential losses of CO: [M+H-H2O-CO]+ (m/z 257) and [M+H-H2O-2CO]+ (m/z
229). Furthermore the C-ring cleavage at bonds 0,2 and 1,3 produced the respective RDA
fragments 0,2B+ (m/z 137) and 1,3A+ (m/z 153) of the protonated molecule.
Table 1.1.13: Accuracy for the quantitative determination of four compounds C. deodara
needles
Added
amount (�g/ml)
Average
Recovery (%) RSD (%)
Taxifoiln 150 99.22 1.27
75 97.92 0.55
37.5 97.41 0.93
Quercitrin 50 99.65 0.66
25 96.34 0.11
12.5 98.76 0.61
Myricetin 100 99.86 0.05
50 99.22 0.97
25 98.86 1.53
Quercetin 50 99.22 0.87
25 97.35 0.13
12.5 95.99 0.13
Table 1.1.14: Quantitative determination of four compounds in the extract of C. deodara
by different extraction techniques
rt
Ultrasonication
(%w/w)
Microwave
(%w/w)
Soxhlet
(%w/w)
Maceration
(%w/w)
Taxifoiln 1.36 1.52 1.80 1.92 1.16
Quercitrin 4.79 0.24 0.26 0.29 0.19
Myricetin 5.11 0.87 0.93 1.25 0.54
Quercetin 6.14 0.21 0.23 0.27 0.17
Table 1.1.15: Identification of major constituents in methanolic extracts of C. deodara
needles
Peak tR
(min)
UV
spectra
Calcd.
MW
(+) ion mode Detected
Compounds [M+H]+ MS/MS (m/z)
a 1.34 282,
327sh 304 305
305, 287, 259, 231, 195, 179, 165, 153, 123, 108
Taxifolin
b 4.88 265, 346 448 449
303, 285, 257, 247, 229,
219, 201, 183, 165, 153,
137, 109, 81, 69
Quercitrin
c 5.11 251, 371 318 319 290, 245, 179, 153, 123,
79 Myricetin
d 6.15 253, 368 302 303 257, 229, 201, 183, 153,
137, 111, 95, 69 Quercetin
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
23
O
OH
HO
OH
O
OH
OH
O
OH
HO
O
O
[M+H-B ring]+ = 195
O
O
O
OH
1,4B+ = 179
O
OH
HO
O
1,3A+ = 153
OH
HO O
O
0,2A+ = 165
[M+H]+ = 305
0
3
2
4
A
B
C
I
O
OH
HO
OH
O
OH
OH
0,2B+ = 137
1,3A+ = 153
0
3
2
4
[M+H]+ = 303
A C
B
II
Figure 1.1.13: Fragmentation pattern of (I) taxifolin and (II) quercetin
1.2 Isolation, characterization and quantification of bioactive molecules from
Albizzia chinensis (Osbek) Merril
1.2.1 Introduction
Albizia or Albizzia is a pantropical genus
(subfamily Mimosoideae) with about 150
species of which 35 occur in Asia, 48 in
Africa and 35 in tropical and subtropical
America [Nielson (1981)]. Most species of
the genus have high biomass production, a
spreading crown and light feathery foliage
in addition to nodulation and nitrogen
fixing capabilities [Allen and Allen (1981)] and consequently are good shade trees for tea
and coffee plantations. These are deciduous woody trees and shrubs. They are easily
identified by their bipinnately compound leaves. Several Albizzia species are planted as
ornamentals or as a source of tannin extracts and are socially significant for producing high
quality timber and as a valuable resource for gum yield. A. julibrissin, A. lebbeck, A.
procera and A. amara are important in ayruvedic medicine. Seeds are regarded as
astringent, and used in the treatment of piles, diarrhea and gonorrhea [Anonymous (1989)].
Many Albizia species such as A. chinensis, A. amara, A. lebbeck, A. odoratissima, A.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
24
procera, A. stipulata, A. thomsonii, A. kalkora, A. lucida and A. orissensis are endemic to
Indian subcontinent.
Albizzia chinensis (Osbek) Merr., belonging to the family Leguminosae, is a large
deciduous tree with feathery foliage and large stipules. It occurs naturally in India,
Myanmar, Thailand, Indo-China, China, Java and the Lesser Sunda Islands (Bali and Nusa
Tenggara). It is a native of mixed deciduous forest in humid tropical and subtropical
monsoon climates with annual rainfall varying from 1000-5000 mm. In India, it occurs
chiefly in moist localities throughout the sub Himalayan tract and valleys up to an
elevation of about 1200 m from Himachal Pradesh eastwards, through Uttaranchal and also
in West Bengal, Assam, Andaman and Nicobar islands. It is excellent for restoration of
degraded lands, produces fuel and small timber, its leaves form excellent fodder for cattle,
and it also serves as the suitable host for Lac insect. The tree is extensively cultivated in tea
gardens for shade and improving the fertility of soil. It is also used for box making
especially tea boxes and heavy packing cases. The tree exudes a gum from the stem, which
is used sometimes for sizing hand made paper. Due to multifarious nature of this species, it
has been overexploited throughout the hills for fuel, fodder and timber requirements
[Dhanari et al. (2003)]. The anticancer and antioxidant activities of the plant were
attributed due to the presnebce of saponinas and flavonoids [Liu et al. (2009)].
1.2.2 Chemical constituents
Many important classes of compounds i.e. saponins, alkaloids and polyphenolic
compounds were reported from Albizzia species. Most of the biological activities of this
plant have been attributed to the presence of saponins, alkaloids and flavonoids. However,
only few saponins and flavonoids were identified from the less explored species i.e. A.
chinensis.
1.2.2.1 Saponins
Five oleanane-type triterpene saponins, albizosides A-E (59-63) were isolated from the
stem bark of A. chinensis [Liu et al. (2009a); Liu et al. (2010)]. The saponins of julibroside
type were isolated from stem bark of other Albizzia sps. and were identified as julibroside
J26 (64), julibroside J1 (65) [Zou et al. (1999)], julibroside J5 (66), julibroside J8 (67),
julibroside J12 (68), julibroside J13 (69) [Zou et al. (2005)], julibroside J28 (70), julibroside
III (71), julibroside J14 (72) [Liang et al. (2005)], julibroside J29 (73), julibroside J30 (74),
julibroside J31 (75) [Zheng et al. (2006)], julibroside II (76) julibroside J16 (77), julibroside
J21 (78) and julibroside J17 (79) [Zou et al. (2010)].
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
25
OOH
OH
R1
OO
OH
O
OR2
OHO
OH
O
OH
OH
OH
O
O
OH
O
O
OH
HO
OO
OHO
Me
OH
O
HO
OH
O
OH
OH
OH
HO
O
O
O
OH
OH
Me
O
O
H
OO
O
OHOR3
OH
Me
O
O
O
OHOH
OH
Me
OH
=S2
R1 R2 R3
Me H S2 (59)
Me Glc2 S2 (60)
H Glc2 H (61)
OOH
OH
R1
OO
OH
O
OR2
OHO
OH
O
OH
OH
OH
O
O
OH
O
O
OH
HO
OO
OHO
Me
OH
O
HO
OH
O
OH
OH
OH
HO
O
O
OH
OH
Me
O
O
H
OO
O
OHOR3
OH
Me
O
O
O
O
OHOH
OH
Me
OH
MT-Q
R1 R2 R3 Me Glc2 H (62)
H H MT-Q (63)
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
26
OOH
OH
OO
OH
O
OH
OHO
OH
O
OH
OH
R1
O
O
O
OH
O
O
OH
HO
OO
OHO
Me
OH
O
HO
OH
O
OH
OH
OH
HO
O
OR2
O
OH
OH
Me
OH
O
O
OH
O
OH
OH
Me
=S
R1 R2
H H (64)
OH S (65)
OOH
OH
Me
OO
OH
O
R
OH
O
OH
O
OH
OH
OH
O
O
O
OH
O
O
OH
HO
OO
OHO
Me
OH
O
HO
OH
O
OH
OH
OH
HO
O
O
O
OH
OH
Me
O
O
OH
O
OH
OH
Me
OH
6
R C(6)
OH (R) (66)
OH (S) (67)
NHAc (R) (68)
NHAc (S) (69)
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
27
OOH
OH
R1
OO
OH
O
R2
OH
O
OH
O
OH
OH
OH
O
O
O
OH
O
O
OH
HO
OO
OHO
Me
OH
O
HO
OH
O
OH
OH
OH
HO
O
O
O
OH
OH
Me
O
O
OH
O
OH
OH
Me
6
R1 R2 C(6) CH3 NHAc (R) (70)
CH3 NHAc (S) (71) H OH (R) (72)
.
OOH
OH
Me
OO
OH
O
R1
OH
O
OH
O
OH
OH
OH
O
O
O
OH
O
O
OH
HO
OO
OHO
Me
OH
O
HO
OH
O
OH
OH
OH
HO
O
OHOH
OH
R2
O
H
R3
R1 R2
R3
NHAc Me OH (73) NHAc H OH (74)
O-glc Me OH (75)
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
28
OH
OHO
HO OH
O
O
OH
HOO
H
H
R1
OHO
HOOH
OO
HOOH
O
O
R2
O
O
OOHO
HO OH
R3
O
O
O
OH
OHO
HO
O
O
OH
OO
HOHO
OH
OH
OH
OHO
HO
OH
6
R1 R
2 R
3 C(6)
Me Me Me (S) (76)
Me Me Me (R) (77)
H HO-CH2 H (R) (78)
OH
OHO
HO OH
O
O
OH
HOO
H
H
OHO
HOOH
OHOO
OH
O
O
O
O
OOHO
HO OH
O
O
O
OH
OHO
HO
O
O
OH
OO
HOHO
OH
OH
OH
OHO
HO
OH
OH
(79)
1.2.2.2 Polyphenols
Recently, eight compounds were isolated from 95% ethanol and methanolic extracts of A.
chinensis leaves and their structures were elucidated as quercetin (25), kaempferol (80),
luteolin (81), quercetin-di-glycoside (82), quercetin-3-O-�-L-rhamnopyranoside (83), rutin
(84), luteolin-7-O-�-D-glucopyranoside (85), kaempferol-3,7-di-O-�-D-glucopyranoside
(86), kaempferol-3-O-�-L-rhamnopyranoside (87), (+)-lyoniresinol-3�-O-�-D-
glucopyranoside (88), (-)-lyoniresinol-3�-O-�-D-glucopyranoside (89) [Liu et al. (2009b);
Ghaly et al. (2010)]. The polyphenols identified from other Albizzia sps. were named as
isoquercitrin (90), sulfuretin (91), hyperoside (92) [Kang et al. (2000); Jung et al. (2003)],
albibrissinosides A (93), and B (94) [Jung et al. (2004)].
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
29
O
O
HO
OH
OH
OH
O
O
HO
OH
OH
OH
(80) (81)
O
OH
HO
O
OH
O
O
O
OH
HO
HO
OHO OH
OH
O
O
OH
HO
OH
HO
O
OH
HO
OH
OH
O
O
O
OH
OH
OH
(82) (83)
O
OH
HO
OH
OH
O
O
O
OH
HO
HO
OHO OH
OH
O
O
OH
O
OH
O
O
HO
OH
HO
OH
OH
(84) (85)
O
OH
O
OH
O
O
OOHHO
HO
OH
O
HO
OH
HO
OH
O
OH
HO
OH
O
O
O
OH
OH
OH
(86) (87)
O
OMe
MeO
MeO
OH
OMe
OH
OGlcHO
O
OMe
MeO
MeO
OH
OMe
OH
OGlcHO
O
OH
HO
OH
O
O
OOHHO
HO
CH2OH
(88) (89) (90)
1.2.2.3 Alkaloids
The CH2Cl2 extract of A. gummifera stem bark yielded four alkaloids, budmunchiamine G
(95), budmunchiamine K (96), 6'�-hydroxybudmunchiamine K (97), and 9-
normethylbudmunchiamine K (98) [Rukunga and Waterman (1996a)] and other
budmunchiamines were characterized from stem bark of A. schimperana [budmunchiamine
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
30
A (99), 6'�-hydroxybudmunchiamine C (100), 5-normethylbudmunchiamine K (101), 6'�-
hydroxy-5-normethylbudmunchiamine K (102) 14-normethylbudmunchiamine K (9)]
[Rukunga and Waterman (1996b)]. The macrocyclic alkaloids, named as budmunchiamines
L4 (104), L5 (105) and L6 (106), were identified from methanolic extract of the seeds of A.
lebbek [Dixit and Misra (1997); Ovenden et al. (1997)].
Syringin (107) was also isoleted from A. chinensis leaves [Liu et al (2009)]
HO O
O
HO OH
O
OH
HO
OH
OH
O
O
O
HO
OHOH
OH
O
O
OH OH
O
OMe
HO
MeO
OHO
HOO
O
OH OMe
R1
R2
(91) (92) R1 R2
OMe OMe (93)
OH H (94)
N
N
N
N
R3 R1
OHR2
(CH2)5 (CH2)n
R
CH3
12
13
11
15
7
9 51
6'
n R R1 R2 R3
6 H H Me Me (95)
8 H Me Me Me (96) 8 OH Me Me Me (97)
8 H Me Me H (98)
4 H Me Me Me (99)
6 OH Me Me Me (100)
8 H H Me Me (101)
8 OH H Me Me (102)
8 H Me H Me (103)
NH HN
HNNH
O( )n
R
O
O
OO
OH
HO
HO
OHH
HO
n R (107) 5 CH2CH(OH)CH2CH2CH3 (104)
9 CH=CHCH2CH2CH3 (105) 7 CH=CHCH2CH2CH3 (106)
1.2.3 Pharmacology and biological activities
1.2.3.1 Cytotoxicity
Two oleanane-type triterpene saponins albizosides D (62) and E (63) from A. chinensis
were evaluated for cytotoxic activities against five human tumor cell lines i.e. HCT-8
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
31
(human colon cancer), Bel-7402 (human hepatoma cancer), BGC-823 (human gastric
cancer), A549 (human lung epithelial cancer), and A2780 (human ovarian cancer) using
the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
(paclitaxel as positive control) and these showed moderate cytotoxicity with IC50 values of
7.70 ± 0.15, 0.70 ± 0.08, 0.08 ± 0.02, 0.30 ± 0.07, 0.90 ± 0.05 �M for albizosides D and
>10, 0.60 ± 0.03, 0.30 ± 0.04, 1.20 ± 0.14, 0.30 ± 0.09 �M for albizosides E, respectively
[Liu et al. (2010)]. Three saponins, albizosides A-C (59-61) from A. chinensis exhibited
cytotoxic activity against above mentioned human tumor cell lines with IC50 value <6 �M
using camptothecin as positive control [Liu et al. (2009a)]. The saponins from A.
adianthifolia i.e., adianthifoliosides A, B and D were found to exhibit a cytotoxic effect on
human leukemia T-cells (Jurkat cells), whereas, the prosapogenins (Pro1 and Pro2) were
found to exert a lymphoproliferative effect on this cell type. These compounds were found
to exert a synergistic lymphoproliferative activity and induced apoptosis and a disrupted
mitochondrial membrane potential in Jurkat cells [Haddad et al. (2004)]. Julibroside J28
(70) displayed significant antitumor activity in vitro against PC-3M-1E8 (prostrate cancer),
Bel-7402, and HeLa cancer (cervical cancer) cell lines using by sulforhodamine B (SRB)
assay; the inhibitory rates were 80.47, 70.26, and 58.53%, respectively, at 10.0 �M dose
[Liang et al. (2005)]. The diastereoisomeric saponins julibroside J8 (67) and J13 (69) from
A. julibrissin showed marked cytotoxic activities against Bel-7402 cancer cell line with
86.66 and 93.33% inhibition respectively at 100 �g/ml concentration using SRB method
[Zou et al. (2005)]. Julibroside J29 (73), julibroside J30 (74), and julibroside J31 (75)
displayed significant in vitro antitumor activities against PC-3M-1E8, HeLa, and
MDAMB-435 (breast cancer) cancer cell lines at 10 �M concentration assayed by SRB and
MTT methods [Zheng et al. (2006b)]. Julibroside J8 (67) significantly inhibited growth in
BGC-823 (gastric cancer), Bel-7402, HeLa cell lines in vitro by MTT and SRB methods at
100 �g/ml dose; with typical apoptotic changes in morphology, nuclear damage and DNA
fragmentation of HeLa cells was observed. The treatment of HeLa cells with 20 �mol/L of
julibroside J8 (67) for 12 h and 24 h induced approximately 72% and 87% of HeLa cell
death [Zheng et al. (2006a)]. The saponins 3-O-[�-D-xylopyranosyl-(1�2)-�-L-
arabinopyranosyl-(1�6)-2-acetamido-2-deoxy-�-D-glucopyranosyl] echinocystic acid, 3-
O-[�-L-arabinopyranosyl-(1�2)-�-L-arabinopyranosyl-(1�6)-2-acetamido-2-deoxy-�-D-
glucopyranosyl] echinocystic acid exhibited cytotoxic effect with IC50 9.13 �g/ml and 10.0
�g/ml, respectively. The presence of the lactone ring in the triterpene and arabinose moiety
induced the inhibition of cytotoxic activity [Melek et al. 2007]. Julibroside II (76),
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
32
julibroside J16 (77), julibroside J21 (78) displayed good in vitro inhibitory activities against
Bel 7402 human cancer cell line at 100 �g/ml concentration with 91.9, 72.4 and 76.6%
cytotoxic activity [Zou et al. (2010)].
The potential cytotoxicity of the nine alkaloids [budmunchiamine G (95), budmunchiamine
K (96), 6'�-hydroxybudmunchiamine K (97), 9-normethylbudmunchiamine K (98),
budmunchiamine A (99), 6'�-hydroxybudmunchiamine C (100), 5-
normethylbudmunchiamine K (101), 6'�-hydroxy-5-normethylbudmunchiamine K (102),
14-normethylbudmunchiamine K (103)] were evaluated using the brine shrimp cytotoxicity
assay (BSCA). The fully N-methylated budmunchiamines, 96 and 99 without side chain
hydroxylation were strongly cytotoxic (<10 �g/ml dose). Two to three times higher
concentrations were required to obtain the same level of activity from the four alkaloids
(95, 96, 101, and 103) where one of the N-methyl groups was lost. The three 6'�-
hydroxylated compounds (97, 100, and 102) showed reduced toxicity by an order of
magnitude [Rukunga et al. (1996a)]. The toxicity of the n-hexane (HX), carbon
tetrachloride (CT), chloroform (CF) and aqueous soluble (AQ) fractions of the methanolic
extract of A. lebbek against brine shrimp was evaluated after 24 h (positive control,
vincristine sulphate, VS) and the LC50 were found to be 2.14, 2.15, 3.14, 6.99 and 0.30
�g/ml for HX, CT, CF, AQ and VS, respectively; n-hexane and carbon tetrachloride
soluble fractions being significantly toxic [Hussain et al. (2008)].
1.2.3.2 Antimicrobial activity
The antibacterial activity of kaempferol-3-O-�-L-rhamnopyranoside (87), quercetin-3-O-�-
L-rhamnopyranoside (83) and luteolin (81) were carried out against gram +ve bacteria,
Bacillus subtilis NRRL B-543 and Staphylococcus aureus NRRL B-313, as well as gram -
ve bacteria, Escherichia coli NRRL B-210 using nutrient agar medium. The results
revealed that these compounds exhibited moderate inhibiting activity against gram +ve and
gram -ve bacteria with zone of inhibition in the range of 16-17.5 mm [Ghaly et al. (2010)].
The nine budmunchiamines alkaloids were tested against gram +ve (B. subtilis, S. aureus)
and gram -ve (Escherichia coli, Pseudomonas aeruginosa) bacteria using the simple disk
diffusion assay. They exhibited zones of inhibition of at least 7 mm diameter against all the
bacteria at a loading of 50 �g/disk; yet found not active as the standard chloramphenicol
[Rukunga et al. (1996a)]. Antibacterial activities of ethyl acetate, ethanol and aqueous
extracts of A. adiantifolia bark and roots showed MIC against B. subtilis, E. coli, Klebsiella
pneumoniae, S. aureus and Micrococcus luteus in the range of 6.250-12.5 mg/ml [Eldeen
et al. (2005)]. Aqueous, ethanol and ethyl acetate extracts of A. gummifera bark showed
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
33
MIC against B. subtilis, E. coli, K. pneumoniae, M. luteus in the range of 0.39-12.5 mg/ml
[Buwa and van Staden (2006)].
n-Hexane and methanolic extracts of stem bark of A. procera showed >12 mm zone of
inhibition against B. subtilis, S. aureus, S. epidermidis, Enterococcus faecalis at
concentration of 5 mg/disc [Duraipandiyan et al. (2006)]. Antibacterial activities of A.
harveii bark against methicillin-sensitive S. aureus were observed at 2 mg/ml MIC
[Heyman et al. (2009)]. The methanol extracts of A. lebbek bark was screened for
antibacterial activity against eight strains of enteropathogenic bacteria, including multi-
drug resistant Vibrio cholerae (SG 24, NB2, PC4 and PC 65), Aeromonas hydrophila and
B. subtilis using broth microdilution method gave minimum inhibitory concentration
(MIC) (16-24 mg/ml) and minimum bactericidal concentration (MBC) (16-24 mg/ml)
[Acharyya et al. (2009)]. The crude hydro-alcoholic extract of stem and leaves exhibited
complete growth inhibition of N. gonorrhoeae strains at 500 �g/ml concentration.
Antimicrobial efficiency of crude leaf extracts of water, benzene and acetone of A. lebbeck
were tested against E. coli, S. aureus, K. pneumoniae, B.cereus, V. cholerae and Candida
albicans. The extracts displayed profound antimicrobial activity (>11 mm inhibition zone),
with 0.40-0.60 mg/ml and 0.50-0.80 mg/ml values of MIC and minimum bactericidal
concentration (MBC) [Maji et al. (2010)]. The carbon tetrachloride, chloroform and
aqueous fractions of methanolic extract of A. lebbek roots exhibited moderate inhibitory
activity against gram+ve bacteria (B. cereus, B. megaterium, B. subtilis, S. aureus, Sarcina
lutea) and gram-ve bacteria (E. coli, P. aeruginosa, Salmonella paratyphi, S. typhi,
Shigella boydii, S. dysenteriae, V. mimicus, V. Parahemolyticus) and fungal strains (C.
albicans, Aspergillus niger, Sacharomyces cerevacae) with the zone of inhibition of 9-17
mm, 9-18 mm and 9-16 mm, respectively at 400 �g/disc concentration. The crude
methanolic extract showed very weak inhibitory activity and the hexane partitionate
remained insensitive to the microorganisms [Hussain et al. (2008)]. The seed pods of A.
lebbeck showed antibactaerial activity with zone of inhibition 7 mm against S. aureus. The
in vitro antibacterial activities of 80% ethanolic and aqueous fractions from A. gummifera
seeds were tested for inhibitory activity against the clinical isolates of six Streptococcus
pneumonae and twenty two S. pyogenes using agar dilution method and it was found to
have antibacterial effects to all assayed bacteria while aqueous fractions did not exhibit any
effect. MIC of 80% ethanolic fractions was ranged from 500 to 1000 �g/ml depicting that
the extracts may contain bioactive compounds of therapeutic interest [Unasho et al.
(2009)].
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
34
1.2.3.3 Antioxidant activity
The antioxidant activity of methanol extract of A. julibrissin stem bark was evaluated for
its potential to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, authentic
peroxynitrites (ONOO-) and to inhibit the generation of the hydroxyl radical (.OH), total
reactive oxygen species (ROS). The antioxidant activity of its further fractions were
observed in the order of EtOAc>n-BuOH>CH2CI2> and H2O Sulfuretin (91) from EtOAc
fraction exhibited good activity in all tested model systems and exhibited five times more
inhibitory activity on the total ROS than Trolox [Jung et al. (2003)]. The methanol extract
of A. chevalieri leaves showed promising DPPH scavenging activity of 59.58, 68.48,
77.24, 85.93 and 94.73% of at 10, 25, 50, 125 and 250 �g/ml concentrations, respectively.
No significant difference (p<0.05) in the antioxidant activity was found between the extract
and standards (ascorbic and gallic acids) at 50, 125 and 250 �g/ml concentrations. The
reducing power of the extract (0.113 ± 0.056 nm) was found to be higher than the gallic
acid standard (0.096 ± 0.035 nm) [Aliyu et al. (2009)].
1.2.3.4 Antimalarial activity
Crude MeOH extract from A. adinocephala stem bark and leaves inhibited the activity of
malarial enzyme plasmepsin II which has profound effect on Plasmodium falciparum
parasite multiplication in vitro. Budmunchiamines L4 (104) and L5 (105) displayed mild
activity against plasmepsin II, with IC50 values of 14 and 15 mM, respectively [Ovenden et
al. (2002)]. The fractions of methanolic extract of A. gummifera showed low activity
against chloroquine sensitive (NF54) and resistant (ENT30) strains of P. falciparum with
IC50 above 3 �g/ml. The alkaloidal fraction exhibited strong activity against NF54 and
ENT30 with IC50 of 0.16 ± 0.05 and 0.99 ± 0.06 �g/ml, respectively. Five spermine
alkaloids [budmunchiamine K (96), 6'�-hydroxybudmunchiamine K (97), 5-
normethylbudmunchiamine K (101), 6'�-hydroxy-5-normethylbudmunchiamine K (102), 9-
normethylbudmunchiamine K (98)] exhibited activities against NF54 and ENT30 strains
with IC50 ranging from 0.09 ± 0.02 to 0.91 ± 0.10 �g/ml. The alkaloids showed percentage
chemosuppression of parasitaemia in mice ranging from 43 to 72% [Rukunga et al.
(2007)]. The methanolic extract of A. zygia stem bark exhibited antiprotozoal activity
against P. falciparum K1 strain with IC50 values of 1.0 �g/ml [Lenta et al. (2007)]. The
larval toxicity and smoke repellent potential of A. amara was evaluated at different
concentrations (2, 4, 6, 8 and 10%) against the different instar (I, II, III and IV) larvae and
pupae of Aedes aegypti (dengue vector). The (Lethal concentration) LC50 values of 5.41,
6.48, 7.11 and 7.52% were observed for I, II, III and IV instar larvae respectively, while,
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
35
the LC50 and LC90 values of 6.79% and 16.93% were found for pupae [Murugan et al.
(2007)].
1.2.3.5 Anti-inflammatory activity
The chloroform extract of A. chinensis bark at 200 mg/kg and 400 mg/kg dose levels
significantly decreased the carrageenan induced paw oedema paw volume in rats (P<0.001)
as compared to control (carboxy methyl cellulose) and positive control (diclofenac sodium)
at the end of 3 h [Perumal et al. (2010)]. A botanical formulation, Aller-7/NR-A2, was
developed for the treatment of allergic rhinitis which is a combination of medicinal plant
extracts from Phyllanthus emblica, Terminalia chebula, T. bellerica, A. lebbeck, Piper
nigrum, Zingiber officinale and P. longum. This formulation demonstrated potent
antihistaminic, anti-inflammatory, antispasmodic, and mast-cell-stabilization activities
[Amit et al. (2003)]. In carrageenan induced rat paw edema model, the equal mixture of
petroleum ether, ethyl acetate and methanol cold extracts at the 200 mg/kg and 400 mg/kg
dose level showed 36.68% and 27.51% inhibition of oedema volume at the end of 4 h
[Saha et al. (2009)]
1.2.3.6 Other activities
The chloroform extract of A. chinensis bark at 200 and 400 mg/kg dose levels showed
significantly reduction in the ulcer index and increase in the ulcer protective effect as
compared to control and positive control [Perumal et al. (2010)]. Albizosides A-C (59-91),
from A. chinensis exhibited hemolytic activity against rabbit erythrocytes [Liu et al.
(2009)]. The sedative activity of flavonol glycosides, quercitrin (83) and isoquercitrin (90)
was evaluated and both compounds increased pentobarbital-induced sleeping time in dose-
dependent manner when administered intraperitoneally (i.p.) to mice. These compounds
were found devoid of any lethal effect in mice and no apparent behavioural change was
observed [Kang et al. (2000)].
However, numbers of reports are available on chemical investigations and biological
activities of different Albizzia species, yet, only few reports are available on chemical
investigations of A. chinensis growing in India. Thus, the present study has been envisaged
to systematically investigate A. chinensis growing in Himachal Pradesh, India for isolation
and characterization of bioactive molecules and development of new analytical methods for
quality assessment. The following pages describe the chemical investigation and biological
activity of A. chinensis performed in the present work. The results and discussion is
followed by experimental section and references are included at the end of the chapter.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
36
1.2.4 Results and discussion
1.2.4.1 Phytochemical studies
For the isolation of bioactive constituents from flowers of A. chinensis, the dried powdered
plant material was extracted with 90% ethanol using percolation. The crude extract was
fractionated with n-hexane, ethyl acetate, n-butanol and water. Different fractions obtained
were chromatographed for the isolation of bioactive constituents. Repeated column
chromatography of ethyl acetate and n-butanol fractions led to the isolation of nine
flavonoids, namely, quercetin (25), kaempferol (80), quercetin-3-O-�-L-rhamnopyranoside
(quercitrin) (83), rutin (84), quercetin-3-O-�-L-galactopyranoside (92), quercetin-3-O-�-L-
arabinofuranoside (108), kaempferol-3-O-�-L-arabinofuranoside (109), myricetin (45) and
myricetin-3-O-�-L-rhamnopyranoside (myricitrin) (110).
The methanolic extract of bark on repeated column chromatography (normal and reverse
phase) led to isolation and characterization of five constituents, namely, catechin (111),
ferulic acid (112), caffeic acid (113) and �-sitosterol (48).
1.2.4.1.1 Quercitrin (83)
36
8
4a
3'
4'
1''
2''
6''
O
OH
HO
OH
OH
O
O
O
OH
OH
OH
Figure 1.2.1: Chemical structure of 83
Compound 83 was isolated as a yellow amorphous powder. The positive HRESI-QTOF-
MS showed a molecular ion peak at m/z 449.3824 [M+H]+ (calcd. 449.3848) indicated the
molecular formula as C21H21O11. UV spectrum showed absorption maxima at 255 and 350
nm, characteristic of flavonols. 1H NMR spectrum showed five aromatic signals at � 7.26
(1H, br s), 7.22 (1H, d, J = 8.7), 6.83 (1H, d, J = 8.1 Hz), 6.26 (1H, br s) and 6.10 (1H, br
s) assignable to H-2', H-6', H-5', H-8 and H-6 protons respectively (Table 1.2.1). 13
C NMR
and HMBC spectra displayed seven oxygenated carbons at � 164.3, 161.7, 157.9, 157.0,
148.3, 144.9, 134.8 and assignable to C-3, C-2, C-8a, C-3', C-4', C-5 and C-7 respectively.
The signal at � 178.2 assigned to C-4 further suggested the presence of flavonol type of
skeleton.
The sugar was identified as �-L-rhamnopyranosyl on the basis of NMR spectral signals at �
5.27, 4.16 3.68, 3.24-3.35, and 0.87 which are characteristic for �-L-rhamnopyranosyl
sugar. The attachment of sugar at C-3 position was deduced on the basis of above spectral
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
37
data and comparison with previously known spectral values. Thus, on the basis of above
spectral data and comparison with previously known spectral values [Manguro et al.
(2004); Yoshioka et al. (2004)], the structure of compound 83 was assigned as quercetin-3-
O-�-L-rhamnopyranoside (Figure 1.2.1).
Table 1.2.1: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 83 in CD3OD
Position �C �H m (J Hz) Position �C �H m (J Hz)
1 - - 2' 115.0 7.26 br s
2 157.9 - 3' 144.9 -
3 134.8 - 4' 148.3 -
4 178.2 - 5' 115.6 6.83 d (8.1)
5 161.7 - 6' 121.5 7.22 d (8.7)
6 98.4 6.10 br s 1'' 102.1 5.27 br s
7 164.3 - 2'' 70.7 3.68 m
8 93.3 6.26 br s 3'' 70.6 3.24-3.35 m
8a 157.0 - 4'' 71.9 3.24-3.35 m
4a 104.5 - 5'' 70.5 4.16 s
1' 121.5 - 6'' 16.3 0.87 d (5.4)
1.2.4.1.2 Quercetin (25)
O
OH
HO
OH
O
OH
OH
Figure 1.2.2: Chemical structure of 25
Compound 25 was isolated as a yellow amorphous powder. Its positive HRESI-QTOF-MS
showed a molecular ion peak at m/z 303.0518 [M+H]+ (calcd. 303.2436) corresponding to
molecular formula C15H11O7. In UV spectrum, the absorption maxima were observed at
256, 372 nm. The UV absorptions were consistent with the presence of a 3,5,7,3',4'-
pentahydroxyflavone structure. 1H and
13C NMR spectra exhibited resonances due to
aromatic systems. In 1H NMR spectrum, the aromatic region showed signals at � 8.66 (1H,
s), 8.14 (1H, d, J = 8.1 Hz), 7.43 (1H, d, J = 8.1 Hz), and 6.43 (1H, br s) and 6.39 (1H, br
s) assignable to H-2', H-6', H-5', H-6 and H-8 protons (Table 1.2.2).
13C and HMBC NMR spectra showed the presence of fifteen aromatic carbon signals
corresponding to ten quaternary and five methine carbons. 13
C NMR signals were assigned
with the help of an HMQC data and it included one flavone carbonyl at � 178.1 (C-4) and
seven oxygen bearing quaternary carbons at � 166.3 (C-7), 163.2 (C-5), 158.3 (C-8a),
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
38
148.5 (C-4'), 147.9 (C-2), 147.9 (C-3') and 138.7 (C-3). Five signals at � 121.9, 117.5,
117.5, 100.0 and 95.1 were assigned to C-6', C-5', C-2', C-6 and C-8 respectively. Thus, on
the basis of above spectral data and comparison with previously known spectral values
[Xiao et al. (2006)], the structure of compound 25 was assigned as quercetin (Figure 1.2.2).
Table 1.2.2: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 25 in C5D5N
Position �C �H m (J Hz) Position �C �H m (J Hz)
1 - - 8a 158.3 -
2 147.9 - 4a 105.3 -
3 138.7 - 1' 123.7 -
4 178.1 - 2' 117.5 8.66 br s
5 163.2 - 3' 147.9 -
6 100.0 6.43 br s 4' 148.5 -
7 166.3 - 5' 117.5 7.43 d (8.1)
8 95.1 6.39 br s 6' 121.9 8.14 d (8.1)
1.2.4.1.3 Rutin (84)
O
OH
HO
OH
OH
O
O
O
OH
HO
HO
OHO OH
OH
O
4
8
4a
8a
3'
4'
1'''6'''1'' 6''
Figure 1.2.3: Chemical structure of 84
Compound 84 was isolated as a yellow amorphous powder. Its positive HRESI-QTOF-MS
showed a molecular ion peak at m/z 611.5237 [M+H]+ (calcd. 611.5254) corresponding to
the molecular formula C27H31O16. The UV spectrum, absorption maxima were observed at
257, 353 nm. In 1H NMR (Table 1.2.3), the aromatic protons exhibited one ABX coupling
system at � 7.68 (1H, d, J = 2.1 Hz, H-2'), 7.64 (1H, dd, J = 2.1, 8.4 Hz, H-6') and 6.89
(1H, d, J = 8.4 Hz, H-5'). The other AX coupling system at � 6.40 (1H, br s) and 6.22 (1H,
br s) was assigned to H-8 and H-6 protons, respectively. 1H NMR spectrum supported the
presence of one rhamnose and one glucose moieties with the � values are similar to that of
rhamnose and glucose proton signals. 13C NMR spectrum displayed twenty seven carbon
signals including fifteen carbon signals due to the flavonol skeleton. By comparison with
the 13
C NMR spectral data of quercetin (25), we found that C-3 (� 135.7) was upfield
shifted, demonstrating glycosylation at C-3 position. The signals due to one flavonol
carbonyl at � 179.5 (C-4) and seven oxygen bearing quaternary carbons at � 166.1 (C-7),
163.0 (C-5), 158.6 (C-8a), 149.8 (C-4'), 159.4 (C-2), 145.9 (C-3') and 135.7 (C-3) were
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
39
observed. Five signals at � 123.6, 117.2, 116.1, 100.0 and 94.9 were assigned to C-6', C-2',
C-5', C-6 and C-8 respectively. The HMBC spectrum was utilized to identify the linkage
between the aglycone and sugar units. Accordingly, the structure of compound 84 was
established as rutin (Figure 1.2.3) on the basis of above spectral data and comparison with
previous reports [Wu et al. (2007)]
Table 1.2.3: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 84 in CD3OD
Position �C �H m (J Hz) Position �C �H m (J Hz)
1 - - 5' 116.1 6.89 d (8.4)
2 159.4 - 6' 123.6 7.64 dd (8.4, 2.1)
3 135.7 - 1'' 104.8 5.12 d (7.4)
4 179.5 - 2'' 75.8 3.43-3.49 m
5 163.0 - 3'' 78.2 3.43-3.49 m
6 100.0 6.22 br s 4'' 71.5 3.25-3.33 m
7 166.1 - 5'' 77.3 3.25-3.33 m
8 94.9 6.40 br s 6'' 68.6 3.82 d (10.4);
3.36-3.38 m
8a 158.6 - 1''' 102.5 4.53 br s
4a 105.7 - 2''' 72.1 3.64-3.66 m
1' 123.2 - 3''' 72.3 3.52-3.57 m
2' 117.2 7.68 d (2.1) 4''' 74.0 3.25-3.33m
3' 145.9 - 5''' 69.8 3.25-3.33 m
4' 149.8 - 6''' 17.9 1.14 d (6.2)
1.2.4.1.4 Quercetin-3-O-�-L-arabinofuranoside (108)
HO
OH
O
O
OH
O
O H
OH
H
H
CH2OH
H
OH
1
3
5
7 8a
4a
3'4'
1''
3''
5''
OH
Figure 1.2.4: Chemical structure of 108
Compound 108 was isolated as a yellow amorphous powder. The negative ESI-QTOF-MS
showed a molecular ion peak at m/z 435 [M+H]+ indicating the molecular formula as
C20H19O11. UV spectrum showed absorption maxima at 255 and 356 nm characteristic of
flavonols. 1H NMR spectrum showed aromatic signals at � 7.45 (1H, br s), 7.41 (1H, br s),
6.84 (1H, d, J = 8.1 Hz), 6.33 (1H, br s) and 6.14 (1H, br s) assignable to H-6', H-2', H-5',
H-8 and H-6 respectively (Table 1.2.4). 13
C and HBMC NMR spectra displayed showed
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
40
seven oxygenated carbons at � 164.7, 161.7, 157.9, 148.4, 157.2, 144.9, 133.5, and
assignable to C-7, C-5, C-3, C-2, C-8a, C-3', and C-4' carbons respectively. The signal at �
178.6 was assigned to C-4 that further suggested the presence of flavonol type of skeleton.
The presence of five signals for sugar included a downfield anomeric signal at � 108.1 (C-
1'') and the sugar was identified as �-L-arbinofuranosyl on the basis of NMR spectral
values at � 5.40, 4.26, 3.83, 3.78-3.80, 3.42-3.43 that are characteristic for �-L-
arbinofuranosyl sugar. The attachment of sugar at C-3 position was deduced on the basis of
above spectral data and comparison with previously known spectral values [Shen et al.
(2009)], compound 108 was assigned as quercetin-3-O-�-L-arabinofuranoside (Figure
1.2.4).
Table 1.2.4: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 108 in CD3OD
Position �C �H m (J Hz) Position �C �H m (J Hz)
1 - - 2' 115.0 7.41 br s
2 157.9 - 3' 144.9 -
3 133.5 - 4' 148.4 -
4 178.6 - 5' 115.4 6.84 d (8.1)
5 161.7 - 6' 121.5 7.45 br s
6 98.5 6.14 br s 1'' 108.1 5.40 br s
7 164.7 - 2'' 81.9 4.26 br s
8 93.4 6.33 br s 3'' 77.3 3.83 m
8a 157.2 - 4'' 86.6 3.78-3.80 m
14a 105.0 - 5'' 61.1 3.42-3.43 m
1' 121.5 -
1.2.4.1.5 Quercetin-3-O-�-L-galactopyranoside (92)
O
OH
HO
OH
OH
O
O
O
HO
OHOH
OH
1
3
5
8
3'
4'
1''
3''
6''
Figure 1.2.5: Chemical structure of 92
Compound 92 was isolated as a yellow amorphous powder. The positive ESI-QTOF-MS
showed a molecular ion peak at m/z 465 [M+H]+ indicated the molecular formula as
C21H21O12. UV spectrum showed absorption maxima at 256 and 354 nm characteristic of
flavonols. 1H NMR spectrum showed five aromatic signals at � 8.45 (1H, br s), 8.12 (1H,
d, J = 8.4), 7.26 (1H, d, J = 8.1 Hz), 6.69 (1H, br s) and 6.63 (1H, br s) and these were
assignable to H-2', H-6', H-5', H-8 and H-6 protons respectively (Table 1.2.5). 13
C and
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
41
HMBC NMR spectra displayed seven oxygenated carbons at � 166.3, 163.0, 158.2, 157.9,
151.1, 147.1, and 135.5 assignable to C-7, C-5, C-2 C-8a, C-4', C-3' and C-3 carbons
respectively. The signal at � 179.2 assigned to C-4 further suggested the presence of
flavonol type of skeleton. The presence of five signals for sugar at � 6.07, 4.78-4.84, 4.60,
4.27-4.43, 4.15-4.18 including a anomeric signal at � 105.5 (C-1'') are characteristic for �-
L-galactopyranosyl. The attachment of sugar at C-3 position was deduced on the basis of
above spectral data and comparison with previously known spectral values [Xiao et al.
(2006); He et al. (2010)], the compound 92 was assigned as quercetin-3-O-�-L-
galactopyranoside (Figure 1.2.5).
Table 1.2.5: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 92 in C5D5N
1.2.4.1.6 Kaempferol-3-O-�-L-arabinofuranoside (109)
HO
OH
O
O
OH
O
O H
OH
H
H
CH2OH
H
OH
1
36
8 4'
1''
3''
5''
Figure 1.2.6: Chemical structure of 109
Compound 109 was isolated as a yellow amorphous powder. The negative ESI-QTOF-MS
showed a molecular ion peak at m/z 419 [M+H]+ indicated the molecular formula as
C20H19O10. UV spectrum showed absorption maxima at 265 and 356 nm characteristic of
flavonols. 1H NMR spectrum revealed two sets of meta-coupled broad singlets at � 6.33
(1H, br s) and 6.14 (1H, br s) assigning to H-8 and H-6 protons respectively. The presence
of a set of A2B2 doublets at � 7.89 (1H d, J = 8.4 Hz) and 6.85 (2H, J = 8.4 Hz) each
Position �C �H m (J Hz) Position �C �H m (J Hz)
1 - - 2' 116.6 8.45 br s
2 158.2 - 3' 147.1 -
3 135.5 - 4' 151.1 -
4 179.2 - 5' 118.2 7.26 d (8.5)
5 163.0 - 6' 123.1 8.12 d (8.4)
6 100.1 6.63 s 1'' 105.5 6.07 d (8.0)
7 166.3 - 2'' 73.7 4.78-4.84 m
8 94.9 6.69 s 3'' 75.8 4.60 m
8a 157.9 - 4'' 70.1 4.15-4.18 m
4a 105.8 - 5'' 78.0 4.78-4.84 m
1' 122.6 - 6'' 62.3 4.27-4.43 m
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
42
integrating for two protons have been assigned to H-2', H-6' and H-3', H-5' respectively
(Table 1.2.6). 13C and HMBC NMR spectra showed six oxygenated carbons at � 164.6,
161.7, 136.6, 160.1, 158.0, and 157.2 assignable to C-7, C-5, C-4', C-8a, C-2, and C-3,
respectively. The signal at � 178.5 assigned to C-4 further suggested the presence of
flavonol type of skeleton. The presence of five signals for sugar (� 5.41, 4.25, 3.83, 3.71-
3.73, 3.40-3.41) including a downfield anomeric signal at � 108.2 (C-1''), clearly suggested
pentose sugar in furanose form. The sugar was identified as �-L-arbinofuranosyl on the
basis of NMR spectral data. The attachment of sugar at C-3 position was deduced on the
basis of above spectral data and comparison with previously known spectral values [Xiao
et al. (2006)], compound 109 was assigned as kaempferol 3-O-�-L-arabinofuranoside
(Figure 1.2.6).
Table 1.2.6: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 109 in CD3OD
Position �C �H m (J Hz) Position �C �H m (J Hz)
1 - - 2' 130.6 7.89 d (8.4)
2 157.2 - 3' 115.1 6.85 d (8.4)
3 136.6 - 4' 160.1 -
4 178.5 - 5' 115.1 6.85 d (8.4)
5 161.7 - 6' 130.6 7.89 d (8.4)
6 98.5 6.14 br s 1'' 108.2 5.41 br s
7 164.6 - 2'' 81.9 4.25 br s
8 93.4 6.33 br s 3'' 77.2 3.83 m
8a 158.0 - 4'' 86.6 3.71-3.73 m
4a 104.0 - 5'' 61.1 3.40-3.41 m
1' 121.4 -
1.2.4.1.7 Myricetin (45)
O
OH
HO
OH
OH
O
OH5
4a
8a
3'
4'
OH
Figure 1.2.7: Chemical structure of 45
Compound 45 was isolated as a yellow amorphous powder. Its positive ESI-MS showed a
molecular ion peak at m/z 319.2411 [M+H]+ (calcd. 319.2430) corresponding to the
molecular formula C15H11O8. In UV spectrum, absorption maxima were observed at 251,
371 nm. 1H and 13C NMR spectra displayed resonances due to aromatic systems. 13C NMR
signals were assigned with the help of HMQC and HMBC data. In 1H NMR spectrum, the
aromatic region showed signals at � 7.35 (2H, br s), 6.38 (1H, br s) and 6.20 (1H, br s)
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
43
assignable to H-2', H-6', H-8 and H-6 protons (Table 1.2.7). 13
C NMR spectrum showed
the presence of fifteen aromatic carbon signals corresponding to eleven quaternary and four
methine carbons; including one flavones carbonyl at � 178.0 (C-4) and eight oxygen
bearing quaternary carbons at � 164.3 (C-7), 161.6 (C-5), 157.2 (C-8a), 145.5 (C-3', 5'),
136.4 (C-4'), 157.2 (C-2), 136.0 (C-3). Four signals at � 107.7, 107.7, 98.4 and 93.7 were
assigned to C-2', C-6', C-6 and C-8 carbons respectively. Based on the above spectral data
and comparison of the data with the literature [He et al. (2009)], the structure of compound
45 was identified as myricetin (Figure 1.2.7).
Table 1.2.7: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 45 in CD3OD Position �C �H m (J Hz) Position �C �H m (J Hz)
1 - - 8a 157.2 -
2 157.2 - 4a 104.5 -
3 136.0 - 1' 122.2 -
4 178.0 - 2' 107.7 7.35 br s
5 161.6 - 3' 145.5 -
6 98.4 6.20 br s 4' 136.4 -
7 164.3 - 5' 145.5 -
8 93.7 6.38 br s 6' 107.7 7.35 br s
1.2.4.1.8 Myricetin-3-O-�-L-rhamnopyranoside (110)
36
8
4a
3'
4'
1''
2''
6''
O
OH
HO
OH
OH
O
O
O
OH
OH
OH
OH
Figure 1.2.8: Chemical structure of 109
Compound 110 was isolated as a yellow amorphous powder. The positive ESI-MS showed
a molecular ion peak at m/z 465 [M+H]+ indicated the molecular formula as C21H21O12. UV
spectrum showed absorption maxima at 258 and 352 nm characteristic of flavonols. 1H
NMR spectrum showed four aromatic signals at � 6.89 (2H, br s), 6.26 (1H, br s), and 6.10
(1H, br s) assignable to H-2', H-6', H-8 and H-6 protons respectively (Table 1.2.8). The
detailed analyses of 13
C and HMBC NMR spectra indicated the presence of eight
oxygenated quaternary carbons at � 164.3, 161.6, 158.0, 157.0, 145.0, 145.0, 136.4, and
134.9 assignable to C-7 C-5 C-2, C-8a, C-3', C-5', C-4', and C-3 carbons respectively. The
signal at � 178.2 assigned to C-4 further suggested the presence of flavonol type of
skeleton. The presence of six signals (� 5.25, 4.19, 3.75-3.77, 3.43-3.57) including a signal
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
44
at � 102.1 (C-1''), suggesting the sugar in pyranose form. The sugar was identified as �-L-
rhamnopyranosyl on the basis of NMR spectral data. The attachment of sugar at C-3
position was deduced on the basis of above spectral data and comparison with previously
known spectral values [Adebayo et al. (2011)], the compound 110 was assigned as
myricetin-3-O-�-L-rhamnopyranoside or myricitrin (Figure 1.2.8).
Table 1.2.8: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 110 in CD3OD
Position �C �H m (J Hz) Position �C �H m (J Hz)
1 - - 2' 107.9 6.89 br s
2 158.0 - 3' 145.0 -
3 134.9 - 4' 136.4 -
4 178.2 - 5' 145.0 -
5 161.6 - 6' 108.3 6.89 br s
6 98.5 6.10 br s 1'' 102.1 5.25 br s
7 164.3 - 2'' 70.7 3.75-3.77 m
8 93.3 6.26 br s 3'' 70.5 3.43-3.57 m
8a 157.0 - 4'' 71.9 3.43-3.57 m
4a 104.5 - 5'' 70.1 4.19 s
1' 121.6 - 6'' 16.3 0.90 d (6.0)
1.2.4.1.9 Catechin (111)
O
OH
HO
OH
OH
OH
4
2
6
8
2'
5'6'
Figure 1.2.9: Chemical structure of 111
Compound 111 was isolated as a white amorphous powder. It showed a molecular ion peak
at m/z 291.2736 [M+H]+ (calcd. 291.2760) in its positive ion HR-ESI-QTOF-MS spectrum,
in accordance with the formula C15H15O6. UV spectra showed �max at 220 and 280 nm. 1H
NMR spectra indicated five aromatic protons at � 6.85 (1H, br s, H-2'), 6.75 (2H, d, J = 8.8
Hz, H-5', H-6'), 5.94 (1H, br s, H-8) and 5.87 (1H, br s, H-6). Four aliphatic protons were
found at � 4.56-4.59 (1H, m, H-2), 3.97-4.00 (1H, m, H-3), 2.82-2.89 (1H, m, H-4) and
2.47-2.55 (1H, m, H-4) (Table 1.2.9). In 13C and HMBC NMR spectra, fifteen carbon
signals including seven oxygen bearing carbons at � 157.9, 157.7, 157.0, 146.4, 146.4,
83.0, and 68.9 due to C-5, C-7, C-8a, C-3', C-4', C-3, C-2 carbons respectively and five
aromatic methine carbons showed peaks at � 120.2, 116.2, 115.4, 96.5, 95.7 assignable to
C-6', C-5', C-2', C-6 and C-8 carbons respectively. Based on the NMR, mass, optical
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
45
rotation data and previous literature [Hye et al. (2009); Cren-Olive et al. (2002)], the
structure of compound 111 was identified as catechin (Figure 1.2.9).
Table 1.2.9: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 111 in CD3OD Position �C �H m (J Hz) Position �C �H m (J Hz)
1 - - 8a 157.0 -
2 83.0 4.56-4.59 m 4a 101.0 -
3 68.9 3.97-4.00 m 1' 132.4 -
4 28.6 2.82-2.89 m; 2.47-2.55 m 2' 115.4 6.85 br s
5 157.9 - 3' 146.4 -
6 96.5 5.87 br s 4' 146.4 -
7 157.7 - 5' 116.2 6.75 d (8.8)
8 95.7 5.94 br s 6' 120.2 6.75 d (8.8)
1.2.4.1.10 Ferulic acid (112)
HO
H3COOH
O
3
5
7
8
Figure 1.2.10: Chemical structure of 112
Table 1.2.10: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 112 in CD3OD Position �C �H m (J Hz)
9 171.0 -
8 116.0 6.31 d (15.8)
7 146.9 7.60 d (15.8)
1 127.9 -
6 124.0 7.06 d (8.1)
5 116.5 6.81 d (8.1)
4 149.4 -
3 150.5 -
2 111.8 7.17 s
3-OCH3 56.3 3.89 s
Compound 112 was isolated as a white amorphous powder. Its positive ESI-QTOF-MS
showed a molecular ion peak at m/z 195.1937 [M+H]+ (calcd. 195.1919) corresponding to
the molecular formula C10H11O4. 1H and
13C NMR spectra exhibited resonances due to
aromatic system. 13
C NMR signals were assigned with the help of an HMQC and HMBC
data. In 1H NMR spectrum, the aromatic region showed signals at � 7.60 (1H, d, J = 15.8
Hz,), 7.17 (s, 1H), 7.06 (1H, d, J =8.1 Hz,), 6.81 (1H, d, J =8.1 Hz,), 6.31 (1H, d, J = 15.8
Hz,) assignable to H-7, H-2, H-6, H-5 and H-8 protons (Table 1.2.10). 13C NMR spectrum
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
46
showed the presence of ten carbon signals including six aromatic carbons at � 150.5 (C-3),
149.4 (C-4), 124.0 (C-6), 116.5 (C-5), 111.8 (C-2) and one carboxylic carbon at � 171.0
(C-9). Three signals of two methines at � 146.9, 116.0 and one methoxy carbon at � 56.3,
were assigned to C-7, C-8 and C3-OCH3 carbons respectively. Based on the above spectral
data and comparison of the data given in the literature [Bunzel et al. (2005); Yoshioka et
al. (2004)], the structure of compound 112 was identified as ferulic acid (Figure 1.2.10).
1.2.4.1.11 Caffeic acid (113)
HO
HOOH
O
1
4
39
7
Figure 1.2.11: Chemical structure of 113
Table 1.2.11: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 113 in CD3OD
Position �C �H m (J Hz)
1 127.9 -
2 115.3 7.04 s
3 149.5 -
4 146.4 -
5 116.6 6.78 d (8.2)
6 123.0 6.93 d (8.2)
7 147.2 7.54 d (15.9)
8 115.6 6.23 d (15.9)
9 171.2 -
Compound 113 was isolated as a white amorphous powder. Its positive ESI-MS showed a
molecular ion peak at m/z 181.1626 [M+H]+ (calcd. 181.1654) corresponding to the
molecular formula C9H9O4. 1H NMR spectrum exhibited signals for three aromatic protons
in a 2,5,6 substitution pattern at � 7.04 (s, 1H, H-2), 6.93 (d, 1H, J = 8.2 Hz, H-6), 6.78 (d,
1H, J = 8.2 Hz, H-5) The two protons of a trans-double bond at � 7.54 (1H, d, J = 15.9 Hz)
and 6.23 (1H, d, J = 15.9 Hz) indicated the presence of a 3,4-dihydroxy-trans-cinnamate
moiety (Table 1.2.11). The 13
C and HBMC NMR spectra indicated the presence of
resonances attributable to a carbonyl group at � 171.2 (C-9), two deshielded oxygen
bearing quaternary carbons at � 149.5 (C-3), 146.4 (C-4), three aromatic methines at �
123.0 (C-6), 116.6 (C-5) and 115.3 (C-2) and two trans doubly bonded carbons at � 147.2
(C-7), 115.6 (C-8). The structure of compound 113 was identified as caffeic acid (Figure
1.2.12) on comparison of the above spectral data and literature values [He et al. (2009);
Hoeneisen et al. (2003)].
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
47
1.2.4.1.12 �-Sitosterol (48)
HO
1
4 6
19
12
14
17
18
2122
24
29
26
27
Figure 1.2.12: Chemical structure of 25
Table 1.2.12: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 113 in CDCl3
Position �C �H m (J Hz) Position �C �H m (J Hz)
1 37.2 1.05-1.08 m 16 28.4 1.75 m
2 31.8 1.40-1.45 m 17 56.2 1.92 m
3 71.9 3.41-3.46 m 18 12.0 0.62 s
4 38.8 2.19 m 19 19.5 0.83-0.95 m
5 140.9 - 20 36.3 2.10 m
6 121.8 5.28 br s 21 18.9 0.83-0.95 m
7 32.0 2.10 m 22 34.2 1.63 m
8 32.0 1.40-1.45 m 23 26.5 1.63 m
9 50.3 0.83-0.95 m 24 46.0 1.40-1.45 m
10 36.9 - 25 29.3 1.40-1.45 m
11 21.2 1.40-1.45 m 26 19.9 0.71-0.75 m
12 39.9 1.92 m 27 19.2 0.71-0.75 m
13 42.4 - 28 23.2 1.40-1.45 m
14 56.9 1.05-1.18 m 29 12.1 0.71-0.75 m
15 24.4 1.40-1.45 m
Compound 48 was isolated as colorless needles. The 1H NMR showed the proton of H3
appeared as a multiplet at � 3.41-3.46 (m, 1H) and revealed the existence of signals for
olefinic proton at � 5.28 (1H, br s) (Table 1.2.12). Angular methyl proton at � 0.62 (s),
0.83-0.85 (m) correspond to H-18 and H-19 protons respectively. 13C NMR displayed
signals at � 140.9 and 121.8, which are assigned C-5 and C-6 double bonded carbons
respectively. The signal at � 19.5 was assignable to angular carbon C-19. The spectra
depicted twenty nine carbon signal including six methyls, eleven methylenes, nine methine
and three quaternary carbons. On the basis of NMR, mass, optical rotation data and
comparison with previously known spectral values [Saxena and Albert (2005); Kamboj and
Saluja (2011)], compound 48 was assigned as �-sitosterol (Figure 1.2.12).
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
48
1.2.4.2 Identification of phenolic compounds by UPLC-DAD-ESI-QTOF-MS in A.
chinensis
Out of varios moblile phase compositions, water (0.05% formic acid) (A) and methanol (B)
was chosen as the best mobile phase based on the chromatographic peak shape. Increasing
flow rates shorten the run time, but have detrimental effects on resolution. A flow rate of
0.275 ml/min was finally chosen for faster separation and better resolution. The column
temperature was set to 28ºC in order to obtain better resolution and appropriate column
pressure. By UPLC-MS analysis the presence of phenolic constituents (flavonols, their
glycosides, procyanidins and galloyl tannins) was detected consisting of fifteen known and
unknown compounds (Figure 1.2.14). The flavonols were found to be glycosides of
quercetin, kaempferol and myricetin. UV spectra showed characteristic absorption bands at
350-367 and 254-266 nm, indicating the presence of flavonols in the extracts. Flavonols
and their glycosides have been identified in other Albizzia species [Kumar et al. (2007);
Lau et al. (2007)]. Four flavonoids were identified by comparison of their retention time
(tR) and UV spectra as myricetin-3-O-rhamnoside, quercetin-3-O-arabinofuranoside,
quercetin-3-O-rhamnoside and quercetin (Figure 1.2.13). The flavonol glycosides (peak 5-
15) were eluted in the order of myricetin, quercetin and kaempferol glycosides,
respectively. The peaks (1-4) show spectral characteristics close to those of procyanidins as
their UV �max values (210 and 262-283 nm) were close to those of catechin [Rohr et al.
(2000); Harris et al. (2007)].
R1 R2 R3
OH OH rha Myricetin-3-O-rhamonside
OH H ara Quercetin-3-O-arabinoside
OH H rha Quercetin-3-O-rhamonside
OH H H Quercetin
Figure 1.2.13: Chemical structures of myricetin-3-O-rhamonside, quercetin-3-O-
arabinoside, quercetin-3-O-rhamonside and quercetin
The phenolics and their glycosides were further characterized by UPLC-ESIMS/MS
analysis in the positive ion mode after tentative identification from UV data allowed the
partial determination of structure. Positive ion UPLC-ESI-MS of ethanolic extracts of A.
chinensis showed protonated [M+H]+ molecules.
O
OH
HO
OH
OR3
O
R1
R2
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
49
Figure 1.2.15: UPLC-DAD chromatograms of (A) standard mixture, (B) flowers, (C)
pods, (D) leaves and (E) bark of A. chinensis methanolic extracts
A
6
11
15
10
2 3
4 20 11
19
D 15
18
8 7
1
10
18
8 7
1
10
E
1
6 7 8
9
10
13
15
14
11 B
2
9
11 12
13
14
15
20
19 3 6
17 16
C
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
50
A difference of 162, 146 and 132 mass units indicated the presence of a hexose (possibly
glucose or galactose), rhamnose, and a pentose (possibly xylose or arabinose), which were
calculated from the difference in mass of molecular ion and fragment ion peaks.
Comparison of retention times and MS data with those of standard compounds revealed the
presence of phenolic compounds; corroborate previous findings [David et al. (1996);
Schiebera et al. (2002)]. ESI-QTOF-MS/MS (in positive mode) of the majority of these
ions showed the characteristic distribution of fragment ions in the A. chinensis extracts
(Figure 1.2.15). Out of eleven flavonoids, six quercetin (peak 7-11, 15), three kaempferol
(peak 12-14), and two myricetin (peak 5-6) derivatives were identified. Other peaks (peak
2-5) were expected to be procyanidins on the basis of UV spectra; however, corresponding
mass signal peaks were not observed for these peaks.
Table 1.2.14: Identification of phenolic compounds in methanolic extracts of different
parts of A. chinensis Peak tR
min)
UV
Spectra
Calculated
MW
Positive ion mode Phenolic compounds
MS MS-MS
1 0.92 210, 272 380 381 219, 201 Procyanidinc
2 1.30 210, 262 382 383 367, 247, 217, 166 Procyanidinc
3 1.50 210, 283 - - - Procyanidinc
4 1.90 210, 262 408 409 300, 247, 185 Procyanidinc
5 3.31 258, 352 486 487 365, 267, 215, 319,
267, 205, 175
Myricetin-3-O-glycoside
6 3.64 259, 352 464 465 361, 341, 319, 205,
175
Myricetin-3-O-
rhamnosidea
7 4.08 256, 354 464 465 383, 367, 303, 229,
205, 175, 121, 109
Quercetin-3-O-galactose
8 4.40 256, 355 434 435 303, 233, 205, 175 Quercetin-3-O-pentosideb
9 4.51 255, 356 434 435 349, 303, 213, 139 Quercetin-3-O-pentosideb
10 4.94 256, 353 434 435 303, 245, 209, 102 Quercetin-3-O-
arabinofuranosidea
11 5.16 256, 350 448 449 303, 147, 129 Quercetin-3-O-
rhamnosidea
12 5.70 266, 356 498 499 287, 175 Kaempferol-3-O-
glycosidec
13 5.99 266, 356 418 419 404, 349, 326, 287 Kaempferol-3-O-
arabinofuranosideb
14 6.27 264, 356 432 433 407, 389, 350, 287,
148, 103, 85, 71
Kaempferol-3-O-
rhamnosideb
15 6.73 256, 367 302 303 285, 257, 247, 229,
201, 183, 153, 137,
111, 95, 69
Quercetina
aCompounds conclusively identified by comparison with authentic standard.
bCompounds tentatively
identified by UV and mass spectral data. cSamples tentatively identified by UV spectral data
Flavonol (quercetin) [M+H]+ molecular ions dehydrated to [M+H-H2O]+ (m/z 285),
followed by two sequential losses of CO: [M+H-H2O-CO]+ (m/z 257) and [M+H-H2O-
2CO]+ (m/z 229). These losses of carbon monoxide were also observed directly from the
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
51
protonated flavonoid: [M+H-CO]+ and [M+H-2CO]
+ (m/z 247). Furthermore the C-ring
was cleaved at bonds 0,2 and 1,3 and produced the respective RDA fragments (0,2A+, 0,2B+
and 1,3A+, 1,3B+) of the protonated molecule (Figure 1.2.15).
0,4A+
O
OH
HO
OH
O
OH
OH
1,4B+ = 179
1,3A+ = 153
0,2A+ = 165
0
3
2
4
A
B
H+
1,3B+
0,2B+ = 137
0,4B+
C
Figure 1.2.15: Fragmentation pattern of quercetin
The plant isolated antioxidant quercitrin has been encapsulated on poly-D,L-lactide (PLA)
nanoparticles by solvent evaporation method to improve the solubility, permeability and
stability of this molecule. The size of quercitrin-PLA nanoparticles was found to be
250±68nm whereas that of PLA nanoparticles was 195±55 nm. The encapsulation
efficiency of nanoencapsulated quercitrin was evaluated by HPLC. The presence of
quercitrin specific peaks on FTIR of quercitrin loaded PLA nanoparticles provides an extra
evidence for the encapsulation of quercitrin into PLA nanoparticles. These properties of
quercitrin nanomedicine provide a new potential for the use of such less useful highly
active antioxidant molecule towards the development of better therapeutic for intestinal
anti-inflammatory effect and nutraceutical compounds.
1.2.4.3 Determination of total flavonoid and phenolic contents of aerial parts of A.
chinensis
Table 1.2.15: Total phenolic and flavonoid contents of different parts of A. chinensis
Plant Part TFCa (mg/g) TPC
b (mg/g)
Flower 8.4 ± 0.09 24.1 ± 0.6
Pods 5.1 ± 0.05 23.1 ± 0.7 Leaf 11.8 ± 0.4 24.6 ± 0.4
Bark 19.6 ± 0.9 26.3 ± 2.0 aTotal flavonoid content; bTotal phenolic content
Various authors have identified and quantified the polyphenols in Albizzia species [Lau et
al. (2007)]. In this study, the total phenol contents (TPC) of different parts of A. chinensis
were measured by the Folin Ciocalteu reagent in terms of gallic acid equivalents (standard
curve equation: y= 0.1194x-0.0438, r2 = 0.9991) (Table 1.2.15). The total phenol content
varied from 23.3 ± 0.7 to 26.3 ± 2.0 mg/g in the extracts in terms of gallic acid equivlents.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
52
Total flavonoid contents (Table 1.2.15) of extracts of aerial parts (flowers, pods, leaves,
bark) of A. chinensis were determined. The flavonoids content of the extracts in terms of
catechin equivalents (standard curve equation: y=0.0131x-0.0149, r2 = 0.9873) were found
between 5.1 ± 0.05 and 19.6 ± 0.9 mg/g; the highest amount was present in the bark extract
(19.6 ± 0.9 mg/g).
1.3 Isolation, characterization and quantification of bioactive molecules from
Podophyllum hexandrum Royle
1.3.1 Introduction
The name Podophyllum was derived
from the Greek words podos and
phyllon meaning foot shaped leaves.
The genus Podophyllum
(Podophyllaceae) has four well known
species, Himalayan, P. hexandrum,
commonly distributed in the Himalayan
regions of Asian continent popularly
known as Himalayan Mayapple,
American, P. peltatum, commonly distributed in Atlantic North America popularly called
as American Mayapple [Chaurasia et al. (2012)] and two of Chinese origin, P. versipelle
and P. aurantiocaule. Several other less-known Chinese species (P. mairei, P. delavayi, P.
difforme and P. pleianthum) have also been identified and chemically investigated
[Rahman et al. (1995)]. They are woodland plants, typically growing in colonies derived
from a single root. Podophyllum rhizomes have a long medicinal history among native
North American tribes who used a rhizome powder as a laxative or an agent that expels
worms (anthelmintic). The dried roots and rhizomes are called podophyllum, which is
enriched in lignans and its first modern botanical name was given by Linnaeus in 1753 [Liu
et al. (2007)]. The natives of the Himalayas as well as the American Indians independently
discovered that the rhizomes extract possessed a cathartic action. The Indians introduced
podophyllin, a resin obtained by ethanolic extraction of the roots and rhizomes, to colonists
for the use as a catharic, an anthelmentic and misuse as a lethal poison. The colonists also
used this resin as an emetic and cholagogue. Podophyllin was included in the first U.S.
Pharmacopoeia in 1820 and the use of this resin was prescribed for the treatment of
venereal warts and as a cathartic. Its first serious chemical investigation was carried out by
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
53
Podwyssotzki [Podwyssotzki (1880)]. Because of its severe toxicity, the drug was removed
from the 12th edition of Pharmacopoeia in 1942 [Ayres and Loike (1990); Horwitz and
Loike (1977)]. In the same year, however, it was reported that venereal warts (Condyloma
acuminata) could be selectively destroyed by the topical application of podophyllin.
Renewed interest in the Podophyllum plant was generated in the 1940s when Kaplan
demonstrated the curative effect of podophyllin, an alcoholic extract of the Podophyllum
rhizomes in C. acuminata.
Podophyllum hexandrum Royle (syn. P. emodi Wall.), Podophyllaceae, is an important
high altitude medicinal plant, commonly known as ‘Indian mayapple’ because its fruits
ripen in spring. It is found in Alpine Himalayas (3000-4000 msl), Jammu and Kashmir,
Himachal Pradesh, Sikkim and Arunachal Pradesh. The plant has been used extensively for
its antitumour, antifungal and immunostimulatory properties [Kamil and Dewick (1986);
Foster (1993); Canel et al. (2000)]. Traditionally, dried rhizomes of the plant were mixed
with liquid and taken as a laxative or to get rid of intestinal worms as a powerful purgative.
Powder of the rhizome was used as a poultice to treat warts and tumorous growth on skin.
Physicians in Missouri, Mississpi and Lousiana used the resin for the treatment of genital
warts. The resin was applied to treat cancerous tumors, polyps and granulations in
traditional medicines. Extracts of Podophyllum species were used as antidotes against
poison, treatments for skin disorders or as purgative, antihelminthic, vesicant, and suicide
agents. P. hexandrum was extensively exploited in Ayurvedic system of medicine for
treating ailments like constipation, cold, biliary fever, septic wounds, inflammation,
burning sensation, mental disorder, genital warts, monocytoid leukemia, Hodgkin’s and
non Hodgkin’s lymphoma [Singh and Shah (1994)].
Two species of the genus Podophyllum, P. hexandrum and P. peltatum are the
commercially most exploited species for the production of podophyllotoxin [Stahelin and
Von Wartburg (1991); Imbert (1998)], that acts as starting material for the preparation of
semisynthetic compounds used in the treatment of lung cancer, a variety of leukemias and
other solid tumours [Van Uden et al. (1989); Canel et al. (2000); Canel et al. (2001)].
Podophyllotoxin is the most abundant lignan isolated from podophyllin. P. hexandrum has
been reported to contain approximately higher podophyllotoxin content (4.3%) in
comparison to the American species, P. peltatum (0.25%) [Jackson and Dewick (1984)].
Podophyllotoxin was also produced in relatively minute quantities by other plant species,
viz., P. aurantiocaule, P. delavayi, P. pleianthum P. peltatum, P. versipelle, Linum flavum,
L. album and Juniperous chinensis.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
54
1.3.2 Chemical constituents
Extensive chemical investigation of roots, rhizomes and leaves of P. hexandrum and P.
peltatum revealed the presence of lignans and their glycosides such as podophyllotoxin
(114) [Podwyssotzki (1880), Liu and Jiao (2006)], �-peltatin (115) [Hartwell (1947)], �-
peltatin (116) [Hartwell and Detty (1948)], 4'-O-demethylpodophyllotoxin (117) [Nadkarni
et al. (1952); Liu et al. (2006)], 4-O-(�-D-glucopyranosy1)-picropodophyllin or
picropodophyllin glucoside (118) [Nadkarni et al. (1953); Liu and Jiao (2006)],
dehydropodophyllotoxin (119) [Kofod and Jorgensen (1954); Liu and Jiao (2006)],
deoxypodophyllotoxin (120) [Kofod and Jorgensen (1955); Dewick and Jackson (1981);
Liu and Jiao (2006)], podophyllotoxone (121) [Dewick and Jackson (1981); Liu and Jiao
(2006)], isopicropodophyllone (122), 4'-O-demethyldeoxypodophyllotoxin (123), 4'-O-
demethylpodophyllotoxone (124), 4'-O-demethylisopicropodophyllone (125) [Dewick and
Jackson (1981); Jackson and Dewick (1984)], picropodophyllotoxin (126) [Chakravarti and
Chakraborty (1954); Liu and Jiao (2006)], 4'-O-demethylpodophyllotoxin-4-O-�-D
glucoside (127), picropodophyllotoxone or picropodophyllone (128) [Singh and Shah
(1994); Chaudhary et al. (2011)], epipodophyllotoxin (129) [Rahman et al. (1995); Liu and
Jiao (2006)], 4'-O-demethyldehydropodophyllotoxin (130) [Rahman et al. (1995)],
podophyllotoxin-4-O-�-D-glucoside (131) and podophyllotoxin-4-O-�-D-glucoside (132)
[Sultan et al. (2010a)].
O
O
O
O
OCH3
OR3
H3CO
R4 R1 R2
O
O
O
O
OCH3
OCH3
H3CO
O
OHO
OHOH
OH
O
O
O
O
OCH3
OR
H3CO
OH
R1 R2 R3 R4 (118) R
OH H Me H (114) CH3 (119)
H H H OH (115) H (130)
H H Me OH (116)
OH H H H (117)
H H Me H (120)
H H H H (123)
H OH Me H (129)
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
55
O
O
O
O
OCH3
OR
H3CO
O
O
O
O
O
OCH3
OR
H3CO
O
O
O
O
O
OCH3
OCH3
H3CO
OH
R R (126) CH3 (121) CH3 (122)
H (124) H (125)
O
O
O
O
OCH3
OCH3
H3CO
O
O
O
O
O
OCH3
OR
H3CO
O
OHO
OHOH
OH
O
O
O
O
OCH3
OCH3
H3CO
O
OHO
OHOH
OH
(128) R (132) CH3 (131)
H (127)
O
OH
HO
OH
O
O
OOHHO
HO
OH
O
OH
HO
OH
O
O
O
OH
HO
HO
OHO OH
OH
O
O
OH
HO
O
OH
OH
(133) (134) (135)
R
O OH
O
H
R
CH3(CH2)10- (136)
CH2=CH-(CH2)10- (137)
CH3(CH2)12- (138)
A few flavonoids such as quercetin (25) [Singh and Shah, (1994)], kaempferol (80) [Shen
and Tian (2006)], quercetin-3-O-�-D-glycoside [Sultan (2010a)], kaempferol-3-O-�-D-
glucopyranoside (133) [Chaudhary et al. (2011)], rutin (84), kaempferol-3-O-rutinoside
(134) [Zhao et al. (2001)] and 8-prenylkaempferol (135) [Shang et al. (2000)] were
reported from roots, rhizomes and fruits. A mixture of three 3-acyl-4-hydroxy-5,6-
dihydropyrones [podoblastin A (136), B (137) and C (138)] were characterized from
chloroform extract of aerial parts of P. peltatum [Miyakado et al. (1982)].
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
56
1.3.3 Chromatographic studies
The most frequently used analytical techniques for determination of lignans and flavonoids
in Podophyllum species so far were high performance liquid chromatography (HPLC), high
performance thin layer chromatography (HPTLC) and micellar electrokinetic
chromatography (MEKC). The majority separations were carried out in the reversed-phase
HPLC mode, however, analysis on conventional silica-gel columns were also reported.
A HPLC method was developed for separation of three lignans namely �- peltatin (115), �-
peltatin (116) and podophyllotoxin (114) in P. peltatum resin using 1.8% ethanol in
chloroform as mobile phase on Perkin-Elmer silica A column at 254 nm with a flow rate of
0.8 ml/min [Treppendahl and Jakobsen (1980)]. Seven diastereoisomers of
podophyllotoxin (114) were successfully separated on silica (Hypersil) and reverse phase
(ODS-Hypersil) column using n-heptane-dichloromethane-methanol (90:10:4) and
methanol-water (45:55) as mobile phase, respectively [Lim and Ayres (1983)].
Furthermore, a method was developed using a Taxsil B column for determination of eight
lignans such as podophyllotoxin (114), epipodophyllotoxin (129), �-peltatin (115), �-
peltatin (116), 4'-O-demethylpodophyllotoxin (117), podophyllotoxin-4-O-�-D-
glucopyranoside (131), epipodophyllotoxin-4-O-�-D-glucopyranoside (139) and 1,2,3,4-
dehydrodesoxypodophyllotoxin (140) by solvent system of (A) reagent alcohol [90.6%
ethanol, 4.5% CH3OH, 4.9% isopropanol)-tetrahydrofuran-methyl-t-butyl ether
(40:30:30)], (B) methanol:water:acetic acid (15:84:1) containing 0.1% ammonium acetate
and (C) acetonitrile in gradient elution in P. peltatum and P. emodi [Bastos et al. (1995);
Bastos et al. (1996)]. In another study, eight lignans and their glucosides [podophyllotoxin
(114), 4'-O-demethylpodophyllotoxin (117), podophyllotoxin-4-O-�-D-glucoside (131), 4'-
O-demethylpodophyllotoxin-�-D-glucoside (127), �-peltatin (115), �-peltatin (116) and
their glucosides] were separated on RP-HPLC (ODS-Hypersil) column with CH3OH-H2O,
CH3CN-H2O, CH3OH-CH3COONH4 or CH3CN-CH3COONH4 as mobile phase at 280 nm
[Lim (1996)].
A microanalytical technique was developed for determination of podophyllotoxin content
(1 to 2 mg) in P. hexandrum resin by RP-HPLC and RP-HPTLC at 230 and 217 nm
respectively using (A) acetonitrile and (B) water as mobile phase [Mishra et al. (2005)].
Another HPLC method was developed for simultaneous determination of nine lignans
[picropodophyllin glucoside (118), 4'-O-demethylpodophyllotoxin (117),
epipodophyllotoxin (129), picropodophyllin (126), picropodophyllone (128),
podophyllotoxin (114), podophyllotoxone (121), deoxypodophyllotoxin (120),
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
57
dehydropodophyllotoxin (119)] and two flavonoids [quercetin (25), kaempferol (80)] in P.
emodi with a mobile phase (A) 25 mM phosphate buffer, pH 2.5 and (B) methanol using
gradient elution [Liu and Jiao (2006)]. Later on, a quantitative LC/MS/MS method was
developed to analyze podophyllotoxin (114), quercetin (25) and kaempferol (80) in
podophyllin. Chromatographic separation was performed on a reversed-phase C18 column
using a gradient of mobile phase of 0.25% formic acid and methanol [Lin et al. (2008)].
Five lignans [podophyllotoxin (114), 4'-O-demethylpodophyllotoxin (117),
podophyllotoxin-4-O-�-D-glycoside (132), podophyllotoxin-4-O-�-D-glycoside (131) and
quercetin-3-O-�-D-glycoside] and two unknown compounds were analyzed in twelve
different accessions of Podophyllum using HPLC-MS on RP-18 column using methanol
and water (60:40) at 290 nm [Sultan et al. (2010a)].
O
O
O
O
OCH3
OCH3
H3CO
O
OHO
OHOH
OH
O
O
O
O
OCH3
OCH3
H3CO
O
O
O
O
OCH3
OR2
H3CO
O
OO
OR1
HOOH
(139) (140) R1 R2
CH3 H (141)
S H (142) Recently, an UPLC-UV-MS was developed for rapid analysis of four lignans [4-O-
demethylpodophyllotoxin (117), podophyllotoxin (114), �-peltatin (115) and �-peltatin
(116)] in P. peltatum leaves on a reversed-phase C18 column using (A) water and (B)
acetonitrile, both containing 0.05% formic acid within 3 min [Avula et al. (2011)]. A
micellar electrokinetic chromatography (MEKC) method was reported for quantitative
analysis of seven lignans [4'-O-demethylpodophyllotoxin (117), epipodophyllotoxin (129),
picropodophyllin (126), podophyllotoxin (114), picropodophyllone (128),
podophyllotoxone (121), deoxypodophyllotoxin (120)] in P. emodi using 10 mM
NaH2PO4-5 mM borate-100 mM sodium dodecylsulfate- 30% isopropanol [Liu et al.
(2001)]. Later, seven pairs of diastereoisomeric (at C-2 and C-4 position) Podophyllum
lignans were separated by MEKC within 35 and 20 min, respectively [Liu et al. (2002a);
Liu et al. (2002b)].
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
58
1.3.4 Pharmacological and biological activities
The plant is used extensively for its medicinal antitumour, antifungal and
immunostimulatory properties of the rhizomes [Kamil and Dewick (1986); Foster (1993);
Canel et al. (2000)]. In the modern allopathic system of medicine, it has been utilized for
the treatment of various metabolic disorders, cancer, bacterial and viral infections [Gowdey
et al. (1995); Cobb (1990)], venereal warts [Beutner and Krogh (1990)], rheumatoid
artharalgia associated with limb numbness and infections of skin tissue [Wong et al.
(2000)], AIDS-associated Kaposis sarcoma and different cancers of brain, lung and bladder
[Blasko and Cordell (1998)]. P. hexandrum extracts has been found to offer radioprotection
by modulating free radical flux involving the role of lignans presents [Chawla et al.
(2006)]. A number of lignans isolated from Podophyllum species have shown a wide range
of biological activities such as antitumor, antimitotic and antiviral. Some of them exhibited
toxicity to fungi, insects and vertebrates.
1.3.4.1 Antitumor activity
The semisynthetic derivatives [etoposide (141) and teniposide (142)] of podophyllin
lignans (podophyllotoxin) were successfully applied as antitumor agents [Farkya et al.
(2004)]. The glucopyranoside derivatives of podophyllotoxin (114) and
deoxypodophyllotoxin (120) were recognized for antihyperlipidimic and antiproliferative
properties [Kusari et al. (2011)]. The ethanol and water extracts of root and rhizome,
podophyllotoxin (114), deoxypodophyllotoxin (120) and 4'-O-
demethyldeoxypodophyllotoxin (123) demonstrated cytotoxicity and inhibitory effects
towards cultured human and mice leukemia, breast, cervical and neuroblastoma cancer cell
lines (K562, MDA468, MCF7, L1210 and L7712, HeLa, SH-SY5Y) [Goel et al. (1998);
Chattopadhyay et al. (2004); Wang et al. (1997); Kumar et al. (2003); Zhang et al. (2005)].
Sequential doses of aqueous extract of P. hexandrum (a daily dose of 34.5 mg/kg b. w. for
15 days) enhanced tumour doubling time (TDT) from 1.94 +/- 0.26 days to 19.1 +/- 2.5
days [Goel et al. (1998)].
1.3.4.2 Radioprotective and antioxidant activities
P. hexandrum was investigated for its radioprotective capabilities, including free radical
scavenging, inhibition of apoptosis and cell cycle arrest-related activities both in vitro and
in vivo models [Arora et al. (2006)]. Methanolic, hydro-alcoholic and chloroform extracts
of P. hexandrum were reported to protect the mice when administered 1-2 h before lethal
whole-body 10 Gy and 20 Gy radiations [Goel et al. (2002); Goel et al. (2007)]. The
radioprotective properties were comparable to synthetic radioprotectors like diltiazem etc
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
59
[Goel et al. (1998)]. Various polyphenols and flavonoids in chloroform extract (REC-2006)
contributed to scavenge the radiation-induced radicals, inhibited superoxide anions,
prevented DNA damage and stimulated DNA repair [Chaudhary et al. (2011)].
Administration of herbal water extract reduced the apoptosis incidence of jejunum villi
cells that underwent radiations [Salin et al. (2001)], protect spermatogenesis disorder
[Samanta and Goel (2002)], physiological markers change [Goel et al. (2002)] and neuron
injury [Sajikumar and Goel (2003)] induced by radiation. Quercetin-3-O-�-D-glycoside
isolated from the hydroalcoholic extract exhibited protective effects on supra-lethal �-
radiation-induced lipids and proteins damage in renal and neuronal systems [Chawla et al.
(2005a); Chawla et al. (2005b)].
Podophyllotoxin (114) also showed protective effect on Co-�-radiation induced damage in
Saccharomyces cerevisiae yeast cells [Bala and Goel (2004)]. The water extracts produced
increased superoxide dismutase activity [Mittal et al. (2001)], decreased reactive oxygen
species and NO production [Gupta et al. (2003)], inhibited radiation-induced decrease in
mitochondrial membrane potential [Gupta et al. (2004)], decreased lipid peroxidation and
increased thiol content [Samanta et al. 2004)], and regulated the protein expressions related
to cell death [Kumar et al. (2005)]. The EtOAc extract showed concentration dependent
superoxide radical scavenging activity, hydrogen peroxide radical scavenging activity
[Ganie et al. (2011)]. The polar fraction (alcoholic) extract exhibited potent antioxidant
activity in terms of reducing power assay [Chawla et al. (2005a)]. The methanolic extract
showed potential antioxidant effects against free radical mediated damages; monitored by
assaying the activities of different antioxidant enzymes viz., superoxide dismutase (SOD),
glutathione peroxidase (GPx), glutathiaone reductase (GR) and glutathiaone S-transferase
(GST) [Ganie et al. (2010)].
1.3.4.3 Hepatoprotective activity
Oral administration of ethanol extracts of the root, rhizome and fruit, as well as
podophyllotoxin (114), inhibited increases in liver index, serum glutamic-pyruvic
transaminase and serum glutamic-oxaloacetic transaminase in CCl4-treated mice, exhibited
hepatoprotective effects. The EtOAc extract of P. hexandrum depicted a liver-protective
effect against CCl4-induced hepatotoxicity in male Wistar rats. Rats pretreated with EtOAc
extract at 20, 30, and 50 mg/kg dose prior to CCl4 administration (1 ml/kg, 1:1 in olive oil)
showed remarkably reduced CCl4-induced toxicity, particularly hepatotoxicity, by
inhibiting lipid peroxidation, suppressing alanine aminotransferase (ALT), aspartate
aminotransferase (AST), and lactate dehydrogenase (LDH) activities [Ganie et al. (2011)].
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
60
1.3.4.4 Other activities
The water extract of P. hexandrum inhibited lipopolysaccharide-induced nitrite production,
interferon-�, interleukin-6 and tumor necrosis factor-� secretion in peritoneal macrophages,
and exhibited anti-inflammatory activities, in vitro [Prakash et al. (2005)].
Deoxypodophyllotoxin (120) demonstrated remarkable potential against Herpes simplex
virus, exhibited antiproliferative, antiplatelet aggregation, in vivo antiasthmatic, insecticidal
and antiallergic activities. Podophyllotoxin (114), deoxypodophyllotoxin (120), 4'-O-
demethyldehydropodophyllotoxin (130) and picropodophyllone (128) demonstrated
insecticidal, antimicrobial activities [Miyazawa et al. (1999); Rahman et al. (1995); Gao et
al. (2004); Kusari et al. (2011)]. The potent immunosuppressive activity of 4'-
demethyldeoxypodophyllotoxin (123) was shown by use of a T-cell-mediated immune
response with respect to their suppression of activated splenocytes [Gordaliza et al.
(1996)].
Numbers of reports are available on biological activities and chemical investigations, still
there is gap of systematic analytical procedures for detailed chemical investigations of the
plant growing in the subtropical regions of Himachal Pradesh. Thus, keeping in view the
importance of P. hexandrum in traditional system of medicine, the present work was
directed towards the phytochemical studies and development of new analytical methods for
quality assessment in P. hexandrum. In the following pages results and discussion followed
by experimental sections are described.
1.3.5 Results and discussion
1.3.5.1 Phytochemical studies
The rhizomes of P. hexandrum were sequentially extracted at room temperature using n-
hexane, chloroform, methanol and water. From chloroform extract, six known lignans
podophyllotoxin (114), 4'-O-demethylpodophyllotoxin (117), deoxypodophyllotoxin (120),
�-peltatin (116), podophyllotoxone (121), and isopicropodophyllone (122) and from
methanolic extract one lignan, podophyllotoxin-4-O-�-D-glucopyranoside (131) and four
flavonoids- kaempferol (80), quercetin (25), quercitrin (83) and rutin (84) were
respectively isolated and characterized.
1.3.5.1.1 Podophyllotoxin (114)
Compound 114 was obtained as colorless crystals. Its positive HRESI-QTOF-MS showed a
molecular ion peak at m/z 415.4124 [M+H]+ (calcd. 415.4132) corresponding to the
molecular formula C22H23O8 indicating 13 degrees of unsaturation. FT-IR spectrum of 114
showed the absorption bands at 3525 (-OH), 3490 (-OH) and 1765 (C=O) cm-1
. The
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
61
absorptions at 2838, 1580 and 1005 cm-1
represented C-H, C=C and C-O functionalities,
respectively. UV spectrum showed absorption maxima at �max 243 and 291 nm.
4a6 3
2'6'
12a
8
O
O
O
O
OCH3
OCH3
H3CO
OH
4a6
3
2'6'
12a
8
O
O
O
O
OCH3
OCH3
H3CO
OH
HMBC
Figure 1.3.1: Chemical structure and selected HMBC correlations of 114
Table 1.3.1: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 114 in CDCl3
Position �C (ppm) �H (ppm) m (J Hz) �C (ppm) �H (ppm) m (J Hz) �C (ppm)
1 44.2 4.48-4.53 m 8a 133.5 -
2 45.5 2.66-2.68 m 1' 135.6 -
2a 174.6 - 2' 108.6 6.34 s
3 40.9 2.66-2.68 m 3' 152.7 -
3a 71.5 3.96-4.00 m; 4.48-4.53 m 4' 137.4 -
4 72.9 4.66-4.69 d (9.0) 5' 152.7 -
4a 131.3 - 6' 108.6 6.34 s
5 106.4 7.10 s -OCH2O- 101.6 5.93 d (1.3), 5.92 d (1.3)
6 147.9 - C-3'-OCH3 56.4 3.64 s
7 147.8 - C-4'-OCH3 60.9 3.70 s
8 109.9 6.47 s C-5'-OCH3 56.4 3.64 s
1H NMR spectrum in CDCl3 indicated the presence of three methoxy groups at � 3.70 (3H,
s), 3.64 (6H, s), two methylene dioxy protons at � 5.93 (1H, d, J = 1.3 Hz), 5.92 (1H, d, J =
1.3 Hz) and three singlets of four aromatic protons at � 7.10 (1H, s), 6.47 (1H, s), 6.34 (2H,
s) (Table 1.3.1). Two singlets resonating at � 7.10 and 6.47 were assigned to the protons of
aromatic ring, placed para to each other. A 2H singlet at � 6.34 was assigned to the
aromatic protons (H-2' and H-6') of the phenyl substituent. The upfield shift of H-2' and H-
6' indicated that this benzene ring bears some electron donating substituent. The signals at
� 4.66-4.69 (1H, d, J = 9.0 Hz), 4.48-4.53 (1H, m), 2.66-2.68 (2H, m) were assigned to H-
4, H-1, H-2 and H-3 protons. Two multiplets at � 3.96-4.00 (1H, m) and 4.48-4.53 (1H, m)
were attributed to the H-3a � and � methylene protons sandwiched between a methine and
oxygen. 13
C NMR and DEPT spectral data revealed the existence of nine quaternary
carbons, eight methine carbons, two methylene carbons and three methoxy carbons. An
ester carbonyl carbon resonated at � 174.6 (C-2a), a methylenedioxy carbon appeared at �
101.6 and a downfield methylene carbon resonated at � 71.5 (C-3a). HMQC spectrum
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
62
showed interactions of H1-5 (� 7.10)/C-5; H1-8 (� 6.47)/C-8; H1-2' (� 6.34)/C-2', H1-6' (�
6.34)/C-6', H1-3a (� 3.96-4.00)/C-3a and H1-3a (� 4.48-4.53)/C-3a. HMBC correlation
displayed interactions of H1-2' (� 6.34)/C-3'; H1-6' (� 6.34)/C-5'; H1-5 (� 7.10)/C-7, C-4a;
H1-3a (� 3.96-4.00)/C-3 and H1-8 (� 6.47)/C-7, C-8a, C-1. Thus, on the basis of above
spectral data and comparison with previously known spectral values [Berkowitz et al.
(2000)], the structure of 114 was assigned as podophyllotoxin (Figure 1.3.1).
1.3.5.1.2 4'-O-Demethylpodophyllotoxin (117)
Compound 117 was isolated as colorless crystals. The molecular formula was determined
as C21H21O8 by HRESI-QTOF-MS with molecular ion peak at m/z 401.3843 [M+H]+
(calcd. 401.3866). UV spectrum showed absorptiom bands at �max 242, 291 and 296 (sh)
nm. IR bands at 3620, 3555, 1774 cm-1
were observed due to the presence of hydroxyl and
carbonyl groups, respectively. 1H NMR spectrum in CD3OD revealed proton signals of two
methoxyl groups at � 3.56 (6H, s) and four aromatic protons at � 6.99 (1H, s), 6.29 (1H, s),
6.28 (2H, s). Broad proton signals of -CH2- at � 5.76 (2H, br s) and the aliphatic proton
signals at � 4.54-4.57 (1H, d, J = 8.4 Hz), 4.39-4.41 (1H, m), 4.30-4.31 (1H, m), 3.90-3.96
(1H, m) and 2.58-2.68 (2H, m) were observed (Table 1.3.2). 13
C NMR spectrum contained
seventeen signals constituting twenty one carbons. Seven of the 13
C signals appeared to be
from saturated carbons, while ten signals appeared to be from unsaturated carbons as
evident from DEPT spectrum. One of the saturated carbons at � 102.7 was exceptionally
deshielded, indicating that it was probably bonded to two oxygens indicating a methylene
oxide group. One of the unsaturated carbon signals at � 177.2 (C-2a) indicated the presence
of a carbonyl group, possibly from an ester or an acid and while signal at � 72.9
represented a downfield methylene carbon. The aromatic ring substituted at C-1 contained
two pairs of identical carbons (the two methoxy carbons appearing at � 56.8, the two
carbons at which the -OCH3 groups are attached resonating at � 148.5 and the two carbons
ortho to the methoxy group resonating at � 109.6). The signals for the C-1' and C-4'
quaternary carbons were appeared at � 135.6 and 132.7, respectively.
4a6 3
2'6'
12a
8
O
O
O
O
OCH3
OH
H3CO
OH
O
O
O
O
OCH3
OH
H3CO
OH
H-H COSY
HMBC
Figure 1.3.2: Chemical structure and selected HMBC and COSY correlations of 117
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
63
HMBC interactions were observed for H1-5 (� 6.99)/C-7; H1-3a (� 3.90-3.96, 4.39-4.41)/C-
2a; H1-8 (� 6.29)/C-1, C-7, C-8a (Figure 1.3.2). Analysis of COSY data supported the
proton-proton network between H-1, H-2, H-3, H-3a and H-4 protons (Figure 1.3.2). Thus,
on the basis of above spectral data and comparison with previously known spectral values
[Xu et al. (2011)], the structure of compound 117 was assigned as 4'-O-
demethylpodophyllotoxin (Figure 1.3.2)
Table 1.3.2: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 117 in CD3OD Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 45.2 4.30-4.31 m 8a 135.4 -
2 46.2 2.58-2.68 m 1' 135.6 -
2a 177.2 - 2' 109.6 6.28 s
3 41.8 2.58-2.68 m 3' 148.5 -
3a 72.9 3.90-3.96 m; 4.39-4.41 m 4' 132.7 -
4 73.0 4.54-4.57 d (8.4) 5' 148.5 -
4a 132.8 - 6' 109.6 6.28 s
5 107.5 6.99 s -OCH2O- 102.7 5.76 br s
6 148.7 - C-3'-OCH3 56.8 3.56 s
7 148.7 - C-5'-OCH3 56.8 3.56 s
8 110.6 6.29 s
1.3.5.1.3 Deoxypodophyllotoxin (120)
4a6 3
2'6'
12a
8
O
O
O
O
OCH3
OCH3
H3CO
Figure 1.3.3: Chemical structure of 120
Compound 120 was obtained as a white amorphous solid. Its positive HRESI-QTOF-MS
showed a molecular ion peak at m/z 399.4122 [M+H]+ (calculated 399.4138) corresponding
to the molecular formula C22
H23
O7. FT-IR spectrum indicated absorption maxima for
lactone carbonyl (1774 cm-1
) and C=C moieties (1590 cm-1
). UV spectrum showed
absorption maxima at �max 210, 240 (sh) and 290 nm. 1H NMR spectrum in CDCl3
indicated the presence of three methoxy groups at � 3.78 (3H, s), 3.73 (6H, s) two of which
were equivalent; two methylene dioxy protons at � 5.91 (2H, br s) and three singlets of four
aromatic protons at � 6.64 (1H, s), 6.50 (1H, s) and 6.34 (2H, s) (Table 1.3.3). Two singlets
resonating at � 6.64 (1H, s) and 6.50 (1H, s) were assigned to protons of the aromatic ring,
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
64
disposed para to each other. A 2H singlet at � 6.34 (1H, s) was assigned to the aromatic
protons (H-2' and H-6') of the phenyl substituent. The multiples at � 4.57 (1H, m) and 3.03-
3.07 (1H, m) were ascribed to H-1 and H-4 respectively. Overlapping multiples at � 2.71
(3H, m) were assigned to H-2, H-3 and H-4. Two multiplets at � 3.88 (1H, m) and 4.42
(1H, m) were attributed to the H-3a � and � methylene protons sandwiched between a
methine and oxygen.
Table 1.3.3: 1H (300 MHz) and 13C NMR (75.4 MHz) data of 120 in CDCl3 Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 43.9 4.57 m 8a 130.9 -
2 47.6 2.71 m 1' 136.4 -
2a 175.0 - 2' 108.7 6.34 s
3 32.9 2.71 m 3' 152.7 -
3a 72.1 3.88 m; 4.42 m 4' 137.5 -
4 33.2 2.71 m;
3.03-3.07 m 5' 152.7 -
4a 128.5 - 6' 108.7 6.34 s
5 108.7 6.64 s -OCH2O- 101.3 5.91 br s
6 147.2 - C-3'-OCH3 56.4 3.73 s
7 146.9 - C-4'-OCH3 60.8 3.78 s
8 110.6 6.50 s C-5'-OCH3 56.4 3.73 s
13C NMR spectrum contained eighteen signals constituting twenty two carbons. Eight of
the 13C signals appeared to be from saturated carbons, while ten signals appeared to be
from unsaturated carbons as evident from DEPT spectrum. The compound (120) possessed
spectroscopic data closely comparable to that of podophyllotoxin (114) except that there is
absence of hydroxyl group at C-4 position as evident from its NMR data (Table 1.3.3).
This was further confirmed by loss of 16 units in mass spectral data (m/z 399). On the basis
of spectral data and comparison with literature data [Hendrawati et al. (2011)], the
structure of compound 120 was assigned as deoxypodophyllotoxin (Figure 1.3.3).
1.3.5.1.4 �-Peltatin (116)
Compound 116 was obtained as a white amorphous solid. Its positive HRESI-QTOF-MS
showed a molecular ion peak at m/z 415.4118 [M+H]+ (calculated 415.4132) corresponding
to the molecular formula C22H23O8. FT-IR spectrum indicated absorption maxima for
hydroxyl (3615 cm-1) and lactone carbonyl groups (1765 cm-1). UV spectrum showed
absorption maxima at �max 271 nm.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
65
4a6 3
2'6'
12a
8
O
O
O
O
OCH3
OCH3
H3CO
OH
O
O
O
O
OCH3
OCH3
H3CO
OH
H-H COSY
HMBC
Figure 1.3.4: Chemical structure and selected HMBC and COSY correlations of 116
Table 1.3.4: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 116 in CDCl3
Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 44.2 4.60 m 8a 137.3 -
2 33.1 2.70 m 1' 136.7 -
2a 175.7 - 2' 108.6 6.37 s
3 47.7 2.70 m 3' 152.8 -
3a 72.8 3.90-3.94 m; 4.49-4.51 m 4' 137.3 -
4 27.3 2.49-2.54 m; 3.19-3.24 m 5' 152.8 -
4a 118.6 - 6' 108.6 6.37 s
5 132.0 - -OCH2O- 101.9 5.92 br s
6 133.3 - C-3'-OCH3 56.5 3.75 s
7 147.6 - C-4'-OCH3 61.1 3.81 s
8 103.7 6.22 s C-5'-OCH3 56.5 3.75 s
1H,
13C NMR and DEPT spectral data revealed the existence of ten quaternary carbons, six
methine carbons, three methylene carbons and three methoxyl carbons. Both 1H and
13C
NMR spectra (Table 1.3.4) revealed close correspondence with those of podophyllotoxin
(114), however, one of the aromatic proton at C-5 carbon was replaced by an aromatic
hydroxyl group. In addition, there is absence of hydroxyl group at C-4 position as in
deoxypodophyllotoxin (120). HMBC interactions showed correlations of methylenedioxy
protons at �H 5.92 (2H, br s) to C-6 and C-7 carbons. The methylene protons at �H 2.49-
2.54 (1H, m), 3.19-3.24 (1H, m) showed correlations with hydroxyl bearing carbon (C-5).
Analysis of COSY data supported the proton-proton network between to H-1, H-2, H-3, H-
3a and H-4 (Figure 1.3.4). Thus, on the basis of above evidences and literature data
[Jackson and Dewick (1984); Schmidt et al. (2006)], the compound 116 was
unambiguously characterized as �-peltatin (Figure 1.3.4).
1.3.5.1.5 Podophyllotoxone (121)
Compound 121 was obtained as a white amorphous solid. The exact mass was obtained by
HRESI-QTOF-MS and was found to be m/z 413.3950 (calculated 413.3973), and
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
66
correspond to the molecular formula C22H21O8. UV spectrum displayed absorption bands at
�max 205, 234, 279 and 315 nm characteristic of a podophyllone skeleton [Gensler et al.
(1960)]. IR spectrum showed strong absorption bands at 1775 and 1665 cm-1, characteristic
of a lactone carbonyl and a ketone moiety conjugated with an aromatic ring, respectively.
The absorptions at 2842, 1576 and 1132 cm-1
represented C-H, C=C and C=O
functionalities, respectively. 1H NMR spectrum displayed signals representing four
aromatic protons, nine protons for three methoxy group and two pairs of methylene protons
(Table 1.3.5). The proton signals of two methoxy group at � 3.79 (3H, s) and 3.73 (6H, s)
four aromatic protons at � 7.52 (1H, s), 6.68 (1H, s) and 6.36 (2H, s), two methylene dioxy
protons at � 6.08 (1H, br s) and 6.06 (1H, br s) were observed. The downfield chemical
shift of H-5 (� 7.52) could be due to the electron withdrawing and magnetic anisotropy
effects of the carbonyl group at C-5. A set of geminally coupled diastereotopic protons
resonating at � 4.51-4.56 (1H, m) and 4.33 (1H, t, J = 9.8 Hz) represented a methylene
group (H-3a) sandwiched between an oxygen and a methine. The signals at � 3.45-3.52
(1H, m) and a doublet of doublet at � 3.27 (1H, dd, J = 15.5, 4.0 Hz) could be assigned to
H-2 and H-3 methine protons. A doublet at � 4.83 (1H, d, J = 3.7 Hz) was assigned to the
H-1 methine, appeared downfield due to the deshielding effect of the carbonyl group. 13
C
NMR and DEPT spectra showed nineteen signals representing twenty two carbons
containing ten quaternary, seven methine, two methylene and three methoxy methyl
carbons. HMBC spectrum showed correlations of H1-3 (�H 3.45-3.52)/C-5 and H1-5 (�H
7.52)/C-5. The other HMBC correlations were found to be similar to that of
podophyllotoxin (114). COSY spectrum displayed crosspeaks between signals at �H 4.51-
4.56 and 4.33 due to their geminal disposition, while the cross-peaks between �H 4.51-4.56,
and 4.33 represented vicinal coupling of methylenic protons with the neighbouring methine
proton at C-3 position (�H 3.45-3.52). Thus, on the basis of above evidences and literature
data [Rahman et al. (1995)], compound 121 was unambiguously characterized as
podophyllotoxone (Figure 1.3.5).
4a6 3
2'6'
12a
8
OO
O
OCH3
OCH3
H3CO
O
O
O
O
O
O
OCH3
OCH3
H3CO
O
H-H COSY
HMBC
Figure 1.3.5: Chemical structure and selected HMBC and COSY correlations of 121
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
67
Table 1.3.5: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 121 in CDCl3
Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 46.8 4.83 d (3.7) 8a 128.4 -
2 44.8 3.27 dd (15.5, 4.0) 1' 132.3 -
2a 173.2 - 2' 107.8 6.36 s
3 43.6 3.45-3.52 m 3' 153.2 -
3a 67.1 4.33 t (9.8);
4.51-4.56 m
4' 137.8 -
4 192.6 - 5' 153.2 -
4a 141.7 - 6' 107.8 6.36 s
5 106.2 7.52 s -OCH2O- 102.6 6.06 br s;
6.08 br s
6 153.3 - C-3'-OCH3 56.4 3.73 s
7 148.3 - C-4'-OCH3 60.9 3.79 s
8 109.8 6.68 s C-5'-OCH3 56.4 3.73 s
1.3.5.1.6 Isopicropodophyllone (122)
Compound 122 was obtained as a white amorphous solid. Its positive HRESI-QTOF-MS
showed a molecular ion peak at m/z 413.3973 [M+H]+ (calculated 413.3973) corresponding
to the molecular formula C22H21O8. In UV spectrum, absorption maxima were observed at
�max 205, 235, 270 and 321 nm, typical of a podophyllotone skeleton. IR spectrum showed
the presence of lactone and conjugated ketone carbonyls through the band at 1765 and
1669 cm-1
, respectively. 1H NMR spectrum showed the presence of four aromatic protons
at � 7.40 (1H, s), 6.67 (1H, s), 6.27 (2H, s) and three methoxy methyls at � 3.78 (3H, s) and
3.70 (6H, s) (Table 1.3.6). Two broad singlets at � 6.06 (1H, br s) and 6.04 (1H, br s) were
assigned to the methylene dioxy protons. A proton resonating at � 4.55 (1H, d, J = 5.3 Hz)
was ascribed to H-1. Overlapping multiplets at � 3.54-3.59 (2H, m) were assigned to H-2
and H-3 protons. Two multiplets at � 3.80-3.86 (1H, m) and 4.47-4.53 (1H, m) were
attributed to the H-3a � and � methylene protons sandwiched between a methine and
oxygen. 13
C NMR spectrum showed the presence of three methoxy, two methylenes, seven
methines and nine quaternary carbons.
OO
O
OCH3
OCH3
H3CO
O
5
8 1
3
3a
2a
2'
3'
6'
O
8a
Figure 1.3.6: Chemical structure of compound 122
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
68
Further investigations by 2D experiments (HMQC and HMBC) led to the conclusion that
the compound (122) has same structure as reported earlier for isopicropodophyllone.
Owing to the deficiency of a hydroxyl group at C-4 position, its protonated molecular ion
(m/z 413) could not produce the ion peak at m/z 397 by elimination of a water molecule.
However, by elimination of a trimethoxybenzene molecule, fragment ion at m/z 245 was
observed in their MS/MS spectra. This ion further produced the ion at m/z 201 after
elimination of a carbon dioxide molecule. Thus, on the basis of above evidences and
literature data [Jackson and Dewick (1984)], compound 122 was unambiguously
characterized as isopicropodophyllone (Figure 1.3.6).
Table 1.3.6: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 122 in CDCl3
Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 44.2 4.55 d (5.3) 8a 128.7 -
2 45.0 3.54-3.59 m 1' 133.9 -
2a 175.4 - 2' 106.5 6.27 s
3 44.7 3.54-3.59 m 3' 153.3 -
3a 69.5 3.80-3.86 m; 4.47-4.53 m 4' 137.6 -
4 194.2 - 5' 153.3 -
4a 139.0 - 6' 106.5 6.27 s
5 106.0 7.40 s -OCH2O- 102.3 6.04, 6.06 brs
6 153.4 - C-3'-OCH3 56.1 3.70 s
7 148.3 - C-4'-OCH3 60.9 3.78 s
8 108.5 6.67 s C-5'-OCH3 56.1 3.70 s
1.3.5.1.7 Podophyllotoxin-4-O-�-D-glucopyranoside (17)
Compound 131 was obtained as white powder. Its molecular formula was determined as
C28H33O13 by HRESI-TOF-MS 599.5329 (calculated 599.5357). UV spectrum showed the
absorption bands at �max 210 and 285 nm, suggesting the existence of the benzene rings. IR
spectrum displayed the absorption bands at 3545, 3355 and 1789 cm-1
, suggesting the
existence of hydroxyl and carbonyl groups, respectively. 1H NMR spectrum revealed
proton signals of three methoxyl groups at � 3.85 (3H, s), 3.62 (6H, s), and four aromatic
protons at � 7.03 (1H, s), 6.84 (2H, s), 6.74 (1H, s), methylenedioxy protons at � 6.02 (2H,
br s), and six aliphatic proton signals at � 5.47 (1H, m), 5.06 (1H, m), 4.84 (2H, m) and
3.30 (2H, m) (Table 1.3.7). 13
C NMR and DEPT spectral data revealed the existence of
nine quaternary carbons, thriteen methine carbons, three methylene carbons and three
methoxyl carbons.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
69
4a63
2'6'
12a
8
1''
6''
O
O
O
O
OCH3
OCH3
H3CO
O
OHO
OHOH
OH
Figure 1.3.7: Chemical structure of compound 131
Table 1.3.7: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 17 in C5D5N
Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 40.6 3.30 m 3' 153.9 -
2 46.2 3.30 m 4' 138.6 -
2a 175.5 - 5' 153.9 -
3 45.1 4.84 m 6' 109.8 6.84 s
3a 72.7 5.06 m -OCH2O- 102.5 6.02 br s
4 79.7 5.47 m C-3'-OCH3 56.7 3.62 s
4a 132.9 - C-4'-OCH3 61.1 3.85 s
5 110.0 7.93 s C-5'-OCH3 56.7 3.62 s
6 148.7 - 1'' 104.1 5.06 m
7 148.4 - 2'' 75.5 4.05 m
8 110.4 6.74 s 3'' 79.4 4.23 m
8a 133.2 - 4'' 72.2 4.23 m
1' 137.4 - 5'' 79.1 4.23 m
2' 109.8 6.84 s 6'' 63.3 4.54-4.58 m
The carbon chemical shifts of the aglycone were very similar to those of podophyllotoxin
(114), in addition, there were signals present due to a sugar moiety. 1H and 13C NMR
spectral data showed one anomeric signal of proton at � 5.06 (1H, m) and a carbon at �
104.1, respectively, indicating it to be a monoglycoside. The sugar was identified as �-D-
glucopyranosyl on the basis of NMR spectral data. The C-1 of glucose was attached to the
4-OH of the aglycone part, as indicated by downfield C-4 chemical shift (� 79.7), and the
correlation of H-1 of glucose and C-4 of the aglycone in HMBC. Based on these findings
and literature data [Sultan et al. (2010); Canel et al. (2000)], the compound 131 was
identified as podophyllotoxin-4-O-�-D-glucopyranoside (Figure 1.3.7).
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
70
1.3.5.1.8 Kaempferol (80)
O
OH
HO
O
OH
OH
Figure 1.3.8: Chemical structure of compound 80
Compound 80 was isolated as a yellow amorphous powder. The positive ESI-QTOF-MS
showed a molecular ion peak at m/z 287.2449 [M+H]+ (calculated 287.2442) indicated the
molecular formula as C15H11O6. UV spectrum showed absorption maxima at �max 265 and
365 nm characteristic of flavonols. 1H NMR spectrum revealed a set of broad singlets at �
6.38 (1H, br s) and 6.17 (1H, br s) assigning to H-8 and H-6 protons respectively. The
presence of a set of A2B2 doublets at � 8.07 (2H, J = 8.5 Hz) and 6.89 (2H, J = 8.6 Hz)
each integrating for two protons were assigned to H-2', H-6' and H-3', H-5' respectively
(Table 1.3.8).
Table 1.3.8: 1H (300 MHz) and
13C NMR (75.4 MHz) data of 80 in CD3OD
Position �C (ppm) �H (ppm) m (J Hz) Position �C (ppm) �H (ppm) m (J Hz)
1 - - 8 94.5 6.38 s
2 148.1 - 8a 158.3 -
3 126.8 - 1' 128.8 -
4 177.4 - 2' 130.7 8.07 d (8.5)
4a 104.6 - 3' 116.4 6.89 d (8.6)
5 162.5 - 4' 160.6 -
6 99.3 6.17 br s 5' 116.4 6.89 d (8.6)
7 165.6 - 6' 130.7 8.07 d (8.5)
13C NMR spectrum showed six oxygenated carbons at � 165.6, 162.5, 160.6, 158.3, 148.1,
and 126.8 assignable to C-7, C-5 C-4', C-8a, C-2, and C-3, respectively. The signal at �
177.4 assigned to C-4 further suggested the presence of flavonol type of skeleton. Thus on
the basis of above spectral data and comparison with previously known spectral values
[Xiao et al. (2006)], the structure of compound 80 was assigned as kaempferol (Figure
1.3.8).
Other compounds were identified as quercetin (25), quercitrin (83) and rutin (84) have
been already discussed in the results and discussion part of Albizzia chinensis
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
71
1.3.5.2 Determination of major lignans and flavonoid by HPTLC
The lignans are interesting group of molecules because of their potent biological activities
[Gordaliza et al. (2000); Gordaliza et al. (2004)]. In recent years, polyphenols have also
become of prominent interest. High intake of flavonoids is associated with a variety of
human health benefits, including prevention of cancer, cardiovascular diseases, and
osteoporosis [Aalvarez et al. (2010); Kimata et al. (2000)].
The lignans and flavonoids were quantified simultaneously by HPLC [Liu and Jiao (2006);
Lin et al. (2008)] and separately by both HPLC [Kumar et al. (2008); Willfor et al. (2006)]
and HPTLC [Bhandari et al. (2007); Mishra et al. (2005)]. Yet no report has been
published till date on simultaneous quantification of lignans and flavonoids in P.
hexandrum by HPTLC that is simple and cost effective and an important tool for the
qualitative, semi-quantitative and quantitative phytochemical analysis of medicinal plants
for the development of TLC fingerprint profiles and estimation of chemical markers and
biomarkers [Bagul et al. (2005)]. Owing to the recent interest and in continuation to our
work on the development of rapid and simple methods for quality assessment of various
medicinal plants of commercial utility [Kaur et al. (2009)], we have optimized, developed
and validated a rapid, sensitive and accurate HPTLC method for the simultaneous
determination of lignans and flavonoid [4'-O-demethylpodophyllotoxin (117),
podophyllotoxin (114), kaempferol (80), podophyllotoxone (121) and
deoxypodophyllotoxin (120)] in P. hexandrum rhizomes.
1.3.5.2.1 Optimization of chromatographic conditions
Five different mobile phases such as acetonitrile:water, ethyl acetate:hexane,
chloroform:hexane and ethyl acetate:methanol:formic acid:water, toluene:ethyl
acetate:acetic acid in different proportions were tested for the separation of lignans and
flavonoids, using silica gel HPTLC plates. The best resolution was achieved with test
mixture of toluene:ethyl acetate:acetic acid (15:7.5:0.5, v/v). The resolution achieved by
the use of these solvent systems provided good separation of the lignans and flavonoid and
offered an advantage over the earlier reported methods [Mishra et al. (2005); Ahmad et al.
(2007)] that described the determination of only one compound i.e. podophyllotoxin in P.
hexandrum by RP-HPTLC using acetonitrile:H2O as mobile phase. In our method, on
applying toluene:ethyl acetate:acetic acid (15:7.5:0.5, v/v) as solvent system, we were able
to quantify four lignans and one flavonoid [4'-O-demethylpodophyllotoxin (117),
podophyllotoxin (114), kaempferol (80), podophyllotoxone (121) and
deoxypodophyllotoxin (120)] simultaneously. Thereafter, the effect of extracting solvents
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
72
was studied with respect to the content of major constituents. Various extracts viz.
chloroform, ethyl acetate, methanol, acetone, methanol:water and water were used to
evaluate the extraction efficiency. The five compounds i.e. 4'-O-demethylpodophyllotoxin
(117), podophyllotoxin (114), kaempferol (80), podophyllotoxone (121) and
deoxypodophyllotoxin (120) were quantified by TLC densitometric methods. The identity
of bands of compounds in the different samples was confirmed by the Rf values and by
overlaying the UV-Vis absorption spectrum with that of standards using the CAMAG TLC
Scanner 3.
1.3.5.2.2 Method validation
Linearity
Working stock solutions containing reference compounds of 4'-O-
demethylpodophyllotoxin (117), podophyllotoxin (114), kaempferol (80),
podophyllotoxone (121) and deoxypodophyllotoxin (120) were prepared in different
dilutions and applied on HPTLC plate for preparing five points linear calibration curves.
The solutions prepared for 4'-O-demethylpodophyllotoxin (117), podophyllotoxin (114),
podophyllotoxone (121) were applied at 1.0, 2.0, 4.0, 6.0 and 8.0 �l, while that of
kaempferol (80) and deoxypodophyllotoxin (120) were applied at 2.0, 4.0, 6.0, 8.0 and 10.0
�l. Sample solutions (5 �l) were applied on TLC plate with similar band pattern. The
calibration curves in this study were plotted between the amounts of analyte versus the
average response (peak area). A linear relationship was found in the calibration range of 1-
8 �g/band for 4'-O-demethylpodophyllotoxin (117), podophyllotoxin (114) and
podophyllotoxone (121) and 2-10 �g/band for kaempferol (80) and deoxypodophyllotoxin
(120). The regression data obtained showed a good linear relationship (r2 = 0.9903-0.9976)
(Table 1.3.9).
Specificity
The specificity of the method was ascertained by analyzing standards and sample. The
bands for four tested lignans [4'-O-demethylpodophyllotoxin (117), podophyllotoxin (114),
podophyllotoxone (121), deoxypodophyllotoxin (120)] and one flavonoid [kaempferol
(80)] in samples were confirmed by comparing the Rf and UV spectra of the spot with that
of standard. The peak purity of individual compounds was assessed by comparing the
spectra at peak start, peak apex, and peak end positions of the band.
Precision
For checking the instrumental precision, five bands of 2 �l for each of five compounds
were applied and analyzed according to the proposed method. The percentage relative
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
73
standard deviation (%RSD) for the instrumental precession were found to be 0.86, 2.85,
1.04, 1.71 and 1.34 for 4'-O-demethylpodophyllotoxin (117), podophyllotoxin (114),
kaempferol (80), podophyllotoxone (121), deoxypodophyllotoxin (120), respectively
(Table 1.3.9). Intraday precision were determined by applying different concentration
levels of five reference compounds five times within 1 day and over a period of 5 days for
interday precision. The intra- and inter-assay precisions were found in the range of 0.97-
2.92% and 0.94-2.87%, respectively in terms of % RSD.
Accuracy
The accuracy was tested by determination of recovery of the compounds in the sample. The
preanalyzed sample, i.e. methanol:water extract, was spiked with three different
concentrations (25, 50, 100%) of compounds, extracted in triplicate and then analyzed by
proposed HPTLC method. Results from measurement of recovery were in the range of
96.38 ± 1.92 to 101.84 ± 1.05% (Table 1.3.10).
Table 1.3.9: Method validation data for five detected compounds in the extract of P.
hexandrum rhizomes 117 114 80 121 120
Rf (± 0.02)a) 0.22 0.37 0.49 0.55 0.7
Regression Equation 2605.3+748.78x 1813.9+589.55x 4464.4+1436x 3327.1+1215.9x 1685.4+463.22x
r2 0.9918 0.9903 0.9976 0.9961 0.9927
Instrument Precisionb)
0.86 2.85 1.04 1.71 1.34
LOD (ng/band) 263 250 617 259 371
LOQ (ng/band) 868 875 1974 856 1188
Robustnessb)
Mobile phase
composition 2.34 1.87 2.35 2.32 2.88
Mobile phase volume 2.98 1.58 1.76 1.85 2.58
Duration of saturation 2.78 1.61 2.35 1.63 1.59
Developing distance 1.26 1.73 2.94 1.72 1.84
Time from spotting to
chromatography 1.22 2.44 2.45 2.04 2.30
Time from
chromatography to
scan
1.58 2.83 1.23 1.63 2.90
a)SD, b)%RSD (n = 5), 117= 4'-O-demethylpodophyllotoxin, 114= podophyllotoxin, 80= kaempferol, 121=
podophyllotoxone, 120= deoxypodophyllotoxin
Robustness
Robustness is a measure of the capacity of a method to remain unaffected by small but
deliberate variations in the method conditions, and is an indication of the reliability of the
method. The robustness was studied by determining the effects of small variation of mobile
phase composition (± 0.1 ml for each component), mobile phase volume (± 5%), chamber
saturation period (20, 30, 40 min) and development distance (80, 85, and 90 cm), time from
spotting to chromatography (5, 10 and 20 min), time from chromatography to scan (5, 10
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
74
and 20 min). The % RSD of peak areas was calculated for each parameter. The overall low
values of %RSD (1.22-2.98%) indicated the robustness of the method (Table 1.3.9).
Quantitation was not significantly effected by changing scanning wavelength by ± 5 nm.
Limits of detection (LOD) and quantification (LOQ)
The limits of detection (LOD) and quantification (LOQ) were determined by injecting a
series of dilute solutions of known concentration. LOD was determined based on the
lowest concentration detected by the instrument from the standard while the LOQ was
found based on the lowest concentration quantified in the sample. In order to estimate the
LOD and LOQ, blank methanol was spotted six times following the same method as
explained. LOD was determined at S/N of 3:1 and LOQ at S/N of 10:1. LOD and LOQ
were in the range of 250-617 and 856-1974 ng/band indicating a high sensitivity for the
investigated compounds (Table 1.3.9).
Table 1.3.10: Results from the recovery analysis to assess accuracy of the method
Amount present
[�g/mg]
Amount added
[�g/mg]
Amount
recovered [%]a)
117 3.99 1.00 96.46 ± 2.60
3.99 2.00 98.78 ± 1.69
3.99 4.00 101.84 ± 1.05
114 8.05 2.00 98.87 ± 1.05
8.05 4.00 97.04 ± 2.31
8.05 8.00 101.37 ± 1.58
80 3.08 0.75 97.04 ± 2.02
3.08 1.50 98.75 ± 0.67
3.08 3.00 96.38 ± 1.92
121 0.94 0.25 96.92 ± 2.18
0.94 0.50 97.45 ± 1.09
0.94 1.00 98.80 ± 1.98
20 1.47 0.35 97.80 ± 2.45
1.47 0.70 98.62 ± 1.24
1.47 1.40 97.79 ± 2.88 a)
Average ± %RSD (n = 3), 117= 4'-O-demethylpodophyllotoxin, 114= podophyllotoxin, 80= kaempferol,
121= podophyllotoxone, 120= deoxypodophyllotoxin
1.3.5.2.3 Quantitative evaluation of extracts
Thin layer chromatographic method was used for quantification of lignans and flavonoids
to resolve the compounds present in the extract. The mobile phase of toluene:ethyl
acetate:acetic acid (15:7.5:0.5, v/v) showed highest selectivity for resolution of lignans and
flavonoids. Five well-separated bands of 4'-O-demethylpodophyllotoxin (Rf 0.22),
podophyllotoxin (Rf 0.37), kaempferol (Rf 0.49), podophyllotoxone (Rf 0.55),
deoxypodophyllotoxin (Rf 0.70) were observed on the F254 TLC plate (Figure 1.3.9) and in
the densitogram (Figure 1.3.10).
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
75
Table 1.3.11: Amounts of detected compounds in P. hexandrum
117 a) 114 80 121 120
Ethyl acetate 3.96 ± 1.54b)
9.30 ± 1.33 3.58 ± 1.18 1.81 ± 0.70 2.51 ± 2.86
Chloroform 4.42 ± 1.06 9.92 ± 1.08 3.37 ± 1.11 1.86 ± 1.27 2.81 ± 1.35
Methanol 3.86 ± 1.91 9.14 ± 0.35 9.02 ± 0.85 1.45 ± 0.55 2.37 ± 1.08
Water 3.69 ± 0.77 4.26 ± 0.53 8.87 ± 0.79 n.d.c)
n.d.
Acetone 4.47 ± 2.12 9.81 ± 0.49 3.16 ± 2.03 1.86 ± 0.74 2.68 ± 1.73
Methanol:water (1:1) 3.99 ± 2.22 8.05 ± 0.90 3.08 ± 1.13 0.94 ± 0.47 1.47 ± 0.80 a)�g/mg of the dry weight of the plant sample, b)mean value (n = 3) ± %RSD, c)not detected, 117= 4'-O-
demethylpodophyllotoxin, 114= podophyllotoxin, 80= kaempferol, 121= podophyllotoxone, 120=
deoxypodophyllotoxin
Figure 1.3.9: CCD image of TLC plate of P. hexandrum at 254 nm. Lines: 1–5: standard
tracks, 6–11: sample tracks of ethyl acetate, chloroform, methanol, water, acetone and
methanol:water (1:1) extracts.
Figure 1.3.10: TLC densitogram of standards 4'-O-demethylpodophyllotoxin,
podophyllotoxin, kaempferol, podophyllotoxone and deoxypodophyllotoxin at 254 nm
All the five compounds were detected in different extracts in the range of 0.94-9.92 �g/mg
(Table 1.3.11) except in water extract in which low polar lignans (podophyllotoxone,
deoxypodophyllotoxin) were not detected. As evident from the table, chloroform was best
extracting solvent for lignans, but as far as extraction of flavonoids was concerned,
podophyllotoxone
podophyllotoxin
kaempferol
4'-demethylpodophyllotoxin
deoxypodophyllotoxin
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
76
methanol was found to be better extracting maximum content of kaempferol (9.02 �g/mg
of plant material). Podophyllotoxin was found in highest (9.92 �g/mg) and
podophyllotoxone in lowest content (0.94 �g/mg) in chloroform and methanol:water
extracts respectively.
1.3.5.3 Simultaneous determination of lignans and flavonoids by UPLC-MS
A simple, sensitive, selective, precise and robust ultra-performance liquid chromatography-
tandem mass spectrometry (UPLC-MS) method was developed and validated for
determination of seven compounds including three lignans [podophyllotoxin (114), 4'-O-
demethylpodophyllotoxin (117), podophyllotoxin-4-O-�-D-glucoside (131)] and four
flavonoids [rutin (84), quercitrin (83), quercetin (25), kaempferol (80)] in the extract of P.
hexandrum rhizomes. All the seven compounds were detected and quantified in the extract.
The chromatographic separation of compounds was achieved in less than 8 min by RP-
UPLC (BEH C18 column, 100 × 2.1 mm i.d., 1.7 �m) using linear gradient elution of water
(0.1% formic acid) and methanol:acetonitrile (25:75, v/v) with flow rate of 0.3 ml/min at
�max 290 nm.
1.3.5.3.1 Optimization of chromatographic conditions
To achieve better resolution in short period for seven compounds, the mobile phase was
standardized after several trials with ACN, methanol and water in various proportions. A
mobile phase consisting of water with 0.1% formic acid (solvent A) methanol:acetonitrile
(25:75, v/v) (solvent B) with a linear gradient elution as follows: 0-1 min, 28% B; 1.0-1.4
min, 28-30% B; 1.4-3.5 min, 30% B, 3.5-4.0 min, 30-50% B, 4.0-5.0 min, 50% B, 5.0-5.5
min, 50-28% B, 5.5-8.0 min, 28% B was finally selected in order to achieve optimal
separation, high sensitivity, and good peak shape. The peak resolution was also recorded
with variation in the column temperature. Column temperature was optimized
systematically from 25 to 40°C, and it was observed that all the components achieved a
baseline resolution at 35°C. Optimal chromatographic conditions were obtained after
running different mobile phases with a reversed-phase C18 column (BEH C18 column, 100
× 2.1 mm i.d., 1.7 �m). Seven compounds viz., rutin (RT: 1.23 min), quercitrin (RT: 1.77
min), quercetin (RT: 3.92 min), 4'-O-demethylpodophyllotoxin (RT: 4.26 min),
podophyllotoxin-4-O-�-D-glucoside (RT: 4.73 min), kaempferol (RT: 5.09 min) and
podophyllotoxin (RT: 5.31 min) were well resolved. The representative chromatograms of
the standard mixture and sample of P. hexandrum rhizome extract have been shown in
Figures 1.3.11 and 1.3.12, respectively. The chromatographic peaks were identified by
comparing their retention times with reference compounds and spiking of samples with the
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
77
reference compounds. The results indicated that compounds were well resolved and their
quantitative determination in P. hexandrum was possible.
Figure 1.3.11: UPLC chromatogram of standard mixture of lignans and flavonoids; peaks
5 = rutin, 8 = quercitrin, 12 = quercetin, 13 = 4'-O-demethylpodophyllotoxin, 14 =
podophyllotoxin-4-O-�-D-glucoside, 16 = kaempferol, 17 = podophyllotoxin
Rhizome extract
Figure 1.3.12: UPLC chromatogram of extract P. hexandrum rhizomes
1.3.5.3.2 Validation parameters
The standard solutions were injected in minimum of nine different concentrations and
linearity was observed with the regression coefficient (r2) > 0.99 presented in Table 1.3.12.
The calibration curves were linear in the range of 6.25-100 �g/ml for rutin, quercitrin,
quercetin, 12.5-200 �g/ml for podophyllotoxin-4-O-�-D-glucoside, 4'-O-
demethylpodophyllotoxin, podophyllotoxin, and 3.13-50 �g/ml for kaempferol (Table
1.3.12).
The LODs for rutin, quercitrin, quercetin, 4'-O-demethylpodophyllotoxin,
podophyllotoxin-4-O-�-D-glucoside, podophyllotoxin and kaempferol were 0.39, 0.39,
0.20, 0.78, 1.56, 0.10 and 0.39 �g/ml and the LOQs for same analytes were found to be
1.25, 1.29, 0.64, 2.03, 4.69, 0.32 and 1.13 �g/ml, respectively (Table 1.3.12). This
indicated that the proposed method exhibited a good sensitivity for the quantification of
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
78
lignans and flavonoids. The intra- and inter-day precisions (expressed in terms of %RSD)
were observed in the range of 0.33-1.10% and 0.78-2.80%, respectively, demonstrating
good precision of the proposed method.
The accuracy of the proposed method was expressed as the recovery of standard
compounds added to the pre-analyzed sample. Samples spiked with 50, 25, 12.5 �g/ml of
rutin, quercitrin, quercetin, 4'-O-demethylpodophyllotoxin, kaempferol and 200, 100, 50
�g/ml of podophyllotoxin-4-O-�-D-glucoside, podophyllotoxin were used in triplicate to
assess accuracy. The amount of compunds was calculated from related linear regression
equation. The percentage recovery ranged from 95.89 to 100.03% with %RSD values in
the range 0.46-2.93%, for the detected compounds (Table 1.3.13).
1.3.5.3.3 Quantification of major constitutes in the extract
The presence of lignans and flavonoids in the extract was confirmed by comparison of their
retention times and overlaying of UV spectra with those of standard compounds. The
methanol extract of P. hexandrum rhizomes showed the presence of twenty compounds out
of which seven compounds were identified and quantified by comparison of their tR and
UV spectra with those of reference standards analyzed under identical chromatographic
conditions (Table 1.3.12). The lignans content in the extract were found to be in higher
amount (5.05%) as compared to flavonoid content (0.67%). Podophyllotoxin was detected
in major amount (2.70%) followed by podophyllotoxin-4-O-�-D-glucoside (1.77%), and 4'-
O-demethylpodophyllotoxin (0.58%). Amongst flavonoids, quercitrin was found in higher
amount (0.24%) and quercetin in very low amount (0.15%).
Table 1.3.12: Method validation data for seven detected compounds in the extract of P.
hexandrum rhizomes
Analytes tR Regression
equation
Linearity
(�g/ml)
r2
LOD
(�g/ml)
LOQ
(�g/ml)
Intraday Precision
(n=5)a
Interday Precision
(n=5) a
%
(w/w)
84 1.23 y = 61.271x - 101.28 6.25-100 0.9987 0.39 1.25 1.05 1.01 0.18
83 1.77 y = 51.457x - 132.26 6.25-100 0.9969 0.39 1.29 0.72 2.68 0.24
25 3.92 y = 156.88x - 44.698 6.25-100 0.9999 0.20 0.64 0.59 2.71 0.10
117 4.26 y = 41.773x - 43.909 12.5-200 0.9996 0.78 2.03 0.65 1.46 0.58
131 4.73 y = 20.964x + 7.2129 12.5-200 0.9991 1.56 4.69 1.10 2.80 1.77
80 5.09 y = 129.16x + 25.646 3.13-50 0.9999 0.10 0.32 0.33 0.78 0.15
114 5.31 y = 36.065x - 35.072 12.5-200 1.0000 0.39 1.13 0.50 1.35 2.70
a%RSD, 84= rutin, 83= quercitrin, 25= quercetin, 117= 4'-O-demethylpodophyllotoxin, 131=
Podophyllotoxin-4-O-�-D-glucoside, 80= kaempferol, 114= deoxypodophyllotoxin
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
79
Table 1.3.13: Accuracy for the quantitative determination of seven compounds in the
extract of P. hexandrum rhizomes
Analytes Added
amount (�g/ml)
Average
Recovery (%) RSD (%)
84 50 97.23 0.46
25 95.89 0.86
12.5 99.17 0.69
83 50 97.56 2.93
25 100.03 2.27
12.5 96.37 1.20
25 50 98.33 1.51
25 97.67 2.12
12.5 97.86 2.19
117 50 97.24 1.17
25 97.36 0.46
12.5 98.31 1.88
131 200 99.61 0.68
100 99.04 0.51
50 98.90 2.03
80 50 98.90 1.41
25 96.52 2.14
12.5 97.52 1.94
114 200 99.31 2.21
100 96.98 2.43
50 99.28 0.85
84= rutin, 83 quercitrin, 25= quercetin, 117= 4'-O-demethylpodophyllotoxin, 131=
podophyllotoxin-4-O-�-D-glucoside, 80= kaempferol, 114= deoxypodophyllotoxin
1.3.5.3.4 Identification of constituents by UPLC-ESI-TOF-MS
Ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole
time of flight mass spectrometry (UPLC-ESI-TOF-MS) was used to study phenolic
composition in the methanolic extract of P. hexandrum rhizomes. The phenolic
constituents were further investigated by ESI-TOF-MS/MS in a positive ion mode. Peaks
5, 8, 12-14, 16, and 17 were identified based on comparison with pure reference
compounds using UPLC, molecular weights and fragment ions by LC–MS/MS and UV
spectra. Peaks 6, 7, 9-11, 15 and 18-20 were identified based on molecular weights,
fragment ions and UV spectra. The molecular weights and the fragment ions were
summarized in Table 1.3.14.
By UPLC-MS analysis presence of twenty known and unknown compounds (lignans,
flavonoids, their glycosides, procyanidins) were detected. The lignans and flavonoids were
found to be glycosides of podophyllotoxin, 4'-O-demethylpodophyllotoxin, quercetin and
kaempferol. The lignans peaks were assigned unambiguously from their characteristic UV
spectra, displaying two absorption bands at �max 240 and 290 nm. The peaks (10, 13-15, 17)
showed characteristic absorption bands at �max 285-290 and 227-243 nm, indicating that the
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
80
presence of podophyllotoxin skeleton type lignans in the extract [Jackson and Dewick
(1984); Bastos et al. (1995)]. The absorption bands at 205, 234, 279, and 315 were found
as the characteristic of podophyllone type skeleton (peaks 18, 20). The UV spectrum
exhibited absorption bands at �max 232, 267, 298 and 361 nm, characteristic of a
tetradehydropodophyllotoxin lignan nucleus (peak 19) [Rahman et al. (1995)]. The UV
spectra suggested that the major flavonoids present in the extracts were flavan-3-ols. The
peaks showed characteristic absorption bands at �max 350-367 and 254-266 nm, indicating
that the flvonols in extracts were substituted in the 3-OH position [Lin et al. (2008); Liu
and Jiao (2006)].
Table 1.3.14: Identification of chemical constituents in methanolic extracts of rhizomes of
P. hexandrum
Peak tR
(min)
UV
spectra
Calcd
MW
(+) ion mode
Detected Compounds [M+H]+/
[M+Na]+
ms/ms (m/z)
1 0.78 210, 262 - - - Procyanidinc
2 0.94 210, 276 - - - Procyanidinc
3 1.02 210, 261 - - - Procyanidinc
4 1.18 210, 274 - - - Procyanidinc
5 1.23 257, 353 610 611 465, 303 Rutina
6 1.39 255, 254 464 465
303, 285, 213, 166,
145, 257, 229, 153,
111
Quercetin-3-O-
glycosideb
7 1.51 254, 352 464 465 303 Quercetin-3-O-
glycosideb
8 1.77 255, 347 448 449 303 Quercitrina
9 2.10 266, 345 449 471 287, 213, 153, 121 Kaempferol-3-O-
glycosideb
10 2.51 243, 285 562 585 383, 229, 333, 299,
267, 85
4'-O-demethyl podo
phyllotoxin glucosideb
11 2.73 265, 347 Kaempferol-3-O-
glycosideb
12 3.92 254, 370 302 303 257, 229, 201, 165,
153, 137, 109, 111, 69 Quercetina
13 4.26 242, 287 400 423 383, 333, 299, 247,
239, 229, 185, 147, 85
4'-O-Demethyl
podophyllotoxina
14 4.73 227, 290 576 599 397, 229, 313, 282, Podophyllotoxin-4-O-
�-glucosidea
15 4.90
235,
268sh,
292
415 397, 247, 229 Epipodophyllotoxinb
16 5.09 265, 365 286 287 287, 258, 213, 153 Kaempferola
17 5.31 235, 291 414 415
397, 351, 313, 282,
247, 229, 195, 169,
147, 85
Podophyllotoxina
18 5.67 236, 287,
324 412 413 245, 201, 353, Picropodophyllone
b
19 5.74 232, 267,
298, 361 410 397 353
4'-O-demethyldehydro
podophyllotoxinb
20 6.14 240, 263, 286, 327
412 413 245, 217, 201, 189,
169, 143, 115 Podophyllotoxone
b
aCompounds conclusively identified by comparison with authentic standard
bCompounds tentatively identified by UV and mass spectral data cSamples tentatively identified by UV spectral data
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
81
Four flavonoids were identified by comparison of their tR and UV spectra with those of
standard flavonoids analyzed under identical chromatographic conditions, namely, rutin
(peak 5), quercetin-3-O-rhamnoside (peak 8), quercetin (peak 12) and kaempferol (peak
16). On the basis of presence of polarity of phenolic groups, these flavonol glycosides were
eluted in the order of quercetin and kaempferol glycosides respectively. The remaining
peaks (peaks 1-4) show spectral characteristics close to those of procyanidins, their UV
�max values were close to with catechin-matching absorption spectra, i.e. �max 210 and 261-
274 nm.
1.3.5.3.5 ESI (+)-MS and ESI (+)-MS/MS analysis
Whilst UV data allowed the partial determination of structure, conclusive structural
information could be obtained from the LC-MS/MS analysis. Therefore, the phenols and
their glycosides were further characterized by UPLC-ESI-MS/MS analysis in positive ion
mode. The fragmentation patterns observed in the mass spectrum were useful in
characterization of lignans and flavonoids. The podophyllotoxin type aryltetralin lignans
(podophyllotoxin, 4'-O-demethylpodophyllotoxin and their glycosides) exhibited a
characteristic fragment ion [A+H]+ formed by loss of the C-4 oxygen substituent along
with the entire lactone ring followed by retro-Diels-Alder rearrangement leading to a
stabilised anthracene derivative (Figure 1.3.13) [Schmidt et al. (2006)].
OO
O
OH
X
O
X
H
OO
O
OH
O
X
H
H
[B + H]+
OO
O
X
O
-H2O
[M + H-H2O]+
O
OX
[A + H]+
H
H
OO
O
H O
H
-H2O
[B + H-H2O]+
X
H
Figure 1.3.13: ESI/MS fragmentation of P. hexandrum lignans
Secondly, the fragments were produced by loss of the pendant phenyl substituent [B+H]+
and subsequent loss of H2O from the lactone showed the dehydration product [B-H2O+H]+
with higher intensity. In case of podophyllone type skeleton (podophyllotoxone,
picropodophyllone), the protonated molecule [M+H]+ could not produce the fragment ion
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
82
by elimination of a water molecule. However, by elimination of a trimethoxybenzene
molecule, fragment ion [B+H]+ was observed in their MS/MS spectra. This ion further
produced the ion [B+H-CO2]+ after elimination of a carbon dioxide molecule. Twenty
compounds were detected in the P. hexandrum extract including five quercetin derivatives
(peaks 5-8, 12), three kaempferol derivatives (peaks 9, 11 and 16), six podophyllotoxin
derivatives (peaks 10, 13-15, 17, 19) and two podophyllone derivatives (peak 18 and 20).
Peaks 1-4 were expected to be procyanidins on the basis of UV spectra; however,
corresponding mass signal peaks were not observed for these peaks.
1.4 Experimental
1.4.1 Isolation, characterization and quantification of bioactive molecules from
Cedrus deodara (Roxb.) Loud.
1.4.1.1 Phytochemical studies
1.4.1.1.1 Instrumentation and conditions
Optical rotation were determined on Horiba Sepa-300 Polarimeter, UV spectra performed
on a Shimadzu UV-2450 instrument (Japan) and IR spectra were obtained on a Nicolet
5700 FTIR (Thermo, USA) spectrophotometer in the region 4000-400 cm-1
using KBr
discs. Mass spectra were recorded on a Waters QTOF-MS with ESI using Waters
MassLynx 4.1 software. 1H and
13C NMR spectra were recorded in Bruker Avance-300.
HPLC analysis was performed on a Shimadzu Prominence HPLC system, equipped with
LC-20AT quaternary gradient pump, SPD-M20A diode array detector (DAD), CBM-20A
communication bus module, CTO-10AS VP column oven, Rheodyne injector and
Shimadzu LC solution (ver. 1.21 SP1) software. Peak purity of compounds was determined
on a LiChroCART® 250-4 LiChrospher
® 100 RP-18 (5 µm) column from Merck
(Darmstadt, Germany). Temperature of the column was set at 30ºC. The mobile phase was
isocratic and composed of water (A) and methanol (B) with a flow-rate of 1.0 ml/min.
Analysis wavelength was set at 290 nm. Column chromatography was carried out with
Merck silica gel 60-120, 230-400 mesh and RP-C18. TLC was run on Merck aluminium
pre-coated silica gel 60 F254 and RP C-18 plates. All the chemicals were purchased from
Merck India Ltd. GC analysis of the oil samples was performed on Shimazdu Gas
Chromatograph (GC 2010) using nitrogen as a carrier gas with a flow rate 1.0 ml/min,
equipped with FID detector and carbowax phase, BP-20 capillary column (30 m × 0.25 mm
i.d. with film thickness 0.25 �m). The injector temperature was programmed from 40-
220ºC @ 4ºC/min rise with 4 min hold at 40ºC and 15 min hold at 220ºC. Injector and
interface temperatures were 250ºC for both. Ion source temperature was 200ºC. Sample (20
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
83
�L) was dissolved in 2 ml GC grade dichloromethane (CH2CI2) and sample injection
volume was 2 �L. Relative percentages of constituents were calculated from the FID, the
automated integrator. GC–MS analysis was conducted on a Shimadzu QP2010 GC–MS
system with 2010 GC. A carbowax phase, BP-20 capillary column (30 m × 0.25 mm i.d.
with film thickness 0.25 �m) was used with helium as a carrier gas at a flow rate of 1.1
ml/min on split mode (1:50) using the same conditions as in GC-FID.
1.4.1.1.2 Plant material
Samples of C. deodara sawdust, woodchips and needles were deposited (PLP 5969) in the
Herbarium of Institute of Himalayan Bioresource Technology (CSIR), Palampur, India.
The plant material was air dried in shade, subjected to grinding to a coarse particle size
powder and stored at ambient temperature before extraction and analysis.
1.4.1.1.3 Isolation and characterization of chemical constituents
1.4.1.1.3.1 Extraction and isolation from sawdust
The sawdust of C. deodara (1.5 kg) was extracted with hexane (4×1L), chloroform (4×1L),
methanol (4×1L) and water (4×1L) at room temperature and filtered. The filtrates were
evaporated under reduced pressure to yield the respective extracts.
The hexane extract (8 g) was chromatographed on a silica gel column, eluting with a
gradient of hexane-EtOAc (1:0�0:1) to give ten fractions (A1-A10). Fraction A3 (1.5 g)
was subjected to silica gel CC with hexane-EtOAc (9.5:0.5�9:1) resulted in the isolation
of (E)-�-atlantone (8) (50.1 mg). The CHCl3 extract (30 g) was loaded on silica gel CC
eluting with a 100% CHCl3 to give eight fractions (B1-B8). The separation of fraction B2
(550 mg) on silica gel CC eluted with isocratic hexane: EtOAc (3:2) to yield fractions B2-1
to B2-7. Fraction B2-1 (230 mg) was further subjected to reverse phase C-18 CC using a
gradient of MeOH: H2O (1:3�1:1) to afford (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57) (61.9
mg). The separation of subfraction B3 (1.91 g) over silica gel column eluting with isocratic
hexane: EtOAc (3:7) to give fractions B3-2 to B3-8. Fraction B3-2 (350 mg) was further
purified by reverse phase C-18 CC using MeOH: H2O (1:5�1:1) to give compound (E)-
(2S, 3S, 6S)-atlantone-2,3,6-triol (58) (57.5 mg). The fraction B8 (250 mg) was
chromatographed over reverse phase C-18 column using MeOH:H2O (0.5:9.5�1:1) to
afford atlantolone (15) (30.6 mg).
The air-dried powdered needles of C. deodara (3.0 kg) were extracted with ethanol (3×1L)
to afford crude extract (350.9 g). The crude extract was suspended in water, and
fractionated with petroleum ether, ethyl acetate, and n-butanol, separately. The petroleum
ether fraction (45.7 g) was chromatographed on a silica gel column by a gradient elution
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
84
with hexane–ethyl acetate (9:1�1:9) to yield protocatechuic acid (53) (20.2 mg). The ethyl
acetate extract (50.1 g) was subjected to silica gel column chromatography and eluted with
a gradient elution of methylene chloride- methanol (36:1�0:100) to give 20 fractions.
Fraction 6 (2.05 g) was applied on silica gel column and Sephadex LH-20 column to give
taxifolin (19) (29.7 mg). Similarly, fraction 10 (2.9 g) was then applied on silica gel
column and Sephadex LH-20 column to give myricetin (45) (31.6 mg).
1.4.1.1.3.2 Essential oil extraction and fractionation
Woodchips (1.5 kg) of C. deodara were subjected to hydrodistillation using a Clevenger-
type apparatus for 6 h. The oil was dried over anhyd. sodium sulphate and stored in airtight
containers at an ambient temperature until analysed. Woodchips (600 g) were extracted by
percolation technique with hexane (3 L) and this process was repeated thrice. After
filtration, the filtrate was evaporated to dryness at 40ºC under reduced pressure. The
essential oil (50 ml) was fractionated between n-pentane and acetonitrile (3×50 ml) (Figure
1.4.1). All the samples were evaporated to dryness at 40ºC under reduced pressure and
stored in refrigerator at 4�C prior to analysis by gas chromatography flame ionization
detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS).
Figure 1.4.1: Fractionation protocol for essential oil of C. deodara
1.4.1.1.3.3 Chromatography and characterization of essential oil fractions
Aliquots of n-pentane and acetonitrile fractions were subjected to chromatography over
silica gel and eluted sequentially with hexane/EtOAc gradients and finally with EtOAc.
Fractions were monitored by TLC and compounds with similar Rf values were pooled
together to give sub fractions. From n-pentane and acetonitrile fractions, mixture of
himachalenes and atlantones were isolated, respectively, and were identified by GC and
GC-MS analyses. Different fractions of essential oil were designated as A2-A6 (A2: crude
oil; A3: atlantones; A4: himachalenes A5: n-pentane fraction and A6: acetonitrile fraction).
(E)-�-Atlantone (8)
Cedrus deodara essential oil
Fractionation
Acetonitrile fraction
Himachalenes mixture Atlantones mixture
Column chromatography Column chromatography
n-Pentane fraction
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
85
Yellowish gum; [�]D25
+21° (c 0.50, MeOH); UV (MeOH): �max 268 nm; IR (KBr, cm-1
):
3139, 2979, 1665, 1614, 1216, 768; 1H NMR (CDCl3, 300 MHz): see Table 1.1.2; 13C
NMR (CDCl3, 75.4 MHz): see Table 1.1.2; HRMS-ESI: m/z [M+H]+ for C15H23O,
calculated 219.3425, observed 219.3411; MS-MS-ESI: m/z 219, 201, 161, 145, 125, 107,
83.
(E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)
Light brownish gum; [�]D25 +10.0° (c 0.50, MeOH); UV (MeOH): �max 296 nm; IR (KBr,
cm-1): 3421, 2923, 1660, 1617, 1440, 1375, 1217, 1048, 769; 1H NMR (CD3OD, 300
MHz): see Table 1.1.3; 13C NMR (CD3OD, 75.4 MHz): see Table 1.1.3; HRMS-ESI: m/z
[M+H]+ for C15H25O3, calculated 253.3572, observed 253.3562; MS-MS-ESI: m/z 275,
253, 235, 217, 199, 189, 135, 119, 83, 59.
(E)-(2S, 3S, 6S)-atlantone-2,3,6-triol (58)
Brownish gum; [�]D25 +19.0° (c 0.50, MeOH); UV (MeOH): �max 293 nm; IR (KBr, cm-1):
3404, 2917, 1650, 1615, 1431, 1369, 1215, 1046, 770; 1H NMR (CD3OD, 300 MHz): see
Table 1.1.4; 13
C NMR (CD3OD, 75.4 MHz): see Table 1.1.4; HRMS-ESI: m/z [M+H]+ for
C15H25O4, calculated, 269.3566, observed 269.3581; MS-MS-ESI: m/z 291, 269, 251, 233,
215, 195, 177, 159, 135, 107, 95, 83.
Atlantolone (15)
Brownish oil; [�]D25 +9° (c 0.50, MeOH); UV (MeOH): �max 241 nm; IR (KBr, cm-1):
3435, 2914, 1664, 1604, 1432, 1371, 1217, 771; 1H NMR (CDCl3, 300 MHz): see Table
1.1.5; 13
C NMR (CDCl3, 75.4 MHz): see Table 1.1.5; HRMS-ESI: m/z [M+H]+ for
C15H25O2, calculated 237.3578, observed 237.3563; MS-MS-ESI: m/z 237, 219, 179, 163,
135, 107, 69.
Protocatechuic acid (53)
White amorphous powder; mp 198-201°C; IR (KBr, cm-1): 3456, 3223, 1680, 1615, 1544,
1254, 1184, 1167; 1H NMR (CD3OD, 300 MHz): see Table 1.1.6; 13C NMR (CD3OD, 75.4
MHz): see Table 1.1.6; HRMS-ESI: m/z [M+H]+ for C7H7O4, calculated 155.1281,
observed 155.1258; MS-MS-ESI: m/z 155, 137, 111.
Taxifolin (19)
Yellow amorphous powder; mp 241-243°C; [�]D25
+50° (c 0.50, MeOH); IR (KBr, cm-1
):
3566, 1663, 1588, 1485, 1443, 1128, 1028; 1H NMR (CD3OD, 300 MHz): see Table 1.1.7;
13C NMR (CD3OD, 75.4 MHz): see Table 1.1.7; HRMS-ESI: m/z [M+H]+ for C15H13O7,
calculated 305.2595, observed 305.2579; MS-MS-ESI: m/z 305, 287, 259, 231, 195, 179,
153, 123, 108.
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Myricetin (45)
Yellow amorphous powder; mp 230-231°C; UV (EtOH): �max 296, 355 nm; IR (KBr, cm-
1): 3556, 1675, 1590, 1535, 1490, 1455, 1130, 1025; 1H NMR (CD3OD, 300 MHz): see
Table 1.1.8; 13
C NMR (CD3OD, 75.4 MHz): see Table 1.1.8; HRMS-ESI: m/z [M+H]+ for
C15H11O8, calculated 319.2430, observed 319.2411; MS-MS-ESI: m/z 319, 273, 245, 217,
179, 165, 153, 137, 111.
Himachalenes (1-3)
Colorless oil; IR (KBr, cm-1): 3056, 1606, 1643, 1362, 720, 945; 1H NMR (CDCl3, 300
MHz): � 5.49, 5.42, 5.36, 4.71, 4.66, 2.83, 2.77, 2.58, 2.56, 2.32, 2.08, 1.80, 1.79, 1.68,
1.65, 1.61, 1.50, 1.38, 1.37, 1.32, 0.94, 0.92, 0.91; 13
C NMR (CDCl3, 75.4 MHz): � 157.7,
137.9, 134.6, 134.0, 133.8, 131.2, 129.0, 126.5, 125.4, 124.5, 122.5, 111.2, 47.7, 47.3,
46.0, 45.0, 42.8, 39.9, 39.1, 38.2, 36.5, 34.7, 34.5, 34.0, 32.0, 31.5, 30.1, 29.4, 26.5, 26.0,
25.0, 24.0, 23.6, 22.6, 20.1; GC-MS: m/z 204, 189, 175, 161, 147, 134, 119, 105, 93, 79,
69, 55.
1.4.1.1.4 Analysis of extract, essential oil and its fractions
Kovats indices (KI) of the compounds relative to a mixture of n-alkanes (C8-C23) were
calculated. Identification of compounds was first attempted using mass spectral libraries
Wiley 7 and NIST 02 [McLafferty (1989); Stein (1990)]. Corroboration of the
identification was conducted by matching the mass spectra of compounds with those
present in our own library and in the literature [Adams (1995); Jennings and Shibamoto
(1980)] and finally by matching the KI of the compounds reported on column having
equivalent binding phase.
1.4.1.1.5 Determination of flavonoids by UPLC-MS in C. deodara needles extract
1.4.1.1.5.1 Chemicals
Standard compounds myretin, quercitrin, quercetin were purchased from Sigma-Aldrich.
Taxifolin was isolated by chromatographic methods, and identified by spectral data (IR,
1D- and 2D-NMR, ESI-MS) that was compared with published spectral data [Sakushima et
al. (2002); Kuspradini and Ohashi (2009)]. Acetonitrile, water and formic acid were of
HPLC grade, purchased from J. T. Baker (USA).
1.4.1.1.5.2 Preparation of standard solutions
An individual stock solution of standard compounds containing taxifolin, quercitrin,
myricetin and quercetin was prepared by dissolving approximately 5 mg each, accurately
weighed, in 50 ml methanol in volumetric flasks.
1.4.1.1.5.3 Sample Preparation
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
87
Comparison of extraction techniques
Comparison of four extraction techniques, i.e., UAE, percolation, soxhlet and maceration
were carried out using single optimized extraction solvent mixture. All the extracts were
analyzed by UPLC for the extracted content of individual flavonoid.
Ultrasound assisted extraction (UAE)
Powdered plant material (200 mg) was sonicated for 20 min at a controlled temperature (40
± 5°C) with 20 ml of methanol in an ultrasonicator bath. The extracts were filtered and
concentrated to dryness under vacuum (temperature, 40-45°C) and then subjected to
lyophilization until a constant weight was obtained.
Microwave-assisted extraction (MAE)
Powdered plant material (200 mg) was extracted with 20 ml of methanol in a domestic
microwave for 20 min at 100 W microwave power. The extracts were filtered and
concentrated to dryness under vacuum (temperature, 40-45°C) and then subjected to
lyophilization until a constant weight was obtained.
Soxhlet Extraction
Powdered plant material (200 mg) was extracted with 50 ml of methanol for 8 h in a
Soxhlet apparatus. The extracts were filtered and concentrated to dryness under vacuum
(temperature, 40-45°C) and then subjected to lyophilization until a constant weight was
obtained.
Maceration
Powdered plant material (200 mg) was packed in a percolator and soaked with 20 ml of
methanol through the powder packing and collected. The collected percolations were
filtered and residues were washed with methanol. The extracts were concentrated to
dryness under vacuum (temperature, 40-45°C) and then subjected to lyophilization until a
constant weight was obtained.
All of the extractions were performed in triplicate. All samples were kept in a nitrogen
atmosphere and at -20°C until further use. For the quantitative determination of compounds
by UPLC, concentrated extracts were dissolved in 2 ml of methanol (analytical grade). The
extracts were filtered through a 0.22 �m membrane filters prior to use.
1.4.1.1.5.4 Instrumentation and chromatographic conditions
The analysis was performed using a Waters ACQUITY™ UPLC system (Waters, Milford,
MA, USA). An ACQUITY UPLC® BEH C18 column (100 × 2.1 mm i.d., 1.7 �m), also
from Waters, was used for achieving separation. The column and sample temperature were
maintained at 35°C and 15°C, respectively. The mobile phase consisted of (A) 0.05%
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
88
formic acid in water and (B) acetonitrile at a flow rate of 0.3 ml/min. Gradient elution was
employed starting at 15% B for 2.5 min, then shifted linearly B to 30% for 1.5 min, held at
30% for 1.5 min, shifted B to 15% for 0.5 min and re-equilibrated for 2.0 min, giving a
total cycle time of 8.0 min. The injection volume was 1 �L with partial loop injection using
needle overfill mode. The peaks were detected at 254 nm. Strong needle wash solution
(90:5, acetonitrile-water) and weak needle wash solution (10:90, acetonitrile-water) were
used. All the solutions mentioned were filtered via 0.22 �m membranes under vacuum and
degassed before their usage. A time-of-flight mass spectrometer with electrospray
ionization (ESI-MS) inter face was used for fingerprinting (Micromass, Manchester, UK).
For UPLC analysis, data acquisition was performed using positive ion mode over a mass
range of m/z 50-1000. The general conditions were: source temperature 80ºC, capillary
voltage 3.1 kV and cone voltage 23 V. Positive ion ESI-MS analysis was performed by
direct infusion with a flow rate of 10 �L/min using a syringe pump. Mass spectra were
acquired and accumulated over 60 sec and spectra were scanned in the range between 50
and 1000 m/z. MassLynx 4.1 (Waters, Manchester, UK) was used for data analysis.
Tandem mass spectrometry for structural analysis of single molecular ion in the mass
spectra from needles extracts was performed by mass-selecting the ion of interest, and in
turn submitted to 15-35 eV collisions with argon in the collision quadrupole.
1.4.1.1.5.5 Method validation
For validation of the developed method, various parameters- linearity, analytical limits,
repeatability, accuracy and recovery were examined using ICH guidelines.
Calibration curves
Stock solution containing four analytes were prepared and diluted to appropriate
concentration in the range of from 0.39-100 �g/ml for establishing calibration curves. For
quantitative analysis, seven different concentrations of four analytes were injected in
triplicate. The calibration curves were constructed by plotting the peak areas versus the
concentration of each analyte.
Selectivity
The selectivity of the method was determined by analysis of standard compounds and
samples. The peaks of compounds were identified by comparing their retention times and
UV spectra with those of the standards.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
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Limit of detection (LOD) and quantification (LOQ)
In order to estimate the LOD and LOQ, blank methanol was spotted six times following the
same method as explained. Limit of detection was determined at a signal to noise ratio of
3:1 and limit of quantitation at a signal-to-noise ratio of 10:1.
Precision and accuracy
The measurements of intra-day and inter-day variability were utilized to assess the
repeatability and reproducibility of the developed method. Intra- and inter-day precisions
(expressed as %RSD) for the four compounds were determined by applying different
concentration levels of five reference compounds five times within 1 day and over a period
of 5 days for interday precision.
Accuracy was evaluated by means of recovery assays carried out by adding known
amounts of the reference compounds to the sample solutions. The amounts of analytes
added correspond to 25, 50, and 100% of compounds concentrations in samples. The
spiked samples were extracted in triplicate and analyzed under the above-mentioned
conditions. The percent recovery and average percent recoveries were calculated.
1.4.2 Isolation, characterization and quantification of bioactive molecules from
Albizzia chinensis (Osbek) Merril
1.4.2.1 Phytochemical studies
1.4.2.1.1 Instrumentation and conditions
Optical rotation were determined on Horiba Sepa-300 Polarimeter, UV spectra performed
on a Shimadzu UV-2450 instrument (Japan). Mass spectra were recorded on a Waters
QTOF-MS with ESI using Waters MassLynx 4.1 software (Micromass, Manchester, UK).
1H and
13C NMR spectra were recorded in Bruker Avance-300. Column chromatography
was carried out with Merck silica gel 60-120, 230-400 mesh and RP-C18. TLC was run on
Merck aluminium pre-coated silica gel 60 F254 and RP-18 plates. TLC plates were
visualized by the UV irradiation (254 and 365 nm), iodine spray and visualising agents. All
the chemicals were purchased from Merck India Ltd.
1.4.2.1.2 Plant material
The plant material was collected from Palampur region of Himachal Pradesh. The samples
were authenticated (PLP 11352) by biodiversity department (CSIR-IHBT) and voucher
specimens were deposited in our herbarium section. The flowers were air dried in shade,
subjected to grinding to a coarse particle size powder and stored at ambient temperature.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
90
1.4.2.1.3 Isolation and characterization of chemical constituents
The air dried powder of A. chiensis flowers (1.5 kg) was extracted with 90% ethanol (1L ×
3) to afford crude extract. The crude extract (200.7 g) was then suspended in H2O (1L × 3),
extracted with n-hexane (1L × 3), EtOAc (1L × 3) and n-BuOH (1 L× 3), successively. The
extracts were concentrated in vacuo to yield semisolid mass. Ethyl acetate soluble part
(45.3 g) was subjected to dry column chromatography on silica gel H. Elution was carried
out in varying percentage of EtOAc:hexane to give fractions A-G. Fractions A-C (01:99,
05:95 and 15:85, v/v, EtOAc:hexane) were pooled and on column chromatography over
silica gel (230-400 mesh) by elution with EtOAc/hexane (05:95, 08:92, 10:90) afforded
three fractions H, I and J. Fraction H was crystallized to yield kaempferol (80) (29.1 mg).
The fractions I and J on silica gel (60-120 mesh) column chromatography on elution with
MeOH:CHCl3 (08:92�12:88, v/v) yielded quercetin (25) (35 mg) and myricetin (45) (33.4
mg).
Fractions D and E on silica gel (230-400 mesh) column chromatography led to fractions K,
L and M (15:85�40:60, v/v, EtOAc/hexane). Fractions K and L resulted in the isolation of
kaempferol-3-O-�-L-arabinofuranoside (108) (17.3 mg) and quercitrin (83) (116.4 mg) and
quercetin-3-O-�-L-arabinofuranoside (107) (22.8 mg) on silica gel (60-120 mesh) column
chromatography on elution with MeOH/CHCl3 (05:95�30:70, v/v). n-Butanol soluble part
(55.7 g) was subjected to dry column chromatography on silica gel H. Elution was carried
out in varying percentage of MeOH:CHCl3 (10:90�70:30, v/v) give fractions N1-N5.
Fractions N1-N3 were pooled and on column chromatography over silica gel (60-120
mesh) by elution with EtOAc/hexane (15:85�40:60, v/v) afforded quercetin-3-O-�-L-
galactopyranoside (92) (16.6 mg), myricetin-3-O-�-L-rhamnopyranoside (myricitrin) (109)
(45 mg) and rutin (84) (27.1 mg).
Dried bark powder of A. chinensis (1.5 kg) were extracted with MeOH (1L � 3) at room
temperature and the solvent was removed under reduced pressure to afford a brown
semisolid mass. One portion (100.5 g) of this mass was suspended in water, and extracted
with petroleum ether, ethyl acetate, and n-butanol, separately. The petroleum ether residue
(35 g) was chromatographed on a silica gel column gradiently eluted with petroleum
ether:EtOAc (9:1�1:9) to yield compounds �-sitosterol (48) (25 mg). The EtOAc fraction
was chromatographed on a silica gel column gradiently eluted with MeOH:CHCl3
(1:10�1:0) to afford catechin (110) (29.7 mg). Second portion of the methanolic extarct
was redissolved in water and adjusted to pH 2.0 with 2 N HCl before being extracted twice
with EtOAc. The EtOAc-soluble acidic phases were combined and partitioned twice
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
91
between a 5% sodium hydrogen carbonate solution. The EtOAc-soluble neutral phases
were combined and concentrated in vacuo. The resulting residue (18.9 g) was first
fractionated by column chromatography on silica gel (EtOAc/hexane 1:5�1:1) to give five
fractions P1-P5. Fraction P5, obtained by elution with 40% EtOAc, was chromatographed
on a silica gel column (CHCl3/MeOH). Fraction P51, obtained by elution with 2%
MeOH/CHCl3, was further chromatographed on a silica gel column (EtOAc/n-hexane
1:3�2:3) to afford ferulic acid (112) (16.3 mg) and caffeic acid (113) (22.3 mg).
Structures of compounds were elucidated by NMR and ESI-MS-MS spectral data and
further confirmed by comparing the spectral data with literature values [Manguro et al.
(2004); Yoshioka et al. 2004; Xiao et al. (2006); He et al. (2009)].
Quercetin-3-O-�-L-rhamnoside (83)
Yellow powder; UV (MeOH): �max 255, 350 nm; 1H NMR (CD3OD, 300 MHz): see Table
1.2.1; 13C NMR (CD3OD, 75.4 MHz): see Table 1.2.1; HRMS-ESI: m/z [M+H]+ for
C21H21O11, calculated 449.3848, observed 449.3824; MS-ESI: m/z 449, 303, 147.
Quercetin (25)
Yellow powder; UV (MeOH): �max 256, 372 nm; 1H NMR (C5D5N, 300 MHz): see Table
1.2.2; 13
C NMR (C5D5N, 75.4 MHz): see Table 1.2.2; HRMS-ESI: m/z [M+H]+ for
C15H11O7, calculated 303.2436, observed 303.0518; MS-ESI: m/z 303, 285, 257, 247, 229,
219, 201, 183, 165, 153, 137, 109, 81, 69.
Rutin (84)
Yellow powder; UV (MeOH): �max 257, 353 nm; 1H NMR (CD3OD, 300 MHz): see Table
1.2.3; 13
C NMR (CD3OD, 75.4 MHz): see Table 1.2.3; HRMS-ESI: m/z [M+H]+ for
C27H30O16, calculated 611.5254, observed 611.5237; MS-ESI: m/z 611, 465, 303, 147.
Quercetin-3-O-�-L-arabinofuranoside (108)
Yellow powder; m. p. 220-224°C; UV (MeOH): �max 255, 356 nm; 1H NMR (CD3OD, 300
MHz): see Table 1.2.4; 13C NMR (CD3OD, 75.4 MHz): see Table 1.2.4; MS-ESI: m/z 303
[M+H] for C20H19O11, 245, 209, 102.
Quercetin-3-O-�-L-galactopyranoside (92)
Yellow powder; m. p. 234-236 °C; UV (MeOH): �max 256, 354 nm; 1H NMR (CD3OD, 300
MHz): see Table 1.2.5; 13
C NMR (CD3OD, 75.4 MHz): see Table 1.2.5; MS-ESI: m/z 465
[M+H] for C21H20O12, 383, 367, 303, 229, 205, 175, 121, 109
Kaempferol-3-O-�-L-arabinofuranoside (109)
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92
Yellow powder; m. p. 229-232 °C; [�]D25
-124° (c 0.50, MeOH); UV (MeOH): �max 265,
356 nm; 1H NMR (CD3OD, 300 MHz): see Table 1.2.6; 13C NMR (CD3OD, 75.4 MHz):
see Table 1.2.6; MS-ESI: m/z 419 [M+H]+ for C20H19O10, 404, 349, 326, 287.
Myricetin (45)
Yellow powder; UV (MeOH): �max 251, 371 nm; 1H NMR (CD3OD, 300 MHz): see Table
1.2.7; 13
C NMR (CD3OD, 75.4 MHz): see Table 1.2.7; HRMS-ESI: m/z [M+H]+ for
C15H11O8, calculated 319.2430, observed 319.2411; ESI-MS: m/z 319, 290, 273, 245, 217,
195, 189.
Myricetin-3-O-�-L-rhamnopyranoside (110)
Yellow powder; m. p. 195-197 °C; UV (MeOH): �max 258, 352 nm; 1H NMR (CD3OD, 300
MHz): see Table 1.2.8; 13
C NMR (CD3OD, 75.4 MHz): see Table 1.2.8; HRMS-ESI:,
calculated 319.2430, observed 319.2411; ESI-MS: m/z 465 [M+H]+ for C21H21O12, 361,
341, 319, 205, 175.
Catechin (111)
White powder; m. p. 172-175 °C; [�]D25
+25° (c 0.50, MeOH); UV (MeOH): �max 220, 280
nm; 1H NMR (CD3OD, 300 MHz): see Table 1.2.9;
13C NMR (CD3OD, 75.4 MHz): see
Table 1.2.9; HRMS-ESI: m/z [M+H]+ for C15H15O6, calculated 291.2760, observed
291.2736. ESI-MS: m/z 291, 273, 165, 139, 123.
Ferulic acid(112)
White powder; 1H NMR (CD3OD, 300 MHz): see Table 1.2.11; 13C NMR (CD3OD, 75.4
MHz): see Table 1.2.11; HRMS-ESI: m/z [M+H]+ for C10H11O4, calculated 195.1919,
observed 195.1937; ESI-MS: m/z 195, 177, 145.
Caffeic acid (113)
White powder; 1H NMR (CD3OD, 300 MHz): see Table 1.2.12;
13C NMR (CD3OD, 75.4
MHz): see Table 1.2.12; HRMS-ESI: m/z [M+H]+ for C9H9O4, calculated 181.1654,
observed 181.1626; ESI-MS: m/z 181, 163, 145.
�-Sitosterol (48)
Colorless needles; m.p. 137-140°C; 1H NMR (CD3OD, 300 MHz): see Table 1.2.13;
13C
NMR (CD3OD, 75.4 MHz): see Table 1.2.13.
1.4.2.2 Identification of phenolic compounds by UPLC-DAD-ESI-QTOF-MS in A.
chinensis
1.4.2.2.1 Plant material
The aerial parts (flower, leaves, pods and bark) of A. chinensis were collected from around
the campus of the IHBT (CSIR), Palampur, India, in May 2007. Voucher specimens were
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
93
deposited (PLP 11352) in the Herbarium of IHBT. After harvest, each of the plant
materials was oven dried at a temperature of 40ºC, pulverized and stored at ambient
temperature (27ºC) before analysis.
1.4.2.2.2 Sample preparation
The powdered plant material (1 g) was sonicated with 25 ml of ethanol in an ultrasonicator
bath (Elma Ultrasonic, Germany) at 35 ± 5ºC for 30-60 min. An extraction time of 40 min
was taken as optimum on a mass yield basis. The extracts were filtered, concentrated to
dryness under vacuum at 40-45ºC, and lyophilized to constant weight.
1.4.2.2.3 Instrumentation and separation conditions
UPLC was performed using a Waters ACQUITY UPLC System (Waters, Milford MA,
USA). Separation was achieved using an ACQUITY UPLC® BEH C18 column (100 mm,
2.1 mm i.d., 1.7 �m particle size; Waters) maintained at 28ºC, with a mobile phase flow
rate of 0.275 ml/min. The system operating pressure was 11000 psi at initial gradient
conditions. The mobile phase contained water, 0.05% formic acid (A) and methanol (B).
Gradient elution was employed starting at 35% B, held for 1.0 min, then shifting linearly B
to 40% over 3.0 min, 50% over 5.0 min, then decreasing to 40% over 6.5 min, 35% over
7.0 min and re- equilibrated for 1.0 min, giving a total cycle time of 8.0 min. The injection
volume was 1 �L with partial loop injection using needle over fill mode. The peaks were
detected at 254 and 360 nm.
A time-of-flight mass spectrometer with electrospray ionization (ESI-MS) inter face was
used for fingerprinting (Micromass, Manchester, UK). For UPLC analysis, data acquisition
was performed using positive ion mode over a mass range of m/z 50-1000. The general
conditions were: source temperature of 80ºC, capillary voltage of 3.1 kV and cone voltage
of 23 V. Positive ion ESI-MS analysis was performed by direct infusion with flow rate of
10 �l min-1 using a syringe pump. Mass spectra were acquired and accumulated over 60 s
and spectra were scanned in the range between 50 and 1000 m/z. Mass Lynx 4.1 (Waters,
Manchester, UK) was used for data analysis. Structural analysis of single molecular ion in
the mass spectra from flower, leaf, bark and pods extracts was performed by mass-selecting
the ion of interest, which was in turn submitted to 15-35 eV collisions with argon in the
collision quadrupole.
1.4.2.3 Determination of total phenolic content
The total phenolic content was measured using Folin-Ciocalteu’s method [Swain and Hillis
(1959)]. For preparation of a calibration curve 20, 40, 60, 80, and 100 �L aliquots of
aqueous gallic acid were mixed with 0.5 ml of 1 N Folin-Ciocalteu’s phenol reagent and
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
94
1.0 ml of 35% Na2CO3 (w/v) in a 25 ml volumetric flask and the solution was made to 25
ml in distilled water. After 35 min of incubation at ambient temperature the absorbance
relative to that of the blank was measured using a XP-2001 Explorer UV
spectrophotometer at 730 nm. The total phenolic content of the sample is expressed as mg
of gallic acid equivalent (GAE)/g of plant material. A total of 50 �L of ethanolic extract of
samples were mixed with the same reagent as described above, and after 35 min of
incubation, the absorption was measured at 730 nm for determination of total phenolics.
All determinations were performed in triplicate.
1.4.2.4 Determination of total flavonoid content
Total flavonoid content (TFC) in the extracts was measured using a modified colorimetric
method [Zhishen et al. (1999)]. For preparation of calibration curve 20, 40, 60, 80,100 �L
of catechin was mixed with 0.3 ml 5% NaNO2 (w/v). After 5 min, 0.3 ml of 10% AlCl3
(w/v) and at 6th min, 2 ml 1M NaOH (w/v) were added in 10 ml volumetric flask. The
absorbance of the reaction mixture relative to blank was measured using spectrophotometer
at 510 nm. The total flavonoid contents of extracts were expressed as mg of catechin
equivalent/g of dry plant material. Aliquots (0.5 ml) of extracts were mixed with the same
reagent as described above, and after 30 min, the absorption at 510 nm was measured for
determination of total flavonoids. All measurements were carried out in triplicate.
1.4.2 Isolation, characterization and quantification of bioactive molecules
from Podophyllum hexandrum Royle
1.4.2.1 Phytochemical studies
1.4.2.1.1 Instrumentation and conditions
UV spectra performed on a Shimadzu UV-2450 instrument (Japan) and IR spectrum was
recorded FTIR (Perkin Elmer). A time-of-flight mass spectrometer with electrospray
ionization (ESI-MS) inter face was used for recording mass spectra (Micromass,
Manchester, UK) using Waters MassLynx 4.1 software. 1H and 13C NMR spectra were
recorded in Bruker Avance-300. Column chromatography was carried out with Merck
silica gel 60-120, 230-400 mesh and RP-C18. TLC was run on Merck aluminium pre-
coated silica gel 60 F254 and RP C-18 plates. All the chemicals were purchased from Merck
India Ltd.
1.4.2.1.2 Plant material and chemicals
The plant material of P. hexandrum was collected from Chamba district, Himachal
Pradesh, in the month of July, 2009. The samples were authenticated (PLP 9763) by
biodiversity department (CSIR-IHBT) and voucher specimens were deposited in the
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
95
herbarium section. The rhizomes of P. hexandrum were air dried in shade, subjected to
grinding to a coarse particle size powder and stored at ambient temperature.
1.4.2.1.3 Isolation and characterization of chemical constituents
The dried powdered plant material (1 kg) was sequentially extracted at room temperature
using n-hexane, chloroform, methanol and water by percolation method and the extracts
were concentrated in vacuo to yield semisolid mass. Chloroform soluble part (59.6 g) was
subjected to dry column chromatography on silica gel H. Elution was carried out in varying
percentage of chloroform in n-hexane to give fractions A-F. Fractions A-C (10:90, 20:80
and 40:60 chloroform:hexane) were pooled and on column chromatography over silica gel
(230-400 mesh) by elution with ethyl acetate/n-hexane (30:70, 40:60, 50:50) afforded three
fractions G, H and I. Fraction G was crystallized to yield deoxypodophyllotoxin (120)
(43.2 mg). The fractions H and I on RP-18 column chromatography on elution with
methanol:water (60:40) yielded isopicropodophyllone (122) (30 mg) and �-peltatin (116)
(19.4 mg) and methanol:water (70:30) afforded podophyllotoxone (121) (33.6 mg).
Fractions D and E on silica gel (230-400 mesh) column chromatography led to fractions J
(50:50 ethyl acetate/n-hexane) and K (55:45 ethyl acetate/n-hexane) and resulted in the
isolation of podophyllotoxin (114) (100 mg) and 4'-O-demethylpodophyllotoxin (117)
(113.8 mg) respectively. The methanolic extract (15 g) was chromatographed over silica
gel (230-400 mesh) by ethyl acetate/n-hexane (60:40, 50:50, 70:30 and 100:0) gave four
fractions L M, N and O. Fraction L yielded podophyllotoxin-4-O- �-D-glucoside (131)
(36.1 mg) and other three fractions (M to O) led to the isolation of kaempferol (80) (18.9
mg), quercetin (25) (32.1 mg), quercitrin (83) (18.2 mg) and rutin (84) (22.7 mg) on
purification by chloroform-methanol mixture (05:95�30:70). Structures of compounds
were elucidated by NMR and ESI-MS-MS spectral data and further confirmed by
comparing the spectral data with literature values [Li et al. (2001); Hadimani et al. (1996);
Rahman et al. (1995)].
Podophyllotoxin (114)
Colorless crystals; mp 180-182°C; [�]D25
-141.3° (c 0.50, CHCl3); UV (EtOH): �max 243,
291, 295 (sh) nm; IR (KBr, cm-1
): 3525, 3490, 2838, 1765, 1580, 1515, 1225, 1005; 1H
NMR (CDCl3, 300 MHz): see Table 1.3.1; 13
C NMR (CDCl3, 75.4 MHz): see Table 1.3.1;
HRMS-ESI: m/z [M+H]+ for C22H23O8 calculated 415.4132, observed 415.4124; MS-MS-
ESI: m/z 415, 397, 313, 247, 229, 169.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
96
4'-O-Demethylpodophyllotoxin (117)
Colorless crystals; mp 248-250°C; [�]D25 -122.3° (c 0.50, CHCl3); UV (EtOH): �max 242,
291, 296 (sh) nm; IR (KBr, cm-1): 3620, 3555, 1774, 1635, 1543; 1H NMR (CD3OD, 300
MHz): see Table 1.3.2; 13
C NMR (CD3OD, 75.4 MHz): see Table 1.3.2; HRMS-ESI: m/z
[M+H]+ for C21H21O8 calculated 401.3866, observed 401.3843; MS-MS-ESI: m/z 401, 383,
339, 299, 247, 229, 185, 155.
Deoxypodophyllotoxin (120)
White powder; C22H22O7; mp 160-163°C; [�]D25 -108.9° (c 0.50, CHCl3); UV (EtOH): �max
210, 240 (sh), 290 nm; IR (KBr, cm-1): 1774, 1590; 1H NMR (CDCl3, 300 MHz): see Table
1.3.3; 13
C NMR (CDCl3, 75.4 MHz) see Table 1.3.3; HRMS-ESI: m/z [M+H]+ for
C22H23O8 calculated 399.4138, observed 399.4122; MS-MS-ESI: m/z 399, 231, 187, 169,
129.
�-Peltatin (116)
White powder; mp 235-238°C; UV (EtOH): �max (log �) 271 nm; IR (KBr, cm-1): 3615,
1765, 1638, 1590, 1512; 1H NMR (CDCl3, 300 MHz): � see Table 1.3.4;
13C NMR (CDCl3,
75.4 MHz): see Table 1.3.4; HRMS-ESI: m/z [M+H]+ for C22H23O8 calculated 415.4132,
observed 415.4118; MS-MS-ESI: m/z 415, 247.
Podophyllotoxone (121)
White powder; mp 184-186°C; mp 178-182°C; UV (EtOH): �max 205, 234, 279, 315 nm; IR
(KBr, cm-1): 2842, 1576, 1665, 1775, 1132; 1H NMR (CDCl3, 300 MHz): see Table 1.3.5;
13C NMR (CDCl3, 75.4 MHz): see Table 1.3.5; HRMS-ESI: m/z [M+H]
+ for C22H21O8
calculated 413.3973, observed 413.3973; MS-MS-ESI: m/z 413, 395, 329, 245, 217, 201,
169, 143.
Isopicropodophyllone (122)
White powder; UV (EtOH): �max 205, 235, 270, 321 nm; IR (KBr, cm-1): 2842, 1765, 1669,
1585, 1126, 1027; 1H NMR (CDCl3, 300 MHz): see Table 1.3.6; 13C NMR (CDCl3, 75.4
MHz): see Table 1.3.6; HRMS-ESI: m/z [M+H]+ for C22H21O8 calculated 413.3973,
observed 413.3950; MS-MS-ESI: m/z 413, 395, 245, 217, 201, 169, 138.
Podophyllotoxin-4-O-�-D-glucopyranoside (131)
White powder; UV (EtOH): �max 210, 285 nm; IR (KBr, cm-1
): 3545, 3355, 1789; 1H NMR
(C5D5N, 300 MHz): see Table 1.3.7; 13C NMR (C5D5N, 75.4 MHz): see Table 1.3.7;
HRMS-ESI: m/z [M+H]+ for C28H33O13 calculated 599.5357, observed 599.5329; MS-MS-
ESI: m/z 599, 397, 353, 313, 229, 203.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
97
Kaempferol (83)
Yellow powder; mp 270-272°C; UV (MeOH): �max 265, 365 nm; 1H NMR (CD3OD, 300
MHz): see Table 1.3.8; 13C NMR (CD3OD, 75.4 MHz): see Table 1.3.8; HRMS-ESI: m/z
[M+H]+ for C15H10O6 calculated 287.2442, observed 287.2449; MS-MS-ESI: m/z 287, 257,
240, 21, 185, 164, 152, 137, 121.
1.4.2.2 Determination of major lignans and flavonoid by HPTLC
1.4.2.2.1 Plant material
The plant material of P. hexandrum was collected from Chamba district, Himachal
Pradesh, in the month of July 2009. The rhizomes of P. hexandrum were air dried in shade,
subjected to grinding to a coarse particle size powder and stored at ambient temperature.
1.4.2.2.2 Preparation of standard solutions
Stock solutions of 4'-demethylpodophyllotoxin, podophyllotoxin, kaempferol,
podophyllotoxone and deoxypodophyllotoxin were prepared by dissolving approximately 5
mg of each in 5 ml methanol in volumetric flasks. These standard solutions were spotted to
HPTLC plates to obtain 4'-O-demethylpodophyllotoxin, podophyllotoxin,
podophyllotoxone in the range of 1.0-8.0 �g/band and kaempferol, deoxypodophyllotoxin
in the range of 2.0-10.0 �g/band.
1.4.2.2.3 Preparation of sample solutions
Powdered rhizomes (500 mg) were extracted with five different solvents- ethyl acetate,
chloroform, methanol, water, acetone and methanol:water (1:1, v/v) (25 ml each) for 20
min at 25°C in ultrasonicator bath (Elma, Germany). All the extractions were performed in
triplicate. The extracts were concentrated under vacuum to remove solvents. Concentrated
extracts were dissolved in methanol (2 ml) and filtered through 0.45 µm membrane.
1.4.2.2.4 Chromatography
A Camag HPTLC system equipped with an automatic TLC sampler ATS4, TLC scanner 3
and an integrated software Win-CATS version 1.2.3 was used for the analysis. HPTLC was
performed on pre-coated silica gel HPTLC 60 F254 (20 cm × 10 cm) plate of 0.2 mm layer
thickness. Samples and standards were applied on precoated plates, as 6 mm bands, with a
Camag automatic TLC applicator (ATS4), ATS4 equipped with 25 �L syringe under N2
gas flow, 10 mm from the bottom and 10 mm from the side and the space between two
bands was 15.4 mm of the plate. Ascending development of the plate, migration distance
90 mm, was performed at 25 ± 2°C with single run using toluene:ethyl acetate:acetic acid
(15:7.5:0.5, v/v) as mobile phase in a Camag twin-trough chamber saturated with mobile
phase vapour. After development, the plate was removed, dried and spots were visualized
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
98
under UV light at 254 nm with Wincats Software, using the deuterium light source, slit
width 6 × 0.45 mm, scanning speed 20 mm/s, and data resolution 100 �m/step.
1.4.2.3 Determination of polyphenols by UPLC-MS
1.4.2.3.1 Plant material and chemicals
The plant material of P. hexandrum rhizomes was collected in the month of July, 2010.
The rhizomes of P. hexandrum were washed, air dried in shade, subjected to grinding to a
coarse particle size powder and stored at ambient temperature. Standard compounds rutin,
kaempferol, podophyllotoxin, quercitrin and quercetin were purchased from Sigma
Aldrich. 4'-O-demethylpodophyllotoxin, podophyllotoxin-4-O-�-D-glucoside were isolated
and identified by spectral data (IR, 1D-, 2D-NMR and ESI-MS) in comparison with
published spectral data [Rahman et al. (1995); Jackson and Dewick (1984)]. Analytical
grade acetonitrile, methanol, water and formic acid were purchased from J. T. Baker
(USA).
1.4.2.3.2 Preparation of standard solutions
An individual stock solution of standard compounds containing rutin, quercitrin, quercetin
(0.1 mg/ml), 4'-O-demethylpodophyllotoxin, podophyllotoxin-4-O- �-D-glucoside,
podophyllotoxin (0.2 mg/ml) and kaempferol (0.05 mg/ml) was prepared in methanol.
1.4.2.3.3 Preparation of sample solution
About 200 mg of powdered plant material was extracted with 20 ml of methanol in an
ultrasonicator bath (Elma Ultrasonic, Germany) at a controlled temperature (40°C) for 20-
40 min. An extraction time of 30 min was taken as optimum on mass yield basis. The
extracts were filtered and concentrated to dryness under vacuum. Concentrated extracts
were redissolved in 2.0 ml methanol (HPLC grade). As the UPLC analysis, the more
sensitive method, the samples were diluted to get a final concentration of 10 mg/ml. The
injection volume was 1 �L.
1.4.2.3.4 Instrumentation and chromatographic conditions
The analysis was performed using a Waters ACQUITY™ UPLC system (Waters, Milford,
MA, USA). An ACQUITY UPLC® BEH C18 column (100 × 2.1 mm i.d., 1.7 �m), also
from Waters, was used for achieving separation. The column and sample temperature were
maintained at 35 and 15°C, respectively. The mobile phase consisted of (A) 0.1% formic
acid and (B) methanol:acetonitrile (25:75, v/v) at a flow rate of 0.3 ml/min. Gradient
elution was employed starting at 28% B for 1.0 min, then shifted linearly B to 30% for 0.4
min, held at 30% for 2.1 min, shifted again to 50% for 0.5 min, held at 50 % for 1.0 min,
shifted B to 28% for 0.5 min and re-equilibrated for 2.5 min, giving a total cycle time of
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
99
8.0 min. The injection volume was 1 �L with partial loop injection using needle overfill
mode. The peaks were detected at 290 nm. The peaks were assigned with respect to the UV
spectra of the compounds and comparison of the retention times.
Strong needle wash solution (90:5, acetonitrile-water) and weak needle wash solution
(10:90, acetonitrile-water) were used. All of the solutions mentioned were filtered via 0.22
�m membranes under vacuum and degassed before their usage.
A time-of-flight mass spectrometer with electrospray ionization (ESI-MS) inter face was
used for fingerprinting (Micromass, Manchester, UK). For UPLC analysis, data acquisition
was performed using positive ion mode over a mass range of m/z 50-1000. The general
conditions were: source temperature 80ºC, capillary voltage 3.1 kV and cone voltage 23 V.
Positive ion ESI-MS analysis was performed by direct infusion with a flow rate of 10
�L/min using a syringe pump. Mass spectra were acquired and accumulated over 60 s and
spectra were scanned in the range between 50 and 1000 m/z. Mass Lynx 4.1 (Waters,
Manchester, UK) was used for data analysis. Tandem mass spectrometry for structural
analysis of single molecular ion in the mass spectra from rhizomes extracts was performed
by mass-selecting the ion of interest, and was in turn submitted to 15-35 eV collisions with
argon in the collision quadrupole.
1.4.2.3.5 Method validation
Calibration curves
Stock solution containing seven analytes were prepared and diluted to appropriate
concentration in the range of 0.39 to 100 �g/ml for rutin, quercitrin, quercetin, 0.78 to 200
�g/ml for 4'-O-demethylpodophyllotoxin, podophyllotoxin-4-O-�-D-glucoside,
podophyllotoxin and 0.20 to 50 �g/ml for kaempferol for establishing calibration curves.
For quantitative analysis, nine different concentrations of seven analytes were injected in
triplicate. The calibration curves were constructed by plotting the peak areas versus the
concentration of each analyte.
Selectivity
The selectivity of the method was determined by analysis of standard compounds and
samples. The peaks of rutin, quercitrin, quercetin, 4'-demethylpodophyllotoxin,
podophyllotoxin-4-O-�-D-glucoside, kaempferol and podophyllotoxin were identified by
comparing their retention times and UV spectra with those of the standards.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
100
Limit of detection (LOD) and quantification (LOQ)
In order to estimate the LOD and LOQ, blank methanol was spotted six times following the
same method as explained. Limit of detection was determined at a signal to noise ration of
3:1 and limit of quantitation at a signal-to-noise ratio of 10:1.
Precision and accuracy
The measurements of intra-day and inter-day variability were utilized to assess the
repeatability and reproducibility of the developed method. Intra- and inter-day precisions
(expressed as %RSD) for the seven compounds were determined by applying different
concentration levels of reference compounds five times within 1 day and over a period of 5
days for interday precision.
Accuracy was evaluated by means of recovery assays carried out by adding known
amounts of the reference compounds to the sample solutions. The amounts of analytes
added correspond to 25, 50, and 100% of compounds concentrations in samples. The
spiked samples were extracted in triplicate and analyzed under the above-mentioned
conditions. The percent recovery and average percent recoveries were calculated.
1.5 Conclusion
For exploration of secondary metabolites from western Himalayan region, three
medicinally important plants were studied for the isolation, characterization and
development of new analytical methods using modern hyphenated techniques. From
Cedrus deodara ten compounds (including two novel) were isolated and characterized. For
the first time, two new sesquiterpenes, (E)-(2S, 3S, 6R)-atlantone-2,3-diol and (2S, 3S, 6S)-
atlantone-2,3,6-triol were identified and their structures were elucidated using 1D and 2D
NMR and mass spectral data. Analyses of essential oil and extracts demonstrated highest
content of himachalenes and atlantones in the extract of C. deodara. Furthermore, a newly
developed UPLC method was applied for identification and quantification of four
flavonoids in Cedrus needles. Analysis of P. hexandrum rhizomes led to isolation and
characterization of seven lignans and four flavonoids. In addition, simple and rapid
analytical procedures (HPTLC, UPLC) have been developed for simultaneous
determination and quantification of lignans and flavonoids in P. hexandrum. From flowers
and bark of A. chinensis fourteen compounds including phenolics and terpenes were
isolated and characterized. This is the first report on complete chemical fingerprinting of
different parts of A. chinensis for identification of secondary metabolites conducted by
UPLC-ESI-QTOF-MS.
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
101
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Spectral data of some compounds
1H NMR (in CDCl3) spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)
220 200 180 160 140 120 100 80 60 40 20 ppm 13
C NMR (in CDCl3) spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)
220 200 180 160 140 120 100 80 60 40 20 ppm DEPT spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)
9 8 7 6 5 4 3 2 1 ppm
O
OHOH
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
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HMBC spectra of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)
HMQC spectra of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)
HRESI-QTOF-MS of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (57)
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
123
1H NMR (in CDCl3) spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)
220 200 180 160 140 120 100 80 60 40 20 ppm 13C NMR (in CDCl3) spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)
220 200 180 160 140 120 100 80 60 40 20 ppm DEPT spectrum of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)
9 8 7 6 5 4 3 2 1 ppm
O
OHOH
OH
Isolation, characterization and quantification of bioactive molecules…… Chapter 1
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HMBC spectra of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)
HMQC spectra of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)
HRESI-QTOF-MS of (E)-(2S, 3S, 6R)-atlantone-2,3-diol (58)