New acylated flavonoid glycosides from flowers of Aerva javanica

Article abstract of DOI:10.1080/10286020.2013.795553

Chromatographic purification of ethyl acetate soluble fraction of the methanolic extract of the flowers of Aerva javanica yielded three new acylated flavone glycosides: kaempferol-3-O-β-d-[4?-E-p-coumaroyl-α-l- rhamnosyl(1 → 6)]-galactoside (1), kaempfero

Full text of DOI:10.1080/10286020.2013.795553

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New acylated flavonoid glycosides from
flowers of Aerva javanica
Sara Mussadiq a , Naheed Riaz a , Muhammad Saleem a ,
Muhammad Ashraf b , Tayaba Ismail b & Abdul Jabbar a
a Department of Chemistry , Baghdad-ul-Jadeed Campus, The
Islamia University of Bahawalpur , Bahawalpur , 63100 , Pakistan
b Department of Biochemistry and Biotechnology , Baghdad-
ul-Jadeed Campus, The Islamia University of Bahawalpur ,
Bahawalpur , 63100 , Pakistan
Published online: 14 Jun 2013.
To cite this article: Sara Mussadiq , Naheed Riaz , Muhammad Saleem , Muhammad Ashraf , Tayaba
Ismail & Abdul Jabbar (2013): New acylated flavonoid glycosides from flowers of Aerva javanica ,
Journal of Asian Natural Products Research, DOI:10.1080/10286020.2013.795553
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Journal of Asian Natural Products Research, 2013
http://dx.doi.org/10.1080/10286020.2013.795553
New acylated flavonoid glycosides from flowers of Aerva javanica
Sara Mussadiqa, Naheed Riaza, Muhammad Saleema*, Muhammad Ashraf b,
Tayaba Ismailb and Abdul Jabbara
*
aDepartment of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur,
Bahawalpur 63100, Pakistan; bDepartment of Biochemistry and Biotechnology, Baghdad-ul-Jadeed
Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
(Received 24 December 2012; final version received 10 April 2013)
Chromatographic purification of ethyl acetate soluble fraction of the methanolic extract
of the flowers of Aerva javanica yielded three new acylated flavone glycosides:
kaempferol-3-O-b-D-[4000-E-p-coumaroyl-a-L-rhamnosyl(1 ! 6)]-galactoside (1),
kaempferol-3-O-b-D-[4000-E-p-coumaroyl-a-L-rhamnosyl(1 ! 6)]-(300-E-p-coumaroyl)-
galactoside (2), and kaempferol-3-O-b-D-[4000-E-p-coumaroyl-a-L-rhamnosyl(1 ! 6)]-
(400-E-p-coumaroyl)galactoside (3), along with p-coumaric acid (4), caffeic acid (5),
gallic acid (6), eicosanyl-trans-p-coumarate (7), hexadecyl ferulate (8), and hexacosyl
ferulate (9). The compounds 1–9 were characterized using 1D (1H, 13C) and 2D NMR
(HMQC, HMBC, and COSY) spectroscopy and mass spectrometry (EI-MS, HR-EI-MS,
FAB-MS, and HR-FAB-MS) and in comparison with the reported data in the literature.
Compound 1 showed weak inhibitory activity against enzymes, such as acetylcholin-
esterase, butyrylcholinesterase, and lipoxygenase with IC50 values 205.1, 304.1, and
212.3 mM, respectively, whereas compounds 2 and 3 were only weakly active against the
enzyme acetylcholinesterase.
Keywords: Amaranthaceae; Aerva javanica; flowers; flavonoid glycosides; charac-
terization; biological activities
1. Introduction
this plant are chewed for teeth cleaning,
the seeds are reported to relieve head-
ache, and the paste of roots is applied to
remove acne from the face [3,4].
Literature survey revealed that the
extracts of different parts of the plant
exhibit antimicrobial activity against
Gram negative bacteria, yeast, and
fungi. Ethanolic and aqueous extract of
A. javanica showed significant antidiar-
roheal [5] and muscle relaxant properties
[6]. The stem leaves and mature fruits
showed antiplasmodic activity [7].
Previous phytochemical investigations
resulted in the isolation of a number of
flavanoids [8]. The ethnopharmacological
and chemotaxonomic importance of A.
javanica prompted us to investigate the
constituents of this species growing in
The genus Aerva of the family Amar-
anthaceae comprises of 28 species,
distributed in the warm parts of Asia
and Africa. In Pakistan, this genus is
represented by six species, being used to
cure ulcer, lithiasis, toothache, headache,
abdominal disorders, and inflammation of
internal organs [1]. The roots and flowers
of various plants of this genus are
reported to possess hypoglycemic, anti-
oxidant, anthelmintic, analgesic, antima-
larial, antivenin activities, and medicinal
properties against rheumatism and kidney
troubles [2]. Aerva javanica is a per-
ennial herb growing in western Hima-
laya, Punjab, and Kashmir. It is
commonly used in folklore for diuretic
and demulcent properties. The roots of
*Corresponding author. Email: m.saleem@iub.edu.pk; nrch322@yahoo.com
q 2013 Taylor & Francis

2
S. Mussadiq et al.
Cholistan Desert. Herein, we report the
isolation and structure elucidation of
three new (Figure 1) acylated flavanol
glycosides, namely kaempferol-3-O-b-D-
[4000-E-p-coumaroyl-a-rhamnosyl(1 ! 6)]
galactoside (1), kaempferol-3-O-b-D-[4000-
E-p-coumaroyl-a-L-rhamnosyl(1 ! 6)]-
(300-E-p-coumaroyl)galactoside (2), and
kaempferol-3-O-b-D-[4000-E-p-coumaroyl-
a-L-rhamnosyl(1 ! 6)]-(400-E-p-coumar-
oyl)galactoside (3), along with p-couma-
ric acid (4) [9], caffeic acid (5) [10],
gallic acid (6) [4], eicosanyl-trans-p-
coumarate (7) [11], hexadecyl ferulate
(8) [12], and hexacosyl ferulate (9) [13].
NMR spectrum of 1 (Table 1) afforded two
pairs of o-coupled doublets [d 7.88 (2H, d,
J ¼ 8.8 Hz), 6.68 (2H, d, J ¼ 8.8 Hz) and
7.18 (2H, d, J ¼ 8.4 Hz), 6.60 (2H, d,
J ¼ 8.4 Hz)] attributed to two p-substituted
benzene rings. The first pair of signals could
be assigned to the ring B of kaempferol
moiety, whereas the other pair of aromatic
protons was identified for a E-p-coumaroyl
unit whose further protons for E-olefin
resonated at d 7.40 (1H, d, J ¼ 16.4Hz) and
6.00(1H, d, J ¼ 16.4Hz). Besides, the same
spectrum displayed two m-coupled doublets
at d 6.17 (1H, d, J ¼ 1.6 Hz) and 6.04
(1H, d, J ¼ 1.6 Hz) corresponding to the
protonsH-6andH-8 ofringA ofkaempferol
nucleus. The 1H NMR spectrum also
showed signals for two sugar moieties
between d 4.68 and 3.33 (Table 1), with
anomeric protons resonating at d 4.58
(1H, d, J ¼ 8.0 Hz) and 4.32 (1H, br s).
Their corresponding carbons appeared in
13C NMR spectrum at d 105.0 and 100.1,
respectively. The amount of coupling
constants of these two protons revealed
that the sugars must be b and a hexoses,
respectively. The coupling constant
(J ¼ 2.8 Hz) of an oxymethine of b-sugar
at d 3.55 due to H-4 indicated it to be a
galactose unit, whereas the resonance of a
doublet methyl at d 1.12 indicated that the
a-sugar could be rhamnose.
2. Results and discussion
Compound 1 was isolated as yellow
amorphous powder whose HR-FAB-MS
(positive mode) exhibited molecular ion
peak at m/z 741.2045 [M þ H]þ corre-
sponding to the molecular formula
C36H36O17 with 19 double bond equival-
ences (DBE). The infrared (IR) spectrum
displayed absorption bands for hydroxyl
(3384 cm21), conjugated ester (1712 cm21),
conjugated ketone (1685 cm21), and phenyl
(1600, 1550, and 1520 cm21) groups,
whereas the UV spectrum showed absorp-
tion maxima at 257, 267, 314, and 357 nm
indicated the presence of an acylated
kaempferol or quercetin glycoside [14,15].
On addition of AlCl3 and AlCl3/HCl, it
showedabathochromicshiftof20–27nmin
The 13C NMR spectrum of 1 (Table 1)
was in full agreement with the mass and
proton data as it displayed signals for
1
various bands. The aromatic region of H
5'
OH
8
3'
HO
O
1'
1
8a
4a
1: R1 = R2 = H, R3 = E-p-coumaroyl
6
3
O
2: R1 = E-p-coumaroyl, R2 = H, R3 = E-p-coumaroyl
3: R1 = H, R2 = R3 = E-p-coumaroyl
OH
O
1''
OH
HO
O
5''
3''
1'''
HO
3'''
5'''
O
OR1
O
R3O
R2O
Figure 1. Structures of the new compounds isolated from A. javanica.

Journal of Asian Natural Products Research
3
d
d
d
d
d
d

4
S. Mussadiq et al.
kaempferol nucleus (d 181.0, 164.2, 161.0,
d
158.0, 157.5, 133.7, 104.5, 98.8, and 93.7),
p-coumarate (d 166.8, 159.8, 145.5, 129.6,
125.4, 115.4, and 113.4), and two sugar
moieties (d 105.0, 100.1, 73.8, 73.6, 73.5,
70.5, 70.3, 69.0, 68.3, 66.4, 66.0, and
16.7). All the assignments were carried out
through HSQC and COSY data. The
connectivity of the sugar units was
accomplished due to HMBC experiment
(Figure 2) in which the anomeric proton of
galactose moiety (d 4.58, H-100) showed a
long-range correlation with a quaternary
carbon at d 133.7 (C-3), whereas the
HMBC correlation of anomeric hydrogen
(d 4.32) of rhamnose suggested its
attachment to C-6 of the galactose which
was also evident from the downfield
chemical shift of C-6 (dC 66.0). The
relatively downfield shift (d 4.68) of H-4
of rhamnose unit and its HMBC corre-
lation with the carbonyl carbon at d 166.8
confirmed the connectivity of coumaroyl
moiety on C-4 of rhamnose.
d
d
Acid hydrolysis of 1 provided three
products that were separated by solvent
extraction. The ethyl acetate layer contains
kaemferol and coumaric acid. The glycone
part was separated from the aqueous layer
and purified using preparative thin-layer
chromatography (TLC) using a solvent
system of EtOAcZMeOHZH2OZAcOH
(4:2:2:2), and thus were identified as D-
galactose and L-rhamnose through their
optical rotation signs and comparison of
the retention time of their trimethylsilyl
(TMS) with that of the standards in gas
chromatography (GC).
d
d
The above discussed data were
further compared with the related pub-
lished compounds and found to be
closely resembled to that of isorhamne-
tin-3-O-b-D-[4000-p-coumaroyl-a-L-rham-
nosyl-(1 ! 6)]galactoside [15], with the
only difference that compound 1 contains
kaempferol nucleus instead of isorham-
netin. Therefore, compound 1 was finally
determined as kaempferol-3-O-b-D-[4000-
p-E-coumaroyl-a-L-rhamnosyl(1 ! 6)]
d

Journal of Asian Natural Products Research
5
OH
OH
OH
HO
O
O
O
HO
HO
O
O
O
O
HO
OH
HO
OH
HO
O
HO
OH
HO
O
HO
OH
OH
OH
O
O
O
O
O
O
OH
O
O
OH
O
O
O
O
O
O
O
O
O
O
HO
HO
O
OH
OH
OH
OH
OH
1
2
3
Figure 2. Key HMBC correlations observed in compounds 1–3.
galactoside, which is a new natural
product.
coumaroyl moiety was placed at C-4 of
rhamnose as observed in 1, whereas the
second moiety was found at C-3 of
galactose unit. Complete assignments of
proton and carbon chemical shifts and acid
hydrolysis of 2 and optical rotation values
of sugar units allowed the characterization
of the compound 2 as kaempferol-3-O-b-D-
[4000-p-E-coumaroyl-a-L-rhamnosyl(1 !
6)]-(300-p-E-coumaroyl) galactoside, which
is also a new natural product.
The molecular formula of compound 2
was established by positive HR-FAB-MS
that showed a quasi-molecular ion peak at
m/z 887.2404 corresponding to the mol-
ecular formula C45H42O19 with 25 DBE.
The UV and IR spectra of 2 were nearly
identical to that of 1, whereas the 1H NMR
spectrum was also similar (Table 1) with
the difference that compound 2 possesses
an additional p-coumaroyl moiety, which
also justified the additional nine carbon
atoms and six DBE in its formula when
compared with that of 1. The protons
resonating at d 7.66 (1H, d, J ¼ 16.0 Hz, H-
70000), 7.55 (1H, d, J ¼ 16.0 Hz, H-700000), 7.36
(2H, d, J ¼ 8.4 Hz, H-20000, 60000), 7.29 (2H, d,
J ¼ 8.8 Hz, H-200000, 600000), 6.76 (2H, d,
J ¼ 8.4 Hz, 30000, 50000), 6.73 (2H, d,
J ¼ 8.8 Hz, H-300000, 500000), 6.32 (1H, d,
J ¼ 16.0 Hz, H-80000), and 6.31 (1H, d,
Compound 3 was also isolated as light
yellow amorphous solid, whose HR-FAB-
MS in positive mode (m/z 887.2410)
depicted the molecular formula as
C45H42O19. The UV and IR data of 3 were
identical to those of 2, and indicated their
1
similar structural features. The H NMR
spectrum of 3 (Table 1) was almost super-
imposed to that of 2 with minor differences.
Analysis of the NMR data (Table 1) clearly
indicated the presence of kaempferol,
galactose, rhamnose, and two coumaroyl
groups. The downfield shift of H-4 (d 5.29)
of galactose and its HMBC correlation
(Figure 2c) with the carbonyl carbon
resonating at d 167.0 confirmed the
attachment of coumaroyl group at C-4 of
galactose moiety instead of C-3as observed
in 2, whereas the other coumaroyl moiety
was observed at C-4 of rhamnose unit.
Detailed analysis of 1H, 13C NMR,
HSQC, HMBC, and COSY data, and acid
hydrolysis confirmed compound 3 as
J ¼ 16.0 Hz, H-800000
) were identified
through COSY, HSQC, and HMBC data
for two coumaroyl moieties in 2. The rest of
the proton signals were attested for
kaempferol unit and two sugar moieties
and are shown in Table 1. Due to the
downfield shifts [d 4.80 (dd, J ¼ 7.6,
2.8 Hz, H-300) and 4.83 (t, J ¼ 7.2 Hz, H-
4000)] of galactose and rhamnose, respect-
ively, and their HMBC correlations
(Figure 2) with the carbonyl carbons
resonating at d 166.9 and 167.2, one

6
S. Mussadiq et al.
kaempferol-3-O-b-D-[4000-p-E-coumaroyl-
a-L-rhamnosyl(1 ! 6)]-400-p-E-coumar-
oylgalactoside, which is a new addition to
the list of natural products.
The acylated flavonoid glycosides 1–3
were evaluated for enzyme inhibition
activity against acetylcholinesterase
(AChE), butyrylcholinesterase (BChE),
and lipoxygenase (LOX) (Table 2). Com-
pound 1 exhibited weak activity against
the three enzymes with IC50 values of
205.1, 304.1, and 212.3 mM, respectively,
whereas compounds 2 and 3 were only
weakly active against AChE with IC50
values of 198.0 and 197.0 mM, respect-
ively. Due to limitation in enzyme
availability and other chemicals, the
known compounds (7–9) were not tested.
The phenolic esters, such as 7–9 are
reported in the literature as antimicrobial
agents [16] and/or as plant growth
inhibitors [11], whereas the phenolic
acids, such as 4–6 are known as potent
antioxidants [17]. Due to lack of facilities,
we could not perform antimicrobial or
antioxidant tests with our isolated com-
pounds, but it is evaluated that the
medicinal potential of A. javanica can be
attributed to these secondary metabolites.
3. Experimental
3.1 General experimental procedures
Optical rotations were measured on a
JASCO DIP-360 polarimeter (Tokyo,
Japan). UV spectra were obtained in
methanol on U-3200 Schimadzu UV-240
spectrophotometer (Duisburg, Germany).
IR spectra were recorded on Shimadzu 460
spectrometer (Duisburg, Germany). The
GC was performed on a Shimadzu gas
chromatograph (GC-9A; Noisiel, France)
(3% OV-1 silanized chromosorb W,
column temperature 1808C, injection port
and detector temperature 275–3008C, flow
rate 35 ml/min, flame-ionization detector).
EI-MS, HR-EI-MS, FAB-MS, and HR-
FAB-MS were recorded on Finnigan
(Varian MAT, Waldbronn, Germany)

Journal of Asian Natural Products Research
7
JMS H £ 110 with a data system and
JMSA 500 mass spectrometers. 1H
(400 MHz), 13C NMR (100 MHz), and
2D NMR (HMQC, HMBC, and COSY;
400 MHz) spectra were recorded on
Bruker spectrometer (Zurich, Switzer-
land). The chemical shift values (d) are
reported in ppm and the coupling constant
(J) is reported in Hz. Chromatographic
separations were carried out using alumi-
num sheets precoated with silica gel 60
EtOAc to get six subfractions (H1–H6).
The subfraction H5 (3.5 g) obtained with n-
hexane:EtOAc (4:6) was further purified
over silica gel column eluted with a mobile
phase of n-hexane:EtOAc in increasing
polarity order. Compound 1 (35.0 mg) was
obtained from this column at n-hexane:
EtOAc (3:7), whereas compounds 2
(28.5 mg) and 3 (25.0 mg) were obtained
at n-hexane:EtOAc (2.5:7.5) from the same
column. Fraction B (12.0 g) obtained from
the main column with n-hexane:CHCl3
(1:1) was chromatographed on silica gel
column to get further four subfractions
(B1–B3), out of which B3 (2.0 g) was
purified on silica gel column eluted with
n-hexane:CHCl3 (6:4) to afford compound
4 (32.0 mg) and a semi-pure fraction
which on further silica gel chromato-
graphic purification yielded compound 5
(22.0 mg). The subfraction B4 (2.5 g)
obtained from n-hexane:CHCl3 (4:6) was
also chromatographed on silica gel column
eluting with n-hexane:CHCl3 (3.5:7.5) to
get compound 6 (15.5 mg). The main
fraction A (45 g) was subjected to silica
gel column chromatography using gradient
of n-hexane:CHCl3 (7:3 to 0:10) as mobile
phase to get three subfractions (A1–A3).
The subfraction A3 (2.5 g) was further
purified on silica gel column eluting with n-
hexane:CHCl3 (6.5:3.5) to get compounds
7 (22.0 mg) and 8 (31.5 mg) in crystalline
form, whereas compound 9 (24.5 mg) was
obtained from the same column with n-
hexane:CHCl3 (6.7:3.3).
F
254 (20 £ 20 cm, 0.2 mm thick; E-Merck,
Darmstadt, Germany) for TLC and silica
gel (230–400 mesh; Darmstadt, Germany)
for column chromatography. TLC plates
were visualized under UV at 254 and
366 nm and by spraying with ceric sulfate
reagent solution (by heating).
3.2 Plant material
The flowers of A. javanica Burm (4 kg)
were collected from Cholistan Desert
(Bahawalpur) in September 2010 and
were identified by Dr. Muhammad Arshad
(late), Plant Taxonomist, Cholistan Insti-
tute for Desert Studies (CIDS), The
Islamia University of Bahawalpur, where
a voucher specimen is deposited
(AJ/CIDS-10-102).
3.3 Extraction and isolation
The shade-dried flowers of A. javanica
(4 kg) were extracted thrice with MeOH (15
liter). The combined MeOH extract was
concentrated, and the residue (160 g) was
divided into n-hexane (30 g), chloroform
(35 g), ethyl acetate (55 g), n-butanol
(20 g), and water-soluble (20 g) fractions.
The ethyl acetate fraction (55 g) was
subjected to column chromatography over
silica gel eluting with n-hexane, n-hexane–
CHCl3, CHCl3, and CHCl3ZCH3OH in
increasing order of polarity, and 10
fractions (A–J) were prepared. Fraction H
(15 g) obtained from CHCl3:CH3OH
(9.5:0.5) was chromatographed over silica
gel eluting with gradient of n-hexane:
3.3.1 Kaempferol-3-O-b-D-[4000-E-p-
coumaroyl-a-L-rhamnosyl(1 ! 6)]
galactoside (1)
26
Yellow amorphous solid (35 mg); ½aꢀD
2
45.5 (0.11, MeOH); UV (MeOH): lmax
(log 1, nm) 257 (4.23), 267 (4.34), 314
(4.35), 357 (4.44); þ AICI3, 273, 305,
405, þ AICl3, –HCI, 277, 305, 405; IR
(KBr): nmax 3384, 1712, 1685, 1600, 1550,
1520 cm21; 1H and 13C NMR spectral data:
see Table 1; HR-FAB-MS: m/z 741.2045

8
S. Mussadiq et al.
[M þ H]þ (calcd for C36H37O17, 741.
3.8 min; b-anomer, 5.2 min; and L-rham-
nose, 8.6 min) with the standards.
2030).
3.5 Enzyme inhibitory assays
3.3.2 Kaempferol-3-O-b-D-[4000-E-p-
coumaroyl-a-L-rhamnosyl(1 ! 6)]-300-E-
p-coumaroylgalactoside (2)
The AChE and BChE inhibition activity
was performed according to the published
method [18] with slight modifications. The
percentage inhibition was calculated by
the following equation:
Yellow amorphous solid (28.5 mg); ½aꢀ2D6 2
55.5 (0.15, MeOH); UV (MeOH): lmax
(log1, nm) 208 (4.33), 228 (4.30), 268
(4.34), 356 (4.45); IR (KBr): nmax 3385,
1715, 1680, 1605, 1550, 1520cm21; 1H and
13C NMR spectral data: see Table 1; HR-
FAB-MS: m/z 887.2404 [M þ H]þ (calcd
for C45H43O19, 887.2399).
Control2Test
Control
Inhibition ð%Þ ¼
£ 100:
3.5.1 LOX assay
LOX activity was assayed according to the
method described in the literature [19–21]
with slight modifications. The percentage
inhibition (%) was calculated by the
following formula:
3.3.3 Kaempferol-3-O-b-D-[4000-E-p-
coumaroyl-a-L-rhamnosyl(1 ! 6)]-400-E-
p-coumaroylgalactoside (3)
26
Yellow amorphous solid (25 mg); ½aꢀD
Control2Test
Control
Inhibition ð%Þ ¼
£ 100;
252.5 (0.11, MeOH); UV (MeOH): lmax
(log 1, nm) 207 (4.33), 226 (4.34), 268
(4.34), 355 (4.43); IR (KBr): nmax 3382,
where control is the total enzyme activity
without inhibitor and test is the activity in
the presence of test compound.
1
1715, 1683, 1604, 1550, 1521 cm21; H
and 13C NMR spectral data: see Table 1;
HR-FAB-MS: m/z 887.2410 [M þ H]þ
(calcd for C45H43O19, 887.2399).
All the measurements were carried out
in triplicate and statistical analysis was
performed by Microsoft Excel 2003.
Results are presented as mean ^ SEM.
IC50 values in all assays were calculated
using EZ–Fit Enzyme Kinetics software
(Perrella Scientific Inc., Amherst, NH,
USA).
3.4 Acid hydrolysis of compounds 1–3
A solution of 1–3 (8 mg each) in MeOH
(5 ml) containing 1 N HCl (4 ml) was
refluxed for 4 h, concentrated under
reduced pressure, and diluted with H2O
(8 ml). It was extracted with EtOAc and
the residue recovered from the organic
phase was found to be kaempferol and
coumaric acid. The aqueous phase was
concentrated and purified using prepara-
tive TLC using the solvent system
(EtOAcZMeOHZH2OZAcOH, 4:2:2:2)
and identified as D-galactose and L-
rhamnose by the sign of their optical
Acknowledgements
The authors thank Higher Education Commis-
sion (HEC) of Pakistan and Director ICCBS,
University of Karachi for providing equipment
access for spectroscopic analysis.
References
[1] E. Nasir and S.I. Ali, Flora of West
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[2] P. Chawla, A. Chawla, N. Vasudeva, and
S.K. Sharma, Acta Pol. Pharm. Drug Res.
69, 171 (2012).
20
20
rotations ½aꢀD þ 81.0 and ½aꢀD þ 8.5,
respectively. These sugars were also
confirmed by the retention time of their
TMS ether (D-galactose a-anomer,

Journal of Asian Natural Products Research
9
[3] L.M. Perry and J. Metzger, Medicinal
Plants of East and Southeast Asia (MIT
Press, Cambridge, 1980), p. 9.
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