Two antifungal active triterpenoid saponins from the seeds of Lathyrus plants

Article abstract of DOI:10.1080/14786419.2011.561205

Two novel triterpenoid glycosides have been isolated from butanolic seeds extract of two varieties of Lathyrus plants, i.e. Lathyrus ratan and Lathyrus aphaca. Their structures were elucidated as 3-O-[β-D-glucuronopyranosyl- (1→4)-α-L-arabinopyranosyl-(1→2)-α-L-arabinopyranosyl] -olean-11,13 (18)-dien-28-oic acid (1) and 3-O-{β-D-xylopyranosyl-(1 → 2)-β-D-glcopyranosyl-(1→4)-[β-D-glucopyranosyl-(1→2)]- α-D-xylopyranosyl}-2, 16-dihydroxy-4-hydroxymethyl urs-12-en-28-oic acid (2) on the basis of spectral evidences, i.e. FTIR, ¹H-NMR, ¹³C-NMR, ESI-MS and FAB-MS data. The isolated saponins were tested for their antifungal activity. Compound 1 showed maximum inhibition against Colletotrichum dematium (77.8%), whereas compound 2 showed maximum inhibition against Alternaria alternata (53.9%).

Full text of DOI:10.1080/14786419.2011.561205

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Two antifungal active triterpenoid
saponins from the seeds of Lathyrus
plants
a
Noor Afshan Khan
a
Department of Postgraduate and Research in Chemistry , R. D.
University of Jabalpur , Jabalpur – 482001, M.P., India
Published online: 08 Sep 2011.
To cite this article: Noor Afshan Khan (2011) Two antifungal active triterpenoid saponins from
the seeds of Lathyrus plants, Natural Product Research: Formerly Natural Product Letters, 25:18,
1687-1694, DOI: 10.1080/14786419.2011.561205
To link to this article: http://dx.doi.org/10.1080/14786419.2011.561205
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Natural Product Research
Vol. 25, No. 18, October 2011, 1687–1694
Two antifungal active triterpenoid saponins from the seeds
of Lathyrus plants
Noor Afshan Khan*
Department of Postgraduate and Research in Chemistry, R. D. University of Jabalpur,
Jabalpur – 482001, M.P. India
(
Received 1 October 2009; final version received 16 August 2010)
Two novel triterpenoid glycosides have been isolated from butanolic seeds
extract of two varieties of Lathyrus plants, i.e. Lathyrus ratan and Lathyrus
aphaca. Their structures were elucidated as 3-O-[ꢀ-D-glucuronopyranosyl-
(
(
1 ! 4)-ꢁ-L-arabinopyranosyl-(1 ! 2)-ꢁ-L-arabinopyranosyl]-olean–11,13
18)–dien–28-oic acid (1) and 3-O-{ꢀ–D–xylopyranosyl–(1 ! 2)–ꢀ-D-glco-
pyranosyl–(1 ! 4)–[ꢀ-D-glucopyranosyl–(1 ! 2)]–ꢀ-D-xylopyranosyl}–2,
1
6ꢁ-dihydroxy–4-hydroxymethyl urs-12-en-28-oic acid (2) on the basis of
1 13
spectral evidences, i.e. FTIR, H-NMR, C-NMR, ESI-MS and FAB-MS
data. The isolated saponins were tested for their antifungal activity.
Compound 1 showed maximum inhibition against Colletotrichum dema-
tium (77.8%), whereas compound 2 showed maximum inhibition against
Alternaria alternata (53.9%).
Keywords: Lathyrus ratan; Lathyrus aphaca; Triterpenoid saponin;
Caesalpiniaceae; antifungal activity
1
. Introduction
Lathyrus is a large genera with 190 species and subspecies being recognised.
The plants are commonly found in Central India (Allkins, Macfarfarlane, &
White, 1983). Lathyrus ratan is a hybrid variety of grass pea developed by scientists
at Plant Breeding Department, JNKVV, Jabalpur. Despite its tolerance to drought,
grasspea is not affected by excessive rainfall; it can be grown on land subject to
flooding and therefore is a popular crop in subsistence farming (Sinha, 1980).
Its seeds are consumed as pulses as it contains a high amount of free L-homoarginine,
which may act as a precursor of lysine in human nutrition (Qureshi, Pilbeam,
Evans, & Bell, 1977). Oil obtained from the seeds of L. ratan is cathartic
(Ambasta, Ramchandran, & Kashgapa, 1986), and it contains sterols, cholesterol,
24-epicholesterol and isoavenasterol and triterpene alcohols (Rotter, Marquarelt,
Low, & Briggs, 1990).
Lathyrus aphaca (Jangli matar) is a common weed of wheat fields and is
commonly used as fodder. The ripen seeds produce narcotic effect and are considered
to be the possible cause of Lathyrism (Anonymous, 2000). The flower contains
*Email: noor_afshan25@yahoo.com
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2011 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2011.561205
http://www.tandfonline.com

1
688
N.A. Khan
flavonol glycosides, mainly larycitrin and syringetin-3-O-rutinoside-7-O-ꢀ-D-gluco-
pyraoside accompanied by a low level of kempherol, quercetine, isorhamnetin
analogous and the respective 3-O-rutinosides (Markham & Hammett, 1994).
The proximate principles of L. aphaca and certain other leguminous seeds have
already been determined by the authors (Dubey, Khan, & Srivastava, 2008; Dubey,
N.K. Saxena, N. Saxena, & Srivastava, 2005).
In this study, two new triterpenoid glycosides were isolated from butanolic seeds
extract of the two plants. Biological screening of both the compounds showed
significant antifungal activity.
2
. Results and discussion
The n-BuOH soluble fractions of methanolic seeds extract of Lathyrus plants were
separated by column chromatography on silica gel. Lathyrus ratan extract afforded
compound 1 whereas compound 2 was obtained from L. aphaca seeds extract. Both
the compounds gave positive result for Libermann–Burchard test for triterpenoid
saponins. The nature of compounds was further confirmed by spectroscopic data.
Compound 1, isolated from L. ratan seeds, yielded aglycone along with three
sugar moieties on acid hydrolysis. The aglycone sapogenin was identified as oleanolic
1
acid by Co-TLC analysis using authentic sample and comparing its NMR data ( H
1
3
and C) with literature (Taketa, Schlager, Guillaume, Gosmann, & Schenkel, 2000).
The sugar components in the hydrolysate were identified to be D-glucuronic acid and
L-arabinose in 1:2 ratio (Sun, Ligne, & Huystee, 1997). The ESI–MS of compound
þ
1
formula to be C H O . The fragments at m/z 718, 586 and 454 attributed to the
showed molecular ion peak [M þ 2H] at m/z 896, suggesting the molecular
4
6
70 17
loss of terminal glucuronic acid unit followed by the loss of two arabinose. The result
of ESI–MS confirms the sequence of sugar units in 1. The presence of glucuronic acid
as terminal sugar was confirmed by partial hydrolysis of compound 1 on TLC in HCl
atmosphere followed by Co-TLC with an authentic sample and HPLC chromato-
gram (Miyase, Shiokawa, Zhang, & Ueno, 1996).
1
In H-NMR spectrum of compound 1, the doublets at ꢂ 4.42 (1H d, J ¼ 6.5 Hz),
H
5
.73 (1H d, J ¼ 5.3 Hz) and 5.71 (1H d, J ¼ 3.51 Hz) were assigned to three anomeric
13
protons of the sugars (Zhao et al., 1999). The proton noise decoupled C-NMR
spectrum of 1 display 46 carbon resonance peaks. The number of hydrogen attached
to each carbon atoms were determined by DEPT technique (Doddrell, Pegg, &
Bendel, 1982). The presence of three anomeric carbon signals at ꢂ 101.34, 101.54 and
c
1
05.32 ppm in C-NMR spectrum further confirms the presence of trisaccharide
3
1
moiety in 1 (Nikaido, Koike, Mitsunaga, & Saeki, 1999). The configuration of
glucuronic acid was determined to be ꢀ and that of arabinose to be ꢁ on the basis of
chemical shift and coupling constant of these signals when were compared with the
reported value (Rios, Berenice, & Guadarrama, 2004).
The inter-glycosidation assignment was further confirmed by the chemical shift
of glycosylated carbon atom. The C-2 of Ara-I was observed at ꢂc 84.76 whereas
C-4 signal of Ara-II at ꢂc 82.36 reveals the deshielding of carbon by 4 and 6 ppm
for these carbon resonance; hence C-2 in Ara-I and C-4 in Ara-II were concluded
to be the glycosidation site. The trisaccharide moiety in 1 was linked at C-3 of
1
3
aglycone as C-3 showed significant downfield shift (ꢂc 89.20) in C-NMR spectra

Natural Product Research
1689
indicating the glycosidation position (Abdel-khader et al., 2000), which was further
confirmed by acid and enzymatic hydrolysis. On the basis of above-mentioned
evidences, the structure of 1 was determined as 3-O-[ꢀ-D-glucuronopyranosyl-
(
2
1 ! 4)-ꢁ-L-arabinopyranosyl-(1 ! 2)-ꢁ-L-arabinopyranosyl]-olean–11,13(18)–dien–
8-oic acid (Figure 1).
Compound 2, obtained from the butanolic extract of L. aphaca seeds, on
acid hydrolysis yields aglycone sapogenins that was identified to be ursolic acid by
Co-TLC with authentic sample and comparing its NMR data with the literature
(Grishkovets, Sobolev, Shashkov, & Chirva, 2000; Weiss & Seebacher, 2002).
The aqueous hydrolysate containing sugar moieties were identified to be
D-glucose and D-xylose in 1:1 ratio, by comparing HPLC chromatogram of sample
with standard. The molecular formula of compound 2 was established to be
C H O by positive FAB-MS, which exhibited pseudo molecular ion peak
5
2
84 24
þ
[
M þ 3H] at m/z 1095 indicating the molecular mass to be 1092. The sequence of
sugar moiety in compound 2 was confirmed by fragment ion peaks at m/z 932, 796,
34 and 506. The partial hydrolysis followed by Co-TLC with authentic sample and
HPLC chromatogram showed xylose to be the terminal sugar.
6
29
3
0
2
0
1
9
21
E
18
22
1
2
1
1
13
17
16
C
26
COOH
D
28
2
5
1
9
14
2
3
10
8
7
15
27
A
B
Ara I
O
O
5
HO
HO
O
4
6
2
3
24
O
30
OH
Ara II
20
29
HOOC
1
9
21
22
O
O HO
E
HO
HO
1
2
18
GlcA OH
1
1
1
13
17
16
C
COOH
D
28
2
5
26
1
9
14
27
HO
OH
2
10
8
15
Glc I
O
B
A
HOH C
2
Xyl I O
HO
3
7
5
O
O
HO
HO
4
6
HOH C
CH
3
2
23
O
O
O
O
24
HO
HO
HOH C
2
OH
Xyl II OH
Glc II
HOHO
2
Figure 1. Structure of triterpenoid saponins 1 and 2.

1
690
N.A. Khan
1
The H-NMR spectrum of 2 showed doublets at ꢂ 6.09 (1H d, J ¼ 7.6 Hz), 6.10
H
(
1H d, J ¼ 4.32 Hz), 5.79 (1H d, J ¼ 3.42 Hz) and 5.71 (1H d, J ¼ 5.312 Hz), which
were assigned to four anomeric protons of the sugars (Tamai et al., 1989).
3
1
The proton noise decoupled C-NMR spectrum of 2 displayed 52 carbon resonance
peaks. The presence of five quaternary carbon atoms, 26ꢀCH, 12ꢀCH , 6ꢀCH and
2
3
2
three sp (CH¼, C¼ and C¼O) hybrid carbon atoms were determined by
DEPT technique. The four anomeric carbon signals at ꢂc 104.78, 105.14, 106.31
and 106.10 ppm further confirmed the presence of tetrasaccharide moiety in 2.
The chemical shift and coupling constant of these signals suggested the ꢀ anomeric
configuration for all sugar moieties (Kovacik et al., 1995).
The chemical shift values of glycosylated carbon atoms in sugar moiety reveals
the peak at ꢂc 81.21, 79.14 and 82.46 ppm. The downfield shift of carbon signal
suggested that C-2 and C-4 in Xyl-I, and C-2 in Glu-I were concluded to be the
glycosidation site. The tetrasaccharide moiety in 2 was linked at C-3 of aglycone as
1
3
C-3 showed significant downfield shift (ꢂc 84.12) in C-NMR spectra indicating the
glycosidation position. The structure of 2 was elucidated as 3-O-{ꢀ-D-xylopyranosyl–
(
1 ! 2)–ꢀ-D-glucopyranosyl–(1 ! 4) –[ꢀ-D-glucopyranosyl–(1 ! 2)]–ꢀ-D-xylopyra-
nosyl}–2,16ꢁ-dihydroxy–4-hydroxymethyl urs-12-en-28-oic acid (2) (Figure 1).
The isolated compounds were tested for their antifungal activity against human
and plant pathogenic fungi. The compounds showed maximum inhibition at
ꢁ1
1
000 mg mL . At this concentration, compound 1 shows maximum inhibition
against Colletotrichum dematium (77.8%) and minimum against Curvularia lunata
27.8%). Similarly, compound 2 showed maximum inhibition of 53.9% against
(
Atlernaria alternata and minimum against Aspergillus flavus (29.5%) (Table 1).
Table 1. In vitro fungicidal bioassay (% inhibition) of compounds 1 and 2.
ꢁ
1
Concentration (mg mL
250 500
)
Positive control
(S)
Name of fungus
100
1000
Aspergillus flavus (FGCC-133)
1
2
Alternaria alternata (FGCC-418)
6.8
–
11.3
–
21.3
16.4
33.5
29.5
98
98
1
22.5
25.2
36.5
35.6
47.5
44.8
59.4
53.9
100
100
2
Collatotrichum dematium
(
FGCC-165)
1
2
22.5
17.6
36.1
35.5
37.7
42.1
77.8
50.5
100
100
Fusarium roseum (FGCC-500)
1
2
Curvularia lunata (FGCC-280)
14.5
–
24.5
10.1
29.4
21.5
36.7
29.6
100
100
1
2
4.6
4.9
10.5
15.1
12.7
19.8
27.8
35.9
100
100
Notes: The results are mean of triplicates reading. The mean were compared by the analysis of
ꢁ1
variance (ANOVA) with 5% significance level. S ¼ Fluconazole (20 mg mL ), Negative
control (10% MeOH) ¼ Shows no inhibition.

Natural Product Research
1691
3
3
. Experimental
.1. General methods
Melting points were measured on an MAC model melting point apparatus and were
uncorrected. Optical rotations were measured on Rudolf Autopol III polarimeter.
HPLC of sugars was done on Shimadzu 10 A Liquid Chromatography system using
EL-SD Detector. UV spectra were measured on Unicam Thremospectronic UV-500
1
13
model Double Beam spectrophotometer in MeOH solution. H-NMR and C-NMR
were recorded on Bruker DRX 300 model operating at 300 and 75 MHz (CD OD or
3
CDCl ). All the NMR spectra were recorded using TMS as internal standard. IR
3
spectra were recorded on a Shimadzu 8400 S spectrophotometer having a range of
4
ꢁ1
000–450 cm and sample was prepared in powered KBr in a ratio 1:3. The ESI-MS
was recorded on a MICROMASS QUATTRO II triple quadrupole mass spectro-
meter. The ESI capillary was set at 3.5 kV and the cone voltage was 40 V. FAB-MS
was recorded on a Jeol SX 102/DA-6000 spectrometer using argon as FAB gas
and accelerating voltage of 10 KV with nitro benzyl alcohol as matrix. Column
chromatography was carried out on silica gel (B.D.H.; 60–120 mesh), TLC and
preparative TLC on 20ꢀ20 cm plates coated with 2-mm thick silica gel (Merk; F254).
ꢂ
Spots were visualised by 10% ethanolic H SO , followed by heating at 110 C. Paper
2
4
chromatography of sugars was performed on Whatman no.1 paper using descending
mode in n-BuOH:AcOH:H O (BAW 4:1:5) and developed with aniline hydrogen
phthalate.
2
3
.2. Plant material
The seeds of plant L. ratan (Commercial variety) and L. aphaca (Wild variety) were
collected from Plant Breeding Department, and wheat fields of Jawaharlal Nehru
Krishi Vishwavidyalaya, Jabalpur, in April 2005. The seeds of L. aphaca were further
identified by Head, National Research Centre for Weed Sciences, Jabalpur, and a
voucher specimen (NRCWS- LA121) was deposited in the herbarium of this
institute.
3
.3. Extraction and isolation
The air-dried and powdered seeds (1 Kg each) were extracted with petroleum ether
ꢂ
(60–80 C) for 12–14 h.The defatted seeds powder was then extracted with MeOH for
18–20 h and combined extract was concentrated in vacuum. The resulting dark yellow
residue (150 g) was suspended in water. The aqueous methanolic extract was then
fractionated successively with n-Hexane, CHCl and n-BuOH to get a total of four
3
fractions. The bioactive n-BuOH fraction (20 g) was subjected to column chroma-
tography on silica gel (100 g, 60–120 mesh) using CHCl –MeOH–H O (v:v ¼
3
2
70 : 25 : 5–50 : 45 : 5) with a 5 mL each as gradient eluent to give 48 fractions and was
monitored by TLC. The fractions 25–36 showing same R on TLC were pooled
f
together and recolumn chromatographed on silica gel with CHCl MeOH (60 : 40–
3
:
50 : 50), followed by preparative TLC in EtOAc: MeOH: H2O (13:8:2) to yield
saponin 1 and 2.

1
692
N.A. Khan
3
.3.1. Oleanolic acid (1)
ꢂ
25
D
A dark yellow amorphous powder; m.p. 190–194 C; ½ꢁꢃ ¼ þ20.8 (MeOH; c ¼ 1.05);
ꢁ
1
UV (MeOH) ꢃ : 286.4 and 341.6 nm; IR (KBr)v max (cm ): 3687, 1714, 1589,
max
1
1
0
5
499,1360; H-NMR (CD OD, 300 MHz) ꢂ : 0.974, 0.961,0.950, 0.931,0.912, 0.882,
3 H
.873 (3H, s, 7ꢀMe), 4.715 (1H, d, J ¼ 5.6 Hz, H-3), 5.364 (1H, br s, H-11),
1
3
.105 (1H, br s, H-12); C-NMR (CDCl , 75 MHz) ꢂ : 38.87 (C-1), 26.21(C-2), 89.20
3
c
(C-3), 40.27 (C-4), 48.52 (C-5), 14.57 (C-6), 33.16 (C-7), 40.41 (C-8), 52.21 (C-9),
3
2
3
1
7
5.12 (C-10), 124.37 (C-11), 127.93 (C-12), 132.61(C-13), 42.23 (C-14), 28.89(C-15),
4.13(C-16), 47.12 (C-17), 138.01 (C-18), 46.30 (C-19), 30.84 (C-20), 34.96 (C-21),
2.77 (C-22), 23.72 (C-23), 17.50 (C-24), 19.70 (C-25), 18.25 (C-26), 25.07 (C-27),
þ
þ
82.90 (C-28), 23.83 (C-29), 16.25 (C-30); ESI-MS m/z 896 [M þ 2H] 895 [M þ H]
,
,
18, 586, 454, 246, 208,201 and 190.
3
.3.2. Ursolic acid (2)
ꢂ
25
D
A light yellow amorphous powder; m.p. 208–211 C; ½ꢁꢃ ¼ ꢁ13.6 (MeOH; c ¼ 0.95);
ꢁ1
UV (MeOH) ꢃ : 224 and 295 nm; IR (KBr)v max (cm ): 3751, 1677, 1571,
max
427,1360; H-NMR (CD OD, 300 MHz) ꢂ : 0.925, 0.892, 0.932, 0.914, 0.964, 0.944
3 H
1
1
(
3 H, s, 6ꢀMe), 3.86 (1H, d, J ¼ 9.81 Hz, H-2), 4.37 (1H, d, J ¼ 7.21 Hz, H-3),
5
H-16); C-NMR (CDCl , 75 MHz) ꢂ : 45.02 (C-1), 71.03 (C-2), 84.12 (C-3), 44.50
.21 (1H, t-like, H-12), 3.67 (1H, br s, J ¼ 7.56 Hz, H-16), 3.52 (1H, d, J ¼ 9.03 Hz,
1
3
3
c
(C-4), 53.20 (C-5), 18.50 (C-6), 31.90 (C-7), 41.23 (C-8), 47.32 (C-9), 37.31 (C-10),
2
4
6
1
3.46 (C-11), 124.01 (C-12), 141.02 (C-13), 43.01 (C-14), 29.69 (C-15), 65.13 (C-16),
8.91 (C-17), 52.60 (C-18), 39.51 (C-19), 40.21 (C-20), 30.90 (C-21), 37.01 (C-22),
3.95 (C-23), 16.42 (C-24), 17.25 (C-25), 18.01 (C-26), 24.02 (C-27), 179.05 (C-28),
8.21 (C-29), 21.08 (C-30); FAB-MS m/z 1095 [M þ 3H] 932, 796, 634, 506, 490, 296
þ
and 265.
3
.3.3. Acid hydrolysis
Saponins 1 and 2 (25 mg each) were refluxed with 10% H SO on a boiling water
4
2
bath for 4 h, respectively. The hydrolysed contents were then neutralised with BaCO3
solution. The neutralised portions were extracted with EtOAc. The EtOAc fraction
when dried afforded the aglycone moiety, whereas the aqueous fraction contains
hydrolysed sugars.
3
.3.4. Identification of sugar moieties of 1 and 2
The neutralised aqueous layer separated after the removal of sapogenin was filtered
and concentrated under reduced pressure. The residue obtained was compared with
standard sugar on TLC and paper chromatography (BAW 4:1:5) indicating them to
be D-glucuronic acid and L-arabinose in compound 1 and D-xylose and D-glucose in
compound 2.
3
.4. Premethylation of 1 and 2
A solution of 1 and 2 (15 mg) in DMSO was treated with NaH (0.2 g) and CH3I
5 mL) at room temperature for 6 h. The usual work up of the reaction mixture
(

Natural Product Research
1693
yielded a residue, which was purified by preparative-TLC in n-hexane-EtOAc (1:1).
Hydrolysis of premethylated 1 and 2 was performed by refluxing with 10 mL of 5%
methanolic HCl. Paper chromatography of the neutralised and concentrated
hydrolysate in benzene:acetone (3:1) showed the presence of 3,4-di-O-methyl-L-
arabinose, 2,3-di-O-methyl-L-arabinose, 2,3,4-tri-O-methyl-D-glucuronic acid in
compound 1, and 3-O-methyl-D-xylose, 3,4,6-tri-O-methyl-D-glucose, 2,3,4-tri-O-
methyl-D-xylose and 2,3,4,6-tetra-O-methyl-D-glucose in compound 2 (paper
chromatography).
4
. Antifungal activity
The antifungal activity was performed on five human and plant pathogenic fungi by
Poison food technique’ (Grover & Moore, 1962). The maximum inhibition by
compound 1 was recorded in C. dematium (77.8%) and minimum in C. lunata
27.8%), whereas for compound 2 maximum inhibition was shown against
A. alternata (53.9%) and minimum against A. flavus (29.5%) at a concentration of
‘
(
ꢁ1
1
000 mg mL . The crude extract was found to be more effective than the pure
compound.
Acknowledgements
The authors are thankful to the Central Drug Research Institute, Lucknow, for providing
instrumental facility and to Head, Department of Bioscience, R.D. University, Jabalpur, for
providing the research laboratory to carry out the antimicrobial assay.
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