A triterpenoid saponin from the seeds of Ricinus communis and its antimicrobial activity

Article abstract of DOI:10.1007/s10600-013-0504-5

A new oleanene triterpenoid was isolated from the butanolic seed extract of Ricinus communis. Its structure was elucidated as 3-O-[β-D- glucuronopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2) β-D-glucopyranosyl]-4α,20α-di(hydroxymethyl)olean-12-en-28-oic acid on the basis of spectral evidence i.e., FTIR, ¹H and ¹³C NMR, and FAB-MS data. The isolated triterpenoid was found to have antimicrobial potential, viz. antibacterial and antifungal activity, where the growth of microbes was found to be an inverse function of the concentration of saponin. The MIC value for E. coli, Klebsiella pneumoniae and Staphylococcus aureus were found to be 260, 235, and 350 μg/mL.

Full text of DOI:10.1007/s10600-013-0504-5

Chemistry of Natural Compounds, Vol. 49, No. 1, March, 2013 [Russian original No. 1, January–February, 2013]
A TRITERPENOID SAPONIN FROM THE SEEDS OF Ricinus communis
AND ITS ANTIMICROBIAL ACTIVITY
1
2*
Chetna Acharya and Noor Afshan Khan
UDC 547.918
A new oleanene triterpenoid was isolated from the butanolic seed extract of Ricinus communis. Its structure
was elucidated as 3-O-[ꢀ-D-glucuronopyranosyl-(1ꢁ3)-ꢂ-L-rhamnopyranosyl-(1ꢁ2)ꢀ-D-glucopyranosyl]-
1
4ꢂ,20ꢂ-di(hydroxymethyl)olean-12-en-28-oic acid on the basis of spectral evidence i.e., FTIR, H and
13
C NMR, and FAB-MS data. The isolated triterpenoid was found to have antimicrobial potential, viz.
antibacterial and antifungal activity, where the growth of microbes was found to be an inverse function of
the concentration of saponin. The MIC value for E. coli, Klebsiella pneumoniae and Staphylococcus aureus
were found to be 260, 235, and 350 ꢃg/mL.
Keywords: Ricinus communis, triterpenoid saponin, Euphorbiaceae, antimicrobial activity.
Plants with antimicrobial properties are being increasingly reported from different parts of the world. Further, the
search for new plants with potential antimicrobial properties has intensified owing to the side effects associated with traditional
antibiotics. Ricinus communis Linn. (Euphorbiaceae) grows as a weed even along roadsides in tropical warm regions [1]. It is
also cultivated in different parts of the country for its oilseeds [2, 3]. The oil is used externally for dermatitis and ailments of
the eye. The leaves have been reported to have flavones and tannins [4], whereas the roots of this plant have been found to
contain alkaloids along with flavonoids and tannins [5]. The seeds, which yield 45–50% of a fixed oil, also contain the
alkaloids ricinine and toxalbumin ricin. It is considered to be purgative and a counter-irritant in scorpion-sting and fish poison
[6]. In continuation of our interest in triterpenoid saponins [7], we have investigated the seeds of the plant and report here a
new oleanene type triterpenoid for the first time.
+
Saponin 1, mp 281ꢄC, C H O [M] – 973, was obtained by repeated CC purification of the n-BuOH fraction
48 77 20
followed by preparative TLC. Its UV spectra contained absorption maxima at 242 nm, while the IR spectrum exhibit peaks at
–1
–1
3400–3750 (OH), 2938 (C-H), 1651 (C=C), and 1044 (C-O) cm . The broadband at 3420 cm indicates its glycosidic nature.
29
30
HOH C
2
19
21
17
11
13
26
25
COOH
1
28
10
15
HOH C
HO
HO
2
3
7
27
O
O
5
O
HOH C
2
24
23
O
O
H C
3
OH
HO
HOOC
HO
HO
O
1
OH
1) Department of Engineering Chemistry, Sagar Institute of Science Technology & Research (SISTec-R), Sikandrabad,
Rathibad, Bhopal, India; 2) Analytical Instruments Division, CSIR-National Environmental Engineering Research Institute,
440020, Nagpur, India, e-mail: noor_afshan25@yahoo.com. Published in Khimiya Prirodnykh Soedinenii, No. 1, January–February,
2013, pp. 48–50. Original article submitted December 12, 2011.
54
0009-3130/13/4901-0054 02013 Springer Science+Business Media New York

13
TABLE 1. C NMR Chemical Shift and DEPT Data of Saponin 1
C atom
DEPT
C atom
DEPT
ꢅC
ꢅC
1
2
3
4
5
6
7
8
39.6
27.2
87.1
44.2
48.1
18.9
32.1
40.2
50.9
36.9
24.6
125.9
131.2
39.8
33.1
24.0
47.5
42.5
46.5
31.9
34.6
33.6
62.4
13.8
24.7
17.5
CH2
CH2
CH
C
CH
CH2
CH2
C
CH
C
CH2
CH
C
27
28
29
30
Glc
1
2
3
4
5
6
Rha
1
2
3
4
22.6
176.0
63.5
CH3
C
CH2
CH3
29.0
104.3
84.5
74.4
69.5
79.9
62.2
CH
CH
CH
CH
CH
CH2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
101.4
72.0
82.7
73.1
69.6
18.4
CH
CH
CH
CH
CH
CH3
C
CH2
CH2
C
CH
CH2
C
CH2
CH2
CH2
CH3
CH3
CH3
5
6
Glc A
1
2
3
4
5
6
104.8
79.5
76.6
76.9
75.5
CH
CH
CH
CH
CH
175.1
C (COOH)
Saponin 1 on acid hydrolysis yields sapogenin 2, mp 192ꢄC, as the aglycone, which was identified by comparison of
physical and spectral properties [8, 9]. A direct comparison with authentic samples (mp, co-TLC) also establishes the identity.
The sugar components in the hydrolysate were identified as D-glucose, L-rhamnose, and D-glucuronic acid in the ratio 1:1:1,
indicating 1 to be a sapogenin triglycoside. The sugar ratio was established by comparing with the HPLC chromatogram of the
+
standard. The position of the FAB-MS showed a molecular ion peak at m/z 996 [M + Na] , indicating a molecular mass of 973,
which is in good agreement with the molecular formula C H O . The fragment at m/z 819 is consistent with the loss of a
48 77 20
+
terminal glucuronic acid unit from the molecular ion, whereas the other fragments at m/z 673 [M + Na – (178 + 146)] and 511
[M + Na – (178 + 146 + 162)] were attributed to the loss of the rhamnose and glucose units, respectively. The results obtained
+
by FAB-MS indicated the sugar sequence in 1. The interglycosidic linkages in 1 were revealed by permethylation followed by
acid hydrolysis, which liberated one molecule each of 3,4,6-O-methyl-D-glucose, 2,4-di-O-methyl-L-rhamnose and 2,3,4-tri-
O-methyl-D-glucuronic acid, as confirmed by HPLC. The presence of glucuronic acid as the terminal sugar was confirmed by
detection on partial hydrolysis of 1 on TLC in HCl atmosphere. Partial hydrolysis of 1 with methanolic H SO also yielded
2
4
+
glucuronic acid and a prosaponin, which display an [M + Na] ion at m/z 819 in FAB-MS. The presence of glucuronic acid in
the hydrolysate was confirmed by co-TLC with an authentic sample and by HPLC.
13
The proton noise decoupled C NMR spectrum of l displayed 48 carbon resonances. The number of attached hydrogens
to each carbon was determined by the DEPT technique [10], which suggested the presence of 6 quaternary C atoms, 19 CH, 13
2
CH , 6 CH , and 4 sp hybrid carbon atoms (CH=, C=, and C=O for aglycone and C=O for sugar), indicating the presence of
2
3
a trisaccharide moiety in triterpene (Table 1). The presence of three anomeric carbon signals at ꢅ 104.3, 104.8, and 101.4 was
in accordance with the presence of the trisaccharide moiety in 1. On the basis of the DEPT spectrum, the molecular formula of
13
1 can be assigned as C H O . A comparison of the C NMR spectral data of the aglycone moiety of 1 with those of the
48 77 20
aglycone of triterpene further confirmed its identity [11, 12].
The inter-glycosidation assignments were further confirmed by the chemical shifts of the glycosylated carbon atoms
at ꢅ 82.7 and 84.5. The C-3 signal of rhamnose was observed at 82.7 whereas the C-2 signal of glucose at 84.5 revealed the
deshielding of carbon by 4 and 6 ppm for these carbon resonances; hence C-2 in glucose and C-3 in rhamnose were concluded
to be the glycosidation site. In addition, the presence of a trisaccharide moiety in 1 was suggested by the absorbance of the
55

proton signals at ꢅ 4.89 (1H, d, J = 7.6 Hz), 6.34 (1H, d, J = 2.7 Hz), and 4.60 (1H, d, J = 7.8 Hz) for glucose, rhamnose, and
glucuronic acid. The chemical shift and coupling constant of these signals suggested the ꢀ-anomeric configuration for glucose
and glucuronic acid and ꢂ for rhamnose when compared with the reported values. The trisaccharide moiety in 1 was linked at
13
C-3 of the aglycone as C-3 showed a significant downfield shift (ꢅ 87.1 ppm) in the C NMR spectra, indicating the glycosidation
position [13]. Further, the glycoside was hydrolyzed with 10% sulfuric acid, which is a specific reagent for hydrolyzing only
the ꢀ-glycosidic linkage without attacking other sugar ester linkages. Thus, sugars are attached through the glycosidic linkage.
13
13
The C NMR spectral data of aglycone is in good agreement with the C NMR data of saponin 1 and other related saponins.
In light of the above observation, the structure of 1 was established as 3-O-[ꢀ-D-glucuronopyranosyl-(1ꢁ3)-ꢂ-L-
rhamnopyranosyl-(1ꢁ2)-ꢀ-D-glucopyranosyl]-4ꢂ,20ꢂ-di(hydroxymethyl)-olean-12-en-28-oic acid.
Seeded broth control showed maximum growth of bacteria; on addition of saponin, growth decreased with increase in
saponin concentration. The MIC values for E. coli, Klebsiella pneumoniae and Staphylococcus aureus were found to be
260, 235, and 350 ꢃg/mL.The growth of fungus was found to be an inverse function of the concentration of saponin. The
relationship was determined by observing the number of fungal colonies that developed.
EXPERIMENTAL
Melting points were measured on a MAC model melting point apparatus and are uncorrected. Optical rotations were
measured on a Rudolf Autopol III polarimeter. UV spectra were measured on a Thremospectronic UV 100 model
1
13
spectrophotometer in MeOH solution. H NMR and C NMR were recorded on a Bruker DRX 300 model operating at 300 MHz
and 75 MHz (CD OD or CDCl ). All the NMR spectra were recorded using TMS as internal standard. IR spectra (KBr disc)
3
3
–1
were recorded on a Perkin–Elmer spectrum RX I spectrophotometer having a range of 4000–450 cm . FAB-MS was recorded
on a Jeol SX 102/DA-6000 spectrometer using argon as FAB gas and an accelerating voltage of 10 kV with nitrobenzyl
alcohol as matrix. Column chromatography was carried out on silica gel (B.D.H.; 60–120 mesh), and TLC and preparative
TLC on 20 ꢆ 20 cm plates coated with 2 mm thick silica gel (Merk; F ). Spots were visualized by 10% H SO , followed by
254
2
4
heating at 110ꢄC. Paper chromatography of sugars was performed on Whatman No. 1 paper in the descending mode in
n-BuOH–AcOH–H O (BAW 4:1:5) and developed with aniline hydrogen phthalate.
2
Plant Material. The seeds of Ricinus communis were collected from the campus of Rani Durgawati University,
Jabalpur, M. P. India. The Head, Department of Biosciences, R. D. V. V. identified the seeds and a voucher specimen was
deposited in the Herbarium of the Department.
Extraction Method. The air-dried and powdered seeds (1 kg) were extracted with petroleum ether (60–80ꢄC) for
12–14 h. The defatted seed powder was then extracted with MeOH for 18–20 h, and the 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 give a total of four fractions.
3
Purification and Isolation of Compound. The n-BuOH fraction (20 g) was subjected to column chromatography on
silica gel (100 g, 60–120 mesh) using CHCl –MeOH–H O (65:25:10 to 50:40:10 v/v), with 5 mL each as gradient eluent to
3
2
give 48 fractions and monitored by TLC. Fractions 25–36 showing the same R on TLC were pooled together and re-colum-
f
chromatographed on silica gel with CHCl –MeOH (60:40 to 50:50), followed by preparative TLC in EtOAc–MeOH–H O
3
2
(13:8:2) to yield saponin 1.
–1
Saponin 1. Amorphous powder (70 mg), mp 281ꢄC, [ꢂ] +26ꢄ (c 1.36; MeOH). IR (ꢇ , cm ): 3755.8, 3420,
2938, 1735.0, 1651, 1411.6, 1381.9, 1251.0, 1044, and 725. H NMR (300 MHz, ꢅ, ppm, J/Hz): 4.89 (1H, d, J = 7.6, H-1 Glc),
D
max
1
6.34 (1H, d, J = 2.7, H-1 Rha), and 4.60 (1H, d, J = 7.8, H-1 GlcA), 1.13, 1.01, 1.04, 1.279, 0.889 (3H, each s, CH -24, 25, 26,
3
+
27, and 30), 1.56 (3H, d, J = 6, CH Rha), 5.35 (t like for 1H at C-12). FAB-MS m/z: 996 [M + Na] , 819, 673, 511.
3
Acid Hydrolysis of 1. Saponin 1 (25 mg) was refluxed with 10% H SO on a boiling water bath for 4 h. The usual
2
4
+
workup of the reaction mixture afforded sapogenin 2, mp 192ꢄC, [ꢂ] +17.6ꢄ (c 1.15; MeOH). FAB-MS m/z: 488 [M] , 303,
D
263, 222, 223, 192, 165, 154.
Identification of Sugar Moiety of 1. The aqueous layer that separated after the removal of sapogenin was neutralized
with barium carbonate, 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-glucose, L-rhamnose, and D-glucuronic acid.
Permethylation of 1 and Acid Hydrolysis of the Product. A solution of 1 (15 mg) in DMSO-d was treated with
6
NaH (0.2 g) and CH I (5 mL) at room temperature for 6 h. The usual workup of the reaction mixture yielded a residue, which
3
56

was purified by preparative TLC in n-hexane–EtOAc (1:1). Hydrolysis of permethylated 1 was performed by refluxing with
10 mL of 3% methanolic HCl. Paper chromatography of the neutralized and concentrated hydrolysate in benzene–acetone
(3:1) showed the presence of 3,4,6-O-methyl-D-glucose, 2,4-di-O-methyl-L-rhamnose, and 2,3,4-tri-O-methyl-D-glucuronic
acid (co-paper chromatography).
Antibacterial Activity. The antimicrobial activity assay was performed with three bacterial strains Staphylococcus
aureus, Klebsiella pneumoniae, and E. coli by the “broth dilution method” [14]. The inoculates of the microorganisms were
prepared from 12-h broth culture, and suspensions were adjusted to 0.5 McFarland Standard turbidity. The tested saponin was
dissolve in distilled water, and different dilutions of it were prepared ranging from 50 to1000 ꢃg/mL. 0.2 mL of different
dilutions of saponin was added to 1.8 mL of seeded broth. A set of seeded broth control was also prepared, and the whole set
was incubated for 24 h.
Antifungal Activity. The fungal strains used for antifungal activity were Aspergillus fumigatus, Alternaria alternata,
and Colletotrichum dematium. The saponin was evaluated in triplicate in a dose response format where the final concentrations
4
were 10, 25, 50, 100, 250, and 500 ꢃg/mL and the final fungal spore concentration was 10 CFU/mL. The antifungal activity
was determined by the “spore dilution method” [15].
ACKNOWLEDGMENT
The authors are thankful to the Director CDRI, Lucknow for providing instrumental facilities and also to the Head,
Department of Biosciences, R. D. University, Jabalpur for providing lab facilities to carry out the antimicrobial assay.
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