Antibacterial activity of a triterpenoid saponin from the stems of Caesalpinia pulcherrima Linn.

Article abstract of DOI:10.1080/14786419.2017.1317772

A new compound 1 was isolated from the methanolic extract of the stems of the Caesalpinia pulcherrima Linn. along with a reported compound (2) 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl hederagenin 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. The new compound 1 has m.p. 272–274°C, m.f. C₄₆H₇₄O₁₇, [M]⁺ m/z 898. It was characterised as 3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranosyl hederagenin 28-O-β-D- xylopyranosyl ester by various colour reactions, chemical degradations and spectral analyses. Antibacterial activity of compound 1 was screened against various Gram-positive and Gram-negative bacteria and showed significant results.

Full text of DOI:10.1080/14786419.2017.1317772

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Antibacterial activity of a triterpenoid saponin
from the stems of Caesalpinia pulcherrima Linn.
Nidhi Asati & R. N. Yadava
To cite this article: Nidhi Asati & R. N. Yadava (2017): Antibacterial activity of a triterpenoid
saponin from the stems of Caesalpinia pulcherrima Linn., Natural Product Research, DOI:
10.1080/14786419.2017.1317772
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Date: 21 April 2017, At: 08:41

Natural Product research, 2017
http://dx.doi.org/10.1080/14786419.2017.1317772
Antibacterial activity of a triterpenoid saponin from the stems
of Caesalpinia pulcherrima Linn.
Nidhi Asati and R. N. Yadava
Natural Products laboratory, department of chemistry, dr. h. s. Gour central university, sagar, India
ABSTRACT
ARTICLE HISTORY
received 6 June 2016
accepted 2 april 2017
A new compound 1 was isolated from the methanolic extract of the
stems of the Caesalpinia pulcherrima Linn. along with a reported
compound (2) 3-O-β-D-glucopyranosyl-(1→4)-β-D-xylopyranosyl-
(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl hederagenin
28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester. The new
compound 1 has m.p. 272–274°C, m.f. C46H74O17, [M]+ m/z 898. It was
characterised as 3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyrano-
syl hederagenin 28-O-β-D- xylopyranosyl ester by various colour re-
actions, chemical degradations and spectral analyses. Antibacterial
activity of compound 1 was screened against various Gram-positive
and Gram-negative bacteria and showed significant results.
KEYWORDS
Caesalpinia pulcherrima
linn.; fabaceae; triterpenoid
saponin; bidesmosidic;
antibacterial activity
1. Introduction
Caesalpinia pulcherrima Linn. (Chatterjee & Pakrashi 1992) belongs to the family Fabaceae,
which is commonly known as ‘Gulutora’ in Hindi. It is small sized tree which grows about
3.7–4.3 m high, its barks are grey. Its leaves are abruptly bipinnate and 1.3–1.9 cm long. Its
flowers are red or yellow in colour. Its Pods are flat, thin, straight, strap-shaped, 5.1–7.6 cm
long. Its leaves are used as emmenagogue, purgative and stimulant. Its barks are used as an
abortifacient and flowers used as pectoral and febrifuge. It is also used in the treatment of
bronchitis, asthma and malarial fevers. Earlier workers (Sivasankari et al. 2010; Pawar et al.
CONTACT Nidhi asati
nidmiss164@gmail.com
supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2017.1317772.
© 2017 Informa uK limited, trading as taylor & Francis Group

2
N. ASATI AND R. N. YADAVA
2011; Pulipati et al. 2012; Kumar et al. 2014; Osayemwenre et al. 2014; Zhao et al. 2014) have
reported various active constituents from this plant. The paper deals with the isolation,
structural elucidation and antibacterial activity of a new triterpenoid saponin 1, 3-O-β-D-
glucopyranosyl-(1→4)-α-L-arabinopyranosyl hederagenin 28-O-β-D- xylopyranosyl ester
from the methanolic extract of the stems of the plant along with a known compound 2.
2. Results and discussion
The methanolic extract of the stems of the plant was concentrated under reduced pressure
to give compound 1, m.p. 272–274°C, m.f. C46H74O17, [M]+ m/z 898 (FABMS). It gave positive
results for Liebermann–Burchard test which ensure the presence of triterpenoid saponin.
(Liebermann & Burchard 1885; Burchard1890) In the IR spectrum, two peaks at 3475,
1669 cm−1 were assigned for hydroxyl group and double bond, respectively. In 1H NMR spec-
trum (300 MHz, CD3OD) singlets at δ 0.80, 1.12, 0.95, 1.24, 1.31 and 1.09, (each 3H, s, Me × 6)
showed the presence of six singlet methyl of an olean-12-ene-type triterpene. A signal at δ
5.29 (1H, t, 3.5 Hz, H-12), showed the presence of trisubstituted olefinic proton, A double
doublets at 3.49(1H, dd, J 11.2,3.8 Hz) was assigned to H-3 and two doublets at 3.52 (1H, d,
J 11.5 Hz, Ha-23), 3.23 (1H, d, J 11.5 Hz, Hb-23) were assigned for H-23. The anomeric proton
signal at δ 4.82 (1H, d, J 6.3 Hz, H-1′), 4.42 (1H, d, J 7.2 Hz, H-1′′) and 4.52 (1H, d, J 7.2 Hz, H-1′′′),
were assigned to H-1′, H-1″ and H-1″′. In 13C NMR spectrum (75 MHz, CD3OD), two signals at
δ 123.2 (C-12), and 147.4 (C-13), showed the presence of double bond between C-12 and
C-13. In 13C NMR spectrum a downfield signal at δ 177.2 showed the presence of carboxylic
group at C-28. Two signal at δ 80.3 (C-3) and 65.2 (C-23) suggested the presence of hydroxyl
group at C-3 and C-23.13C NMR spectrum of compound 1 exhibited 46 signals, out of them
30 signals could be assigned to the aglycone and 16 signals to the sugar moieties, suggested
the nature of compound 1 as bidesmosidic.
In the mass spectrum of the compound 1, characteristic ion peaks at [M]+ m/z 898, 736,
604 and 472 were found by subsequent losses from the molecular ion of each molecule of
D-xylose, L-arabinose and D-glucose showing D-xylose as terminal sugar, L-arabinose was
linked at C-3 position of aglycone.
Acid hydrolysis of compound (1) (Figure 1) with ethanol and 10% H2SO4 gave compound
1a (Figure 2). Compound 1a has m.p. 231–233°C, molecular formula C30H48O4 [M]+ m/z 472
(FABMS). It responded to all the characteristic colour reactions of triterpenoid (Barton 1951;
Fieser & Fieser 1966). Thus it was identified as hederagenin. The aqueous hydrolysate
obtained after acid hydrolysis of the compound 1 was neutralised with BaCO3 and the BaSO4
was filtered off. The filtrate was concentrated under reduced pressure and subjected to paper
chromatography examination using nBAW (4:1:5) as solvent and aniline hydrogen phthalate
as detecting agent yielded D-xylose (Rf 0.28), L-arabinose (Rf 0.21) and D-glucose (Rf 0.18)
by (Co-Pc and Co-TLC) (Lederer & Lederer 1957).
Permethylation (Kuhn et al. 1955)of compound 1 yielded methylated aglycone identified
as 23 α-methoxy hederagenin confirmed that glycosidation was involved at C-3 and C-28
position and methylated sugars were identified as 2,3,4-tri-O-methyl-D-xylose (RG 0.94),
2,3-di-O-methyl-L-arabinose (RG 0.64) and 2,3,4,6-tetra-O-methyl-D-glucose (RG 1.0) which
showed that C-1″ of D-glucose was attached to C-4′ of L-arabinose and C-1′ of L-arabinose
was attached to C-3 position of aglycone and C-1″′ of D-xylose attached to carboxylic group
at C-28 position of aglycone thus the interlinkage (1→4) was found between D-glucose and

NATURAL PRODUCT RESEARCH
3
Figure 1. compound 1.
Figure 2. compound 1a.
L-arabinose and L-arabinose directly attached to C-3 position of aglycone. This linkage and
the linkage between D-xylose attached to carboxylic group at C-28 position of aglycone
were further confirmed by 13C NMR spectral data. Periodate oxidation (Hirst & Jones 1949)
of compound 1 confirmed that three sugars were present in pyranose form.
Enzymatic hydrolysis (Harborne 1965) of compound 1 with almond emulsin enzyme
liberated D-glucose (Rf 0.18) first, followed by D-xylose (Rf 0.28) and proaglycone identified
as 3-O-α-L-arabinopyranosyl hederagenin confirming the presence of β-linkage between
D-glucose and Proaglycone as well as between D-xylose and Proaglycone. Proaglycone was
further hydrolysed with enzyme takadiastase liberated L-arabinose (Rf 0.21) and aglycone
confirming the presence of α-linkage between L-arabinose and aglycone.
Quantitative examination (Mishra & Rao 1960) of the sugars present in the hydrolysate
revealed that the three sugars were present in equimolecular proportions indicating that
the triterpenoid saponin consists of one molecule of each, aglycone (sapogenin), D-xylose,
L-arabinose and D-glucose.

4
N. ASATI AND R. N. YADAVA
Antibacterial activity of compound 1 was screened against various Gram-positive and
Gram-negative bacteria. The antimicrobial activity of compound 1 was carried out by agar
disc diffusion method at different concentrations (25, 50, 75 and 100 μg/mL). Tetracyclin was
used as a standard antibacterial drug. It was concluded that compound 1 showed maximum
zone of inhibition (20.1 0.32 mm) against Bacillus subtilis at 100 μg/ml concentration and
minimum (13.8 0.43 mm) against Escherichia coli at same concentration. (Table 1) and from
Table 2, it was concluded that compound 1 had lowest MIC value of 1.56 μg/ml against
Bacillus subtilis and highest MIC value of 6.25 μg/ml against E. coli.
On the basis of above, the structure of compound 1 was established as 3-O-β-D-
glucopyranosyl-(1→4)]-α-L-arabinopyranosyl hederagenin 28-O-β-D- xylopyranosyl ester
and exhibited antibacterial activity against various Gram-positive and Gram-negative
bacteria.
3. Experimental
3.1. General experimental procedure
All the melting points were determined on a thermo electrical melting point apparatus and
are uncorrected. The IR spectra were measured on Shimadzu 84,005 FTIR spectrophotometer
using KBr matrix. UV spectra were recorded on Systronics-2201 UV/Vis Double Beam spec-
trophotometer in MeOH. The NMR spectral data were recorded at 300 MHz for 1H NMR and
75 MHz for 13C NMR on Bruker DRX 300 NMR spectrometer using TMS as internal standard
and CD3OD as solvent. The FABMS were recorded on a Joel SX (102) Mass spectrometer.
3.2. Plant material
The stems of the plant were collected locally around Sagar region and were taxonomically
authenticated by Taxonomist, Department of Botany, Dr H. S. Gour Central University, Sagar
(M.P.) India. A voucher specimen (Bot/Her/A/3157) has been deposited in the Natural Products
Laboratory, Department of Chemistry of this university.
Table 1. antibacterial activity of compound 1.
Concentration of compound 1 (μg/ mL)
25
50
75
100
Tetracycline (stand.
drug) (10 μg/ mL)
Pathogenic bacteria
Zone of inhibition (mm)
Escherichia coli
Bacillus subtilis
Staphylococcus aureus
Pseudomonas
aeruginosa
6.9 0.05**
10.2 0.09**
7.3 0.05**
8.2 0.23*
9.6 0.12*
15.8 0.18*
10.1 0.08**
10.8 0.12*
11.3 0.10**
17.1 0.07**
12.9 0.14*
13.8 0.43*
20.1 0.32*
16.5 0.23*
15.1 0.17*
22.8 0.15**
24.2 0.12**
27.3 0.91*
23.1 0.13**
13.01 0.09**
Notes: data are expressed as Mean s.e.
experiments were conducted in triplicates.
aNoVa followed by dunnett’s test.
p value indicate calculated probability.
*p < 0.05, **p < 0.01 considered as significant.
*p indicates that when p is less than 0.05 showed statistically significant values.
**p indicates that when p is less than 0.01 showed statistically highly significant values.

NATURAL PRODUCT RESEARCH
5
Table 2. Minimum inhibitory concentration of compound 1.
MIC (μg/mL) of tetracycline (positive
control)
S. No.
Bacteria
MIC (μg/mL) of compound 1
1.
2.
3.
4.
Escherichia coli
6.25
1.56
3.12
3.12
0.78
0.19
0.04
0.39
Bacillus subtilis
Staphylococcus aureus
Pseudomonus aueruginosa
3.3. Extraction and isolation
Air-dried and powdered stems (4.00 kg) of the plant were extracted with 95% ethanol (60–
80°C) in Soxhlet apparatus for 74 hThe total ethanol extract was concentrated under reduced
pressure and successively partitioned with petroleum ether (40–60%), CHCl3, CH3COOC2H5,
CH3COCH3 and CH3OH. The methanol soluble fraction was concentrated under reduced
pressure by rotary vacuum evaporator to yield brown viscous mass. It gave two spots on TLC
examination using chloroform: methanol: water (6:4:2) as solvent and I2 vapours as visual-
ising agent indicating it to be mixture of two compounds 1 and 2. These were separated by
column chromatography over a silica gel column using CHCl3 :MeOH (3:6) as eluent and
purified by TLC and studied separately.
3.4. Study of compound 1
It was crystallised from methanol to give light brown crystalline compound (1.75 gm), m.p.
272–274°C, m.f. C46H74O17 [M]+ m/z 898(FABMS). Found (%): C 60.62, H 8.13, O 30.62 calcd.
for m.f. C46H74O17 :C 61.46, H 8.24, O 30.28%, IR (KBr): 3475, 2935, 1669, 1715, 1038, 1353,
1220, 960 cm−1; 1H NMR (300 MHz, CD3OD): δ 0.80 (3H, s, Me-24), 1.24 (3H, s, Me-25), δ 1.09
(3H, s, Me-26), 1.31 (3H, s, Me-27), 0.95 (3H, s, Me-29), 1.12 (3H, s, Me-30),1.52 (1H, m, H-1a),
1.02 (1H, m, H-1b), 1.83 (1H, m, H-2a), 1.66 (1H, m, H-2b), 3.49(1H, dd, J 11.2,3.8 Hz, H-3), 1.35
(1H, m, H-5), 1.48 (1H, m, H-6a), 1.36 (1H, m, H-6b), 1.59 (1H, m, H-7a), 1.71 (1H, m, H-7b), 1.62
(1H, m, H-9), 1.94 (2H, m, H-11), 5.29 (1H, t, 3.5 Hz, H-12), 1.79 (1H, m, H-15a), 1.18 (1H, m,
H-15b), 1.85 (1H, m, H-16a), 1.57 (1H, m, H-16b), 2.78 (1H, m, H-18), 1.64 (1H, m, H-19a), 1.14
(1H, m, H-19b), 1.39 (1H, m, H-21a), 1.21 (1H, m, H-21b), 1.69 (1H, m, H-22a), 1.53 (1H, m, H-22b),),
3.52 (1H, d, J 11.5 Hz, Ha-23), 3.23 (1H, d, J 11.5 Hz, Hb-23)2.83 (1H, d, J 11.2 Hz, H-28), δ 4.82
(1H, d, J 6.3 Hz, H-1′), 3.68 (1H, dd, J 8.2,3.6 Hz, H-2′), 3.64 (1H, dd, J 8.3,3.0 Hz, H-3′), 3.75 (1H,
m, H-4′), 3.83 (1H, dd, J 11.8,2.0 Hz, H-5a′), 3.27 (1H, dd, J 11.2,3.0 Hz, H-5b′); δ 4.42 (1H, d,
J 7.2 Hz, H-1′′), 3.35 (1H, dd, J 7.3, 9.0 Hz, H-2′′), 3.54 (1H, t, J 9.0, Hz, H-3′′), 3.46 (1H, t, J 9.0,
Hz, H-4′′), 3.40 (2H, dd, J 3.5,4.5 Hz, H-5′′); 3.77 (1H, dd, J 3.5,12.0 Hz, H-6a′′), 3.67 (1H, dd,
J 11.4,12.2 Hz, H-6b′′); δ 4.52 (1H, d, J 7.2 Hz, H-1′′′), 3.29 (1H, dd, J 7.2,9.2 Hz, H-2′′′), 3.48(1H,
t, J 9.0 Hz, H-3′′′), 3.69 (1H, m, H-4′′′), 4.02 (1H, dd, J 5.0,11.2 Hz, H-5a′′′); 3.34 (1H, t, J 11.5 Hz,
H-5b′′′); 13C NMR (75 MHz, CD3OD) δ 38.8 (C-1), 27.1 (C-2), 80.3 (C-3), 42.9 (C-4), 49.2 (C-5),
18.9 (C-6), 34.2(C-7), 41.2 (C-8), 50.2 (C-9), 36.8 (C-10), 25.5 (C-11), 123.2 (C-12), 147.4 (C-13),
43.5(C-14), 29.2 (C-15), 25.3 (C-16), 48.2 (C-17), 40.8 (C-18), 45.8 (C-19), 30.8 (C-20), 34.8 (C-21),
32.6 (C-22), 65.2 (C-23), 13.5 (C-24), 19.2(C-25), 17.2 (C-26), 24.4 (C-27), 177.2 (C-28), 31.5
(C-29), 24.8 (C-30), δ 105.2 (C-1′), 75.5 (C-2′), 73.2 (C-3′), 68.4 (C-4′), 70.2(C-5′); δ 103.8 (C-1′′),
76.2 (C-2′′), 78.5(C-3′′), 72.4 (C-4′′), 74.2 (C-5′′),65.6 (C-6′′), δ 104.3 C-1′′′), 76.1 (C-2′′′), 79.3(C-
3′′′), 71.4 (C-4′′′), 67.5(C-5′′′).

6
N. ASATI AND R. N. YADAVA
3.5. Study of compound 2
It was crystallised from methanol to give colourless crystalline compound (0.710 gm), m.p.
251–253°C, m.f. C64H104O31[M]+ m/z 1368 (FABMS). Found (%):C 55.92%, H 7.23%, O 35.93%,
calcd. for C64H104O31 :C 56.14%, H 7.61%, O 36.25%, IR (KBr): 3150, 2946, 2881, 1730, 1689,
1453, 1370, 1225, 1012, 739 cm−1. It was identified as 3-O-β-D-glucopyranosyl-(1→4)-β-D-
xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl hederagenin 28-O-β-
D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester by comparison of its spectral data with
reported literature values (Gulcemal et al. 2014).
3.6. Acid hydrolysis of compound 1
Compound 1 (500 mg) was dissolved in ethanol (30 mL) and refluxed with 10% H2SO4 (20 mL)
on water bath for 7 h. The reaction mixture was concentrated and allowed to cool and filtered.
The residue was extracted with diethyl ether. The ether layer was washed with water and
evaporated to dryness. The residue was subjected to column chromatography over silica
gel column using CHCl3 :MeOH (5:10) as solvent to give compound 1a as aglycone, which
was identified as hederagenin. The aqueous hydrolysate was neutralised with BaCO3 and
the BaSO4 was filtered off. The filtrate was concentrated and subjected to paper chromatog-
raphy examination using n-Butanol:Acetic Acid:Water [nBAW, 4:1:5] as solvent and aniline
hydrogen phthalate as spraying reagent which showed the presence of D-xylose (Rf 0.28),
L-arabinose (Rf 0.21) and D-glucose (Rf 0.18) by (Co-Pc and Co-TLC).
3.7. Study of compound 1a
It had m.p. 231–233°C, molecular formula C30H48O4 [M]+ m/z 472 (FABMS). Found: C, 76.22;
H, 10.21%; O 13.49 calcd. for m.f. C30H48O4, C 76.27%; H, 10.16%, O 13.55. IR (KBr): 3478,
1
2940,1725,1670, 1120 cm −1, H NMR (300 MHz, CD3OD): 0.83 (3H, s, Me-24), 1.23 (3H, s,
Me-25), δ 1.02 (3H, s, Me-26), 1.32 (3H, s, Me-27), 0.97 (3H, s, Me-29), 1.12 (3H, s, Me-30),1.49
(1H, m, H-1a), 1.13 (1H, m, H-1b), 1.78 (1H, m, H-2a), 1.64 (1H, m, H-2b), 3.51(1H, dd, J 11.2,3.8 Hz,
H-3), 1.39 (1H, m, H-5), 1.46 (1H, m, H-6a), 1.33 (1H, m, H-6b), 1.54 (1H, m, H-7a), 1.73 (1H, m,
H-7b), 1.67 (1H, m, H-9), 1.91 (2H, m, H-11), 5.31 (1H, t, 3.5 Hz, H-12), 1.85 (1H, m, H-15a), 1.12
(1H, m, H-15b), 1.82 (1H, m, H-16a), 1.59 (1H, m, H-16b), 2.76 (1H, m, H-18), 1.69 (1H, m, H-19a),
1.18 (1H, m, H-19b), 1.42 (1H, m, H-21a), 1.24 (1H, m, H-21b), 1.69 (1H, m, H-22a), 1.58 (1H, m,
H-22b),3.51 (1H, d, J 11.5 Hz, Ha-23), 3.28 (1H, d, J 11.5 Hz, Hb-23), 2.79 (1H, d, J 11.2 Hz, H-28),
13C NMR (75 MHz, CD3OD) 38.2 (C-1), 28.2 (C-2), 81.1 (C-3), 43.2 (C-4), 48.9 (C-5), 18.5 (C-6),
33.7(C-7), 39.8 (C-8), 49.5 (C-9), 35.1 (C-10), 24.8 (C-11), 122.5 (C-12), 146.3 (C-13), 44.1(C-14),
30.1 (C-15), 26.4 (C-16), 46.8 (C-17), 38.2 (C-18), 43.9 (C-19), 29.5 (C-20), 35.1 (C-21), 30.5 (C-22),
64.8 (C-23), 13.1 (C-24), 18.3(C-25), 17.5 (C-26), 23.7 (C-27), 176.5 (C-28), 30.4 (C-29), 25.5
(C-30).
3.8. Permethylation of compound 1
Compound 1 (35 mg) was dissolved in DMF (25 mL) and treated with MeI (5 mL) and Ag2O
(15 mg) in 150 mL round bottomed flask fitted with condenser and refluxed for two days.
The reaction mixture was filtered and washed with DMF. The filtrate was concentrated under

NATURAL PRODUCT RESEARCH
7
reduced pressure and treated with CHCl3 (25 mL) and washed with water. After removal of
solvent a syrupy mass was obtained which was hydrolysed with 7% H2SO4 (5 mL) to give
aglycone, identified as 23α-methoxy hederagenin. The aqueous hydrolysate obtained after
the removal of aglycone was neutralised with BaCO3 and the BaSO4 was filtered off. The filtrate
was concentrated under reduced pressure and subjected to paper chromatography exam-
ination on Whatman filter paper No.1 using n-Butanol:Ethanol:Water (5:1:4) as solvent
system and aniline hydrogen phthalate as spraying agent. The methylated sugars were iden-
tified as 2,3,4-tri-O-methyl-D-xylose (RG 0.94), 2,3-di-O-methyl-L-arabinose (RG 0.64) and
2,3,4,6-tetra-O-methyl-D-glucose (RG 1.0).
3.9. Enzymatic hydrolysis of compound 1
Compound 1 (30 mg) was dissolved in MeOH (20 mL) and hydrolysed with equal volume of
enzyme almond emulsin. The content was allowed to stay at room temperature for two days
and filtered. The hydrolysate was concentrated and subjected to paper chromatography
examination using nBAW (4:1:5) as solvent system and aniline hydrogen phthalate as a
spraying reagent which showed the presence of D-glucose and D-xylose suggesting the
presence of β linkage between D-glucose (Rf 0.18) and Proaglycone as well as between
D-xylose (Rf 0.28) and Proaglycone. Proaglycone on further hydrolysis with enzyme takadi-
astase yielded L-arabinose (Rf 0.21) and aglycone suggesting the presence of α linkage
between L-arabinose and aglycone.
3.10. Antibacterial activity of compound 1
The antibacterial activity of compound 1 was performed by agar disc diffusion method
(Doughari 2006). Compound 1 was dissolved in water and further diluted to obtain desired
concentrations (25, 50, 75 and 100 μg/mL). Tetracyclin was used as a standard antibacterial
drug. The bacterial strains were activated by inoculating a loopful of the strains in the nutrient
broth (30 mL) and incubated for 6 h to maintain standard turbidity (106 cells/mL). After that
1 mL of inoculum of bacterial strains was swabbed in Muller Hinton agar plate. Disc (6 mm)
was dipped in different concentrations (25, 50, 75 and 100 μg/mL) of compound 1 and
allowed to dry. Then the disc was impregnated on the seeded agar plates. The plates were
allowed to stand for 1 h for pre-diffusion of the compound 1 and incubated at 37°C for 24 h.
The antimicrobial activity was taken on the basis of diameter of zone of inhibition (mm)
around each paper disc. All the experiments were done in triplicates. The results obtained
from experimental findings are recorded in the Table 1.
3.11. Minimum inhibitory concentrations of compound 1
The minimum inhibitory concentrations of compound 1 were determined by two fold broth
micro-dilution method. Bacterial cultures were activated by transferring a loopful of strains
from stock culture to tubes and inoculated in Nutrient-broth (NB) medium then incubated
for 24 h at 37°C. The bacterial cultures were diluted with fresh NB media to achieve optical
density (600 nm) of 107 CFU/ml. Compound 1 was incorporated into NB medium to get a
concentration of 100 μg/ml which was further serially diluted to 50, 25, 12.5, 6.25, 3.12, 1.56
and 0.78 μg/ml, respectively. The tubes were then inoculated with 20 μl microbial suspension

8
N. ASATI AND R. N. YADAVA
and incubated at 37°C for 24 h. Minimum concentrations of the assayed samples (Compound
1), which inhibits the visible growth of the tested microorganisms were considered as MIC
of the compound 1. The results obtained from experimental findings are recorded in the
Table 2 (Clinical & Laboratory Standards Institute 2012).
4. Conclusion
Thus, on the basis of above evidences the structure of compound 1 was established as
3-O-β-D-glucopyranosyl-(1→4)-α-L-arabinopyranosyl hederagenin 28-O-β-D- xylopyranosyl
ester from the methanolic extract of the stems of the plant. Compound 1 showed significant
antibacterial activity and thus it may be used as a potent antibacterial agent against diseases
caused by these bacteria.
Acknowledgement
Authors are thankful to Department of Chemisty, Dr H. S. Gour Central University, Sagar (M.P.) for
providing necessary laboratory facilities.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
Nidhi Asati is thankful to UGC for the financial support.
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