Asian Journal of Biochemical and Pharmaceutical Research Issue 4 (Vol. 2) 2012
ISSN: 2231-2560
CODEN (USA): AJBPAD
Research Article
Asian Journal of Biochemical and Pharmaceutical Research
Isolation, Characterization & Qunatitation Of Phytocontituents From Two Plants Of
Fabaceae
D.S. Pokle1 , S.C.Pal2 & Khan Subur W3*
1.. Retired Professor of Botany, presently Head, Yashvantrao Chavan Open University
2.Associate Professor, Dept. of Pharmacognosy, NDMVPS’s College of Pharmacy, Nashik
3.Dept. of Pharmacognosy. Y.B. Chavan College of Pharmacy, Rauza Bagh, Aurangabad.
Received: 15 September 2012; Revised: 29 September 2012; Accepted: 09 October 2012
Abstract: Two sterols and Lupeol is isolated from whole herb of Teramnus labialis. These compounds are
isolated from petroleum ether soluble portion of the plant. The plants are collected from wild source and
petroleum ether extract is prepared which showed presence of steroids in preliminary phytochemical studies.
TLC studies are performed on petroleum ether extract to optimize solvent system for column chromatography.
Column chromatography yielded three compounds Beta-sitosterol, Stigmasterol and Lupeol structures of which
are established by available spectroscopic studies viz UV, IR, MS and NMR. These isolated compounds are
quantified in leaves, stem and roots of T. labialis as well as from whole herb of Alysicarpus vaginalis by
HPTLC studies.
Keywords: Sterol, Lupeol, Fabaceae, HPTLC,
INTRODUCTION:
Alysicarpus vaginalis is a more or less prostrate, somewhat hairy branching herb, commonly
found in lawns, by roadside ditches, and in waste ground exposed to the sun. Leaves are of two types,
closely spaced elliptic, and narrower, lanceolate once spaced farther apart. Leaves are about 3-10 mm
long, rounded at tips, obtuse, or truncate at base, with short stalks, about 1-5 mm long. Flowers are
reddish purple, in racemes up to 13 cm long, at the end of branches. Flower are 6 mm long. Pods are
cylindric, jointed, nearly 2 cm long, 5-7-seeded. Seeds are pale brown, 1.5 mm long, oval.[3]
Teramnus labialis (L.f.) Spreng. Syn: Glycine labialis: Widely spreading twinning herbs; stems
slender, appressed hairy. Leaves three foliate; petioles 1-3.5cm long; stipules ovate-lanceolate, 2.53mm long, acute deciduous. Leaflets 2-6x 1-4.5cm acute or rounded at base, acute, apiculate,
appressed hairy beneath; petiolules 2-3mm long, hairy; stipels filiform, 1-1.5mm long. flowers in
axillary, few flowered, lax racemes 2-15 cm long, solitary or fascicled along the slender hairy rachis;
pedicels 3-4mm long; bracts linear-lanceolate, 2mm long; bracteoles subulate, 1-1.5mm long. Calyx 34mm long, silky outside; teeth lanceolate as long as the tube. Corolla white, turning reddish, 4-6mm
long. Pods narrowly linear, 3.5-0.3-0.4 cm, slightly curved, apiculate. Seeds 8-12, oblong, 3-4 mm
long, brown, polished. It is common in hedges around fields and under shade of trees. Flowering and
fruiting September to November. [4] It is one of the component of Chavyanprash.[6]
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Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
MATERIAL AND METHODS
Collection Authentication and Drying of Plant Material: Both plants are collected from wild in
Aurangabad (Maharashtra) authenticated by Herbarium Department, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad and dried under shade then size reduction is done in grinder to
coarse powder. Teramnus labialis is separated into roots stem and leaves and then subjected to
extraction.
Extraction of Plant Material: Plant material is extracted by hot continuous soxhlet extraction by
using petroleum ether (bp 60-800C) as extracting solvent. Plant material is whole herb in case of A.
vaginalis and Parts viz. roots, stems and leaves in case of T. labialis. The extracts thus obtained are
filtered and concentrated by solvent evaporation at room temperature. Preliminary phytochemical
studies of both extracts showed presence of steroids and triterpenoids. This extracts is redissolved in
petroleum ether and TLC studies are performed.
TLC Studies of Petroleum Ether Extract: TLC is carried out precoated silica gel G layer by Merck.
Various solvent systems were tried to optimize solvent system which gives better resolution. Detection
was carried out by Anisaldehyde sulphuric acid reagent. For T. labialis leaves extract petroleum ether:
ethyl acetate (9:1) gave better resolution and its polarity can be increased by grediently increasing
portion of ethyl acetate viz 8:2, 7:3 and so on in column chromatography.
Column Chromatography: Column chromatography of leaves extract of T. labialis is carried out in
glass column of internal diameter 34mm and stationary phase used is silica gel 230-330 mesh size
(particle size- 0.037-0.063 mm). Column is set in Petroleum Ether (60-80): Ethyl acetate (9:1) and
eluted. Fractions 1-5 eluted 9:1 proportion of solvent system and 6-10 with 8:2 and 11-15 with 7:3 of
20 ml each are collected. Fraction No. 7-11 showed same TLC pattern and combined and subjected to
further purification by again column chromatography in column of 12 mm diameter and eluted in 1ml
fractions with solvent system (7:3). Out of 25 fractions first ten fractions yielded a white coloured
crystalline compound and from next ten it yielded two white compounds.
Spectroscopic Studies of Compounds: UV spectroscopy is carried out at Y.B. Chavan College of
Pharmacy, Aurangabad on Jasco UV spectrophotometer and IR spectroscopy is carried out at Y.B.
Chavan College of Pharmacy, Aurangabad on Shimadzu (Japan) by KBr disc method. Mass and NMR
spectroscopy is carried out at IIT, Powai.
Compound 1: White crystalline solid, M.P: 214-216 °C, IR ν max cm-1 (CHCl3): 3455 (OH), 3070,
1645 and 880 (C = C), 1HNMR (CDCl3) (300MHz) (δ ppm): δ 4.69, 4.56 (2H, br s, 1H each, H-29),
3.18 (1H, dd, J = 9.9, 4.5, H-3), 2.39 (1H, d, J = 10.5, 5.4 Hz, H-19), 1.65 (3H, d, J = 1.3 Me-30), 1.05
(3H, s, Me-26), 0.96 (6H, s, Me-25, Me-27), 0.90 (3H, s, Me-24), 0.85 (3H, s, Me-28), 0.76 (3H, s,
Me-23). EI-MS m/z: 426 [M+], 411 [M-CH3]+, 408 [M-H2O]+, 393 [M-CH3-H2O]+, 220 [MC15H28]+, 218 [M-C14H24O]+, 207 [M-C16H23]+, 133
Compound 2: White amorphous powder, UV λ max nm (log ε) (MeOH): 208, IR ν max cm-1 (CHCl3):
3450 (OH), 3055, 1650, 810 (C=C), 1HNMR (CDCl3) (300MHz) (δ ppm): δ 5.13 (1H, m, H-6), 3.33
(1H, m, H-3), 1.44 (1H, t, H-8), 1.14 (1H, d, H-17), 1.12 (1H, d, H-14), 1.01 (3H, s, Me-19), 0.93 (1H,
m, H-24), 0.92 (3H, d, J = 6.2 Hz, Me-21), 0.91 (1H, d, H-9), 0.84 (3H, t, J = 7.0 Hz, Me-29), 0.83
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Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
3H, d, J=6.5 Hz, Me-26). 0.81 (3H, d, J = 6.5 Hz, Me-27), 0.67 (3H, s, Me-18). EI-MS m/z: 414, 399,
396, 381, 329, 303, 275, 273, 255
Compound 3: White Crystalline powder, IR ν max cm-1 (CHCl3): 3434 (OH), 1648 (C=O) 1HNMR
(CDCl3) (300MHz) (δ ppm): δ 5.33 (1H, s, H-6), 5.12 (1H, dd, J=15.5, 8.5Hz, H-22), 4.98 (1H, dd,
J=15.5, 8.5Hz, H-23), 3.45 (1H, s, H-3), 1.01 (3H, d, J= 6.5Hz, H-21), 0.97 (3H, s, H-19), 0.83 (3H, d,
J= 6.0Hz, H-26), 0.78 (3H, t, J= 7.5Hz, H-29), 0.77 (3H, d, J= 6.0Hz, H-27) and 0.68 (3H, s, H-18).
EI-MS m/z: 43, 55, 69, 81, 91, 105, 121, 133, 145, 159, 173, 185, 199, 207, 213, 229, 255, 271, 281,
300, 314, 327, 341, 351, 369, 394, 412,
After systematic analysis of spectroscopic data Compound 1 is found to be Luepol, Compound 2 is
Beta sitosterol and Compound 3 is Stigmasterol. These compounds are used as standards for HPTLC
studies.
HPTLC studies: HPTLC is carried out to estimate percentage of above compounds from different
parts of T. labialis like root stem and leaves and whole herb of A. vaginalis. It is carried out on
CAMAG Linomat 5 "Linomat5_160449" S/N 160449 (1.00.12) instrument at NDMVP Samaj’s
College of Pharmacy, Nasik.
Steps and parameters for HPTLC studies:
Preparation of standard solution: Standard solutions are prepared by dissolving 10mg in 10 ml of
Petroleum ether. Concentration of standard solution 1mg/ml, out of this series of volumes is applied as
tracks.
Preparation of test solution: Test solution is prepared by dissolving 10mg of petroleum ether extract
of T. labialis leaves, stem and roots individually and 10mg of A. vaginalis whole herb extract in 10ml
of petroleum ether as solvent.
Stationary phase: Precoated layer silica gel 60 F 254 from Merck of plate size 20.0 x 10.0 cm is used.
Mobile phase: Petroleum ether: Ethyl acetate (9:1)
Development: It is done in Twin Trough Chamber (20x10cm) made up of glass of volume 10.0 ml
Procedure: Standard solution thus prepared was having concentration of 1mg/ml. This standard
solution was applied on TLC plates at specific volumes say 2µl, 4µl, 8µl, 10µl etc. which means 2µg,
4µg, 8µg, 10µg of standard respectively is applied on TLC plates. Along with these test solutions were
applied at specific constant concentration 10µl. this TLC plate is then subjected to densitometric scan
and data obtained is analyzed by regression. Standard Calibration graph is prepared and amount of
these compounds in test sample is determined.
HPTLC Studies:
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Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
Calculation of amount of Beta sitosterol in Teramnus labialis:
Standard: β sitosterol @ 520nm
Regression via area: Linear
Y = 763.2 + 190.9 * X
r = 0.99892
sdv = 1.70
RESULT AND DISCUSSION:
In the present study three compounds are isolated from petroleum ether soluble portion of T.
labialis. Spectroscopic studies of these compounds revealed that these compounds are triterpenoid
Lupeol, sterols Beta sitosterol and Stigmasterol. HPTLC studies were carried out on extracts to
estimate percentage of these compounds for above mentioned two plants showed following results.
Thus from calculations Teramnus labialis contains 2.95% of Lupeol, 0.96% of Beta sitosterol and
0.85% of Stigmasterol in leaves, 2.66% of Lupeol, 1.21% of Beta sitosterol 0.56% of Stigmasterol in
Stems and 1.29% of Lupeol and 0.33% of Stigmasterol and very negligible amount of Beta sitosterol is
present in roots. Alysicarpus vaginalis contains 3.25% of Lupeol, 0.98% of Beta sitosterol and 1.07%
of Stigmasterol in whole herb.
CONCLUSION:
From the above discussion it can be concluded that as these plants contains Sterols and Lupeol
they will possess various pharmacological activities which is shown by these phytoconstituents like
antiiflammatory, anticancer, estrogenic, gonadotropic, hepatoprotective etc. Potency of these activities
will be proportional to amount of respective chemical present in them of any of their part.
Lupeol
Beta sitosterol
Stigmasterol
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Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
Table1: Calculation of amount of Beta sitosterol in T. labialis by regression:
Sample
Rf
Amount
Area
Beta sitosterol
0.91
24.00µg
5380.30
Beta sitosterol
0.90
20.00 µg
4594.42
Beta sitosterol
0.90
16.00 µg
3745.03
Beta sitosterol
0.90
12.00 µg
3011.51
Beta sitosterol
0.90
08.00 µg
2354.33
Leaves 1
0.90
2605.00
9.65
Stem1
0.90
3076.3
12.12
Roots1
0.90
-
X (Calc.)
-
Fig1: Linear regression of standard and Teramnus labialis samples
Calculation of percentage of Stigmasterol in Teramnus labialis by regression:
Substance: Stigmasterol @ 254 nm
Regression via area: Linear
Y = -585.3 + 258.2 * X
r = 0.91728
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sdv = 32.55
Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
Table 2: Calculation of amount of stigmasterol in T. labialis by regression:
Sample
Rf
Amount
Area
X (Calc)
1.
Stigmasterol
0.39
4.00µg
1673.6
2.
Stigmasterol
0.39
8.00 µg
1271.8
3.
Stigmasterol
0.36
12.00 µg
2014.9
4.
Stigmasterol
0.37
24.00 µg
4624.3
5.
Stigmasterol
0.37
28.00 µg
5376.5
6.
Stigmasterol
0.36
32.00 µg
7062.0
7.
Stigmasterol
0.36
36.00 µg
11060.4
8.
Leaves 1
0.36
1467.4
7.950 μg
9.
Leaves 2
0.36
1624.2
8.557 μg
10.
Leaves 3
0.36
1746.6
9.031 μg
11.
Stem1
0.36
871.5
5.642 μg
12.
Stem2
0.36
840.9
5.523 μg
13.
Stem 3
0.37
925.2
5.850 μg
14.
Roots1
0.39
279.64
3.315 μg
15.
Roots2
16.
Roots 3
X average
8.513 μg
5.672 μg
3.315 μg
Fig 2: Linear regression of Stigmasterol and samples of T. labialis
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Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
Calculation of amount of Lupeol in T. labialis by regression:
Substance: Lupeol @ 520 nm
Regression via area: Linear
Y = -417.9 + 53.05 * X
r = 0.99450
sdv = 7.24
Table 3: Calculation of amount of Lupeol in T. labialis by regression
Sample
Rf
Amount
Area
X (Calc.)
1.
Lupeol
0.94
12.00 µg
193.47
2.
Lupeol
0.94
16.00 µg
472.38
3.
Lupeol
0.94
20.00 µg
671.53
4.
Lupeol
0.94
24.00 µg
792.80
5.
Lupeol
0.95
32.00 µg
1297.52
6.
Leaves
0.94
1151.11
29.58 μg
7.
Stem
0.95
993.93
26.61 μg
8.
Roots
0.96
271.06
12.99 μg
Fig 3: Linear regression of Lupeol and of samples
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Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
Calculation of amount of Beta sitosterol by regression:
Substance: Beta sitosterol @ 366 nm
Regression via area: Linear
Y = 19.26 + 150.6 *
X r = 0.99833
sdv = 2.01
Table 4: Calculation of amount of Beta sitosterol in A. vaginalis by regression:
Sample
Rf
Amount
Area
X (Calc)
β sitosterol
0.88
6.000 μg
933.12
β sitosterol
0.88
8.000 μg
1204.21
β sitosterol
0.89
10.00 μg
1535.66
Leaves 1
0.90
1325.93
8.675 μg
Leaves 2
0.92
1667.28
10.94 μg
X average
9.80μg
Fig 4: Linear regression of standard and A. vaginalis samples
Calculation of amount of Stigmasterol in A. vaginalis by regression:
Substance: Stigmasterol @ 366 nm
Regression via area: Linear
Y = -7913 + 1230 * X r = 0.99713 sdv = 5.78
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Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
Table 5: Calculation of amount of Stigmasterol in A. vaginalis by regression
Sample
Rf
Amount
Area
X (Calc)
1.
Stigmasterol
0.39
9.00 µg
2997.39
2.
Stigmasterol
0.39
12.00 µg
7173.19
3.
Stigmasterol
0.38
15.00 µg
10378.50
4.
AVPE
0.38
5409.47
10.83 μg
5.
AVPE
0.37
5379.14
10.80 μg
6.
AVPE
0.37
4983.34
10.48 μg
X (Avg.)
10.71 μg
Fig 5: Linear regression of Stigmasterol and samples of A. vaginalis
Calculation of amount of Lupeol in A. vaginalis by regression:
Substance: Lupeol @ 520 nm
Regression via area: Linear
Y = -417.9 + 53.05 * X
r = 0.99450
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sdv = 7.24
Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
Table 6: Calculation of amount of Lupeol in A. vaginalis by regression
Sample
Rf
Amount
Area
X (Calc)
1.
Lupeol
0.94
12.00 µg
193.47
2.
Lupeol
0.94
16.00 µg
472.38
3.
Lupeol
0.94
20.00 µg
671.53
4.
Lupeol
0.94
24.00 µg
792.80
5.
Lupeol
0.95
32.00 µg
1297.52
6.
AVPE
0.95
1307.66
32.53 μg
Fig 6: Linear regression of Lupeol and A. vaginalis samples
Amount of isolated phytoconstituents in Teramnus labialis
Sample
Amount in micrograms
Lupeol
Beta sitosterol
Stigmasterol
Leaves
29.58
9.65
8.513
Stem
26.61
12.12
5.672
Roots
12.99
Negligible
3.315
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Asian Journal of Biochemical and Pharmaceutical Research
Issue 4 (Vol. 2) 2012
CODEN(USA) : AJBPAD
Amount of isolated phytoconstituents in Alysicarpus vaginalis
Sample
Whole herb
Amount in micrograms
Lupeol
Beta sitosterol
Stigmasterol
32.53
9.80
10.71
REFERENCES:
1. J.B. Harborne., Phytochemical Methods, Chapman and Hall Ltd., London.
2. K. R. Khandelwal, Practical Pharmacognosy, Techniques and Experiments, Nirali Prakashan
25.1-25.8
3. V.N. Naik., Flora of Marathwada., Amrut Prakashan, Pune; Vol. 1, 250,
4. V.N. Naik., Flora of Marathwada., Amrut Prakashan, Pune; Vol. 1, 308
5. R. Silverstein, X. Francis, Spectroscopic Identification of Organic Compounds, VI
th
edn,
Wiley India Ltd.
6. The Wealth of India raw materials. Publications and Information Directorate, CSIR, New
Delhi. 1948-1976. Vol. 01
7. H. Wagner, S. Bladt, Plant Drug Analysis, A Thin Layer Chromatography Atlas, IInd edn,
Springer.
*Correspondence Author: Khan Subur W.* Dept. of Pharmacognosy. Y.B. Chavan College of
Pharmacy, Rauza, Bagh, Aurangabad.
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