Determination of natural compounds in dashmool extracts by thin layer chromatography and high-pressure liquid chromatography

Abstract

The determination of the natural compounds lupeol, beta sitosterol and stigmasterol in plant extracts using HPLC Shimadzu Model LC2010 AHT Auto Sampler (UV -VIS Detector) is reported. The methods were applied to the analysis of lupeol, beta sitosterol and stigmasterol in petroleum ether extract originating from Dashmool. The result of TLC of Dashmool petroleum ether extract showed the presence of β- sitosterol, stigmasterol and lupeol in Dashmool plant extract at different Rf. HPLC profile of petroleum ether extract of Dashmool have characteristics peaks at retention time 2.888, 2.971, 3.135, 3.442, 4.018, 4.220, 4.406, 4.885, 5.388, 5.657, 6.083 (Stigmasterol), 7.848, 9.137, 10.012, 17.656 (Lupeol), 18.138 (β-Sitosterol) and 23.096. These peaks showed that there are different compounds and characteristic fingerprints of drug to judge in a herbal formulation.

Full text

Mittal Sandhya et al / IJRAP 3(6), Nov – Dec 2012
814
Research Article
www.ijrap.net
DETERMINATION OF NATURAL COMPOUNDS IN DASHMOOL EXTRACTS BY
THIN LAYER CHROMATOGRAPHY AND HIGH-PRESSURE LIQUID CHROMATOGRAPHY
Mittal Sandhya1*, Rao Nidhi1, Sudhanshu1, Menghani Ekta2
1Suresh Gyan Vihar University, Jaipur, India
2Mahatma Gandhi Institute of Applied Sciences, JECRC University, Jaipur, India
Received on: 11/08/12 Revised on: 19/10/12 Accepted on: 04/11/12
*Corresponding author
E-mail: guddan_mitt@ya hoo.co.in
DOI: 10.7897/2277-4343.03626
Published by Moksha Publishing House. Website www.mokshaph.com
All rights reserved.
ABSTRACT
The determination of the natural compounds lupeol, beta sitosterol and stigmasterol in plant extracts using HPLC Shimadzu Model LC2010 AHT Auto
Sampler (UV –VIS Detector) is reported. The methods were applied to the analysis of lupeol, beta sitosterol and stigmasterol in petroleum ether
extract originating from Dashmool. The result of TLC of Dashmool petroleum ether extract showed the presence of β- sitosterol, stigmasterol and
lupeol in Dashmool plant extract at different Rf. HPLC profile of petroleum ether extract of Dashmool have characteristics peaks at retention time
2.888, 2.971, 3.135, 3.442, 4.018, 4.220, 4.406, 4.885, 5.388, 5.657, 6.083 (Stigmasterol), 7.848, 9.137, 10.012, 17.656 (Lupeol), 18.138 (β-
Sitosterol) and 23.096. These peaks showed that there are different compounds and characteristic fingerprints of drug to judge in a herbal formulation.
Keywords: Lupeol, β-Sitosterol and Stigmasterol, high-performance liquid chromatography.
INTRODUCTION
Dashmool is a combination of ten ayurvedic herbs; Aegle
marmelos L., Oroxylum indicum (Linn) Vent, Gmelina
arborea L., Stereospermum suaveolens L., Premna
integrifolla L., Desmodium gangeticum (L.) DC., Uraria
lagopoides L., Solanum indicum L., Solanum
xanthocarpum SCHRAC&WENDLE and Tribulus
terrestris L. which is used in pacifying vata dosha. The
herbs are used in making certain therapeutic massage oils,
and are also used in the form of tea to treat certain
conditions. In the Ayurvedic system of medicine it is used
as analgesic, antiarthritic, against cough and rheumatism
etc1.
Considering the fact that Dashmool is easily available and
more useful, some herbal industries tend to use Dashmool
for different purpose. So it is critical to build up a
appropriate and consistent classification system to
corroborate the value of extracts and herbal drugs.
Separation and detection of diverse constituents in plants
have been always convoluted. While conventional
research mostly focuses on fortitude of the active
components, fingerprinting can offer characterization of a
complex system with a degree of quantitative reliability,
so it has gained increasing interest for quality control
systems over the past years 2. Chromatography methods
including TLC and HPLC techniques are mainly used for
fingerprinting3-5. TLC is a common quick and cost-
efficient method used for fingerprinting plant extracts.
Moreover, several samples can be chromatographed
concurrently on a single plate and complex instruments
are not necessary4-6. In this examination, TLC and HPLC
chromatograms of essential oil and extract of Dashmool
were prepared and their patterns were compared with each
other to specify the similarities and differences between
them.
MATERIALS AND METHODS
Collection: Plant sample was collected with the help of
various tribes living in tribal pockets of Mount Abu, arid
zone of Rajasthan, in the month of Feb, 2010. This plant
was used by these tribes in their daily lives to cure various
ailments.
Identification: This sample was authenticated and
submitted in Ethnomedicinal Herbarium, Centre of
Excellence (funded by DST), MGIAS, Jaipur (Rajasthan)
with herbarium voucher number Oroxylum indicum Linn
Vent (MP0089), Gmelina arborea L. (MP0055),
Stereospermum suaveolens L. (MP00156), Premna
integrifolla L. (MP00102), Desmodium gangeticum (L.)
DC. (MP0032), Uraria lagopoides L. (MP00178),
Solanum indicum L. (MP00139), Solanum xanthocarpum
Schrac & Wendle (MP00148) and Tribulus terrestris L.
(MP00165), Aegle marmelos L. (MP0020)
Preparation of test extracts: Crushed powder of species
was soxhlet extracted. Later the homogenate was filtered
and the residue was re-extracted twice for complete
exhaustion and extract was cooled individually. Filtrate
was concentrated to dryness in vitro and re dissolved in
respective solvent which was stored at 4°C in a
refrigerator, until screened for TLC and HPLC profiling.
Thin layer chromatography (TLC)
Extraction procedure
For TLC profile of selected species, dried and powdered
(100 gm) test samples was soxhelt extracted in petroleum
ether for 6 hours. These extract were filtered, evaporated
to dryness and weighed. Extract (10 mg) was dissolved to
make a concentration of 1mg/ml used for further studies.
TLC plates
Extract was applied on silica gel G Thin Layer
Chromatography (TLC) coated plates (Merck: 20x20 cm;
with thickness 0.2-0.3mm) which were activated at 100°C
for 30 minutes and brought to room temperature, just
before use. Extract of species was applied 1cm above the

Mittal Sandhya et al / IJRAP 3(6), Nov – Dec 2012
815
edge of the chromatographic plates along with the
reference compounds and developed in air-tight chamber
already saturated with 200 ml of solvent system7.
TLC solvent system
Extract of test sample was subjected to different solvent
systems for identification of any significant bio
molecules. After having used different solvent systems,
on the basis of better resolution of spots for generating
“Thin Layer Chromatography (TLC) fingerprints” for
chemical libraries of the test drugs, following solvent
system were used in the present study- Acetone : Hexane
(1:3) for Petroleum ether extract.
TLC spraying reagents
During the work of present studies, different visualizing
reagents i.e. 10% sulphuric acid (10 ml conc. Sulphuric
acid dissolved in 100ml absolute alcohol), I2 vapour
(Saturated iodine chamber) and Dragendorff reagent were
used.
Qualitative TLC
Thin glass plates were coated (0.2-0.3 mm) with silica gel
G (30 g/60 ml distilled water) and dried at room
temperature. The coated plates were activated in an oven
at 100°C for 30 minutes and cooled. The plates were then
placed in developing tanks having 150 ml of an organic
solvent mixture of Acetone: Hexane (1:3) for Petroleum
ether extracts. The lid of the developing tanks was sealed
with vaccum grease. The plates were removed after
making the solvent front and were air-dried. The dried
plates were sprayed with 10% sulphuric acid (10 ml
concentrated Sulphuric acid dissolved in 100ml absolute
alcohol) and further addition of 28 g (KI) and alkaloid.
Positive spot (Rf value) was calculated.
Preparative TLC
Silica gel G thick layer plates were activated at 100̊ C for
30 min. The petroleum ether extract and the reference
compound β- sytosterol8, Stigmasterol and Lupeol were
applied separately as a streak 1 cm above the edges of the
plates and developed in an organic solvent mixture of
Acetone: Hexane (1:3). A portion of the plate containing
the applied standard reference and the extract was
visualized under 10 % sulphuric acids (10 ml conc.
Sulphuric acid dissolved in 100ml absolute alcohol) and
also exposed to I2 vapour for 10 min. The spots coinciding
to the reference compounds were marked and compared
with that of standard reference compound.
HPLC Analysis
The HPLC analysis was performed using a Shimadzu
Model LC2010 AHT Auto Sampler (UV –VIS Detector)
set at 254nm, column Hypersil BDS C18 (250 x 4.6 mm;
5 Micron), flow rate: 1ml/min, injection volume 20μl in
methanol (HPLC grade).
Standard Preparation
One mg of standard compound that is lupeol,
stigmasterol, β- sitosterol were isolated from TLC
fingerprinting of plant and authenticated by spectral
analysis. They were dissolved in methanol and volume of
which was raised to 1ml and used.
Table 1: Rf values of Dashmool Pet.ether extract with standard
Pet. ether
Plant Extract
β- Sitosterol
Stigmasterol
Lupeol
Rf(A)
.72
-
-
.72
Rf(B)
.62
-
.62
-
Rf(C)
.60
.60
-
-
Table 2: HPLC retention time and area of Lupeol, β- Sitosterol, Stigmasterol pure compound
Peak
Retention Time
Area
1.
2.833
357078
2.
6.714
765257
3.
17.656
965560
4.
18.136
970432
Table 3: HPLC retention time and area of Dashmool petroleum ether extract
Peak
Retention Time
Area
Height
Area%
Height%
K’
1.
2.888
348101
76298
3.267
7.995
0.000
2.
2.971
662293
68659
6.216
7.195
0.029
3.
3.135
240598
54072
2.258
5.666
0.085
4.
3.442
4556016
299958
42.758
31.433
0.192
5.
4.018
28142
4315
0.264
0.452
0.390
6.
4.220
474367
67956
4.452
7.121
0.461
7.
4.406
3826856
348190
35.915
36.487
0.525
8.
4.885
21577
3192
0.203
0.334
0.691
9.
5.388
31955
2576
0.300
0.270
0.866
10.
5.657
99463
10712
0.933
1.123
0.959
11.
6.083
1667
1205
0.156
0.126
1.106
12.
7.848
15617
1372
0.147
0.144
1.717
13.
9.137
21400
1388
0.201
0.145
2.164
14.
10.012
72482
5592
0.680
0.586
2.466
15.
17.656
21210
976
0.199
0.102
3.681
16.
18.138
27163
1334
0.255
0.140
6.097
17.
23.096
191459
6478
1.797
0.679
6.996

Mittal Sandhya et al / IJRAP 3(6), Nov – Dec 2012
816
Figure 1: Presence of β- Sitosterol, Stigmasterol an d Lupeol in plant extract
Figure 2: TLC plate of Dashmool Pet.ether extract
Figure 3: HPLC chromatograms of Lupeol, β- Sitosterol and
Stigmasterol pure compound
Figure 4: HPLC chromatograms of Dashmool petroleum ether extract.
Figure 5: Normalized fingerprints of alcohol soluble Dashmool extract

Mittal Sandhya et al / IJRAP 3(6), Nov – Dec 2012
817
Figure 6: Normalized fingerprints of alcohol soluble Lupeol, β- Sitosterol and Stigmasterol
Quantification of natural compounds in Dashmool by
HPLC
Pet. ether extract (piperine-rich fraction) of Dashmool
was weighed and dissolved in methanol. 20 μl of
concentration of Dashmool was injected onto HPLC and
the peak which appeared at the same retention time as that
of standard was recorded. This value was used to
calculate the amount of standard in the extract by using
the linear equation obtained from the composite standard
curve. In the present work, various calculations were
achieved by Height/ Area method. This method utilizes
the fact that the area of a peak is a function of its height
and standard deviation. To determine efficiency, values
for peak height and area are used in a different formula: N
= 2π (htr) 2/A2
(Where h =peak height; tr= retention time; N= number of
theoretical plates; A=area). A computer is usually
necessary to use this method in order to calculate the area
and height.
RESULTS AND DISCUSSION
Dashmool Petroleum ether extract when run in solvent
system Acetone: Hexane (1:3) with standard showed 3
spots by visible / naked eye at Rf .72 (Purple), Rf
.62(Purple), Rf .60 (Pink) by spraying with 10% H2SO4.
Graph showing the result of TLC of Dashmool Pet.ether
extract. It showed the presence of β- Sitosterol,
Stigmasterol and Lupeol in Dashmool plant extract.
+In the present study, HPLC was performed for Lupeol,
β-Sitosterol and Stigmasterol run in methanol under 254
nm, the time recorded at 18.138, 6.714 and 17.656, which
showed that as the column size increases it affects on
retention time (column size α rt). It also affects the peak
sharpness.
HPLC profile of petroleum ether extract of Dashmool
have characteristics peaks at retention time 2.888, 2.971,
3.135, 3.442, 4.018, 4.220, 4.406, 4.885, 5.388, 5.657,
6.083 (Stigmasterol), 7.848, 9.137, 10.012, 17.656
(Lupeol), 18.138 (β-Sitosterol), 23.096. These peaks
showed that there are different compounds and
characteristic fingerprints for each drug to judge in an
herbal formulation. There normalized fingerprints are
principal markers that can check the purity/impurity of
drug at very low concentration.
CONCLUSION
In present investigations attempts were made to isolate
various pure bioactives from Dashmool and it is
noteworthy that beta sitosterol, stigmasterol and lupeol
were isolated which are potentials source as anti HIV
agents. Thus, these plants have potentials role in future as
drug or therapeutic targets.
ACKNOWLEDGEMENT
Author acknowledge with thanks the financial support
from Department of Science and Technology,
Government of Rajasthan, in the form of Centre with
Potentials for Excellence in Biotechnology, sanction no F
7(17) (9) Wipro/Gaprio/2006/7358-46(31/10/2008).
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Cite this article as:
Mittal Sandhya, Rao Nidhi, Sudhanshu, Menghani Ekta. Determination
of natural compounds in Dashmool extracts by Thin layer
chromatography and High-pressure liquid chromatography. Int. J. Res.
Ayur. Pharm. 2012; 3(6):814-817 .
Source of support: Nil, Conflict of interest: None Declared