PHYTOCHEMICAL ANALYSIS

Phytochem. Anal. 12, 91–95 (2001)

DOI: 10.1002/pca.569

Determination of Marker Constituents from

Cissus quadrangularis Linn. and their
Quantitation by HPTLC and HPLC

Manisha Mehta, Navneet Kaur and K. K. Bhutani*

Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S. Nagar (Mohali), Near
Chandigarh, India

Four marker constituents, namely, onocer-7-ene-3a,21b-diol, d-amyrin, d-amyrone and 3,3',4,4'-

tetrahydroxybiphenyl of an Ayurvedic crude drug Cissus quadrangularis Linn. are defined for
standardisation purposes. 3,3',4,4'-Tetrahydroxybiphenyl has been isolated for the first time from this
drug. The contents of the marker constituents were quantitatively determined by HPTLC and HPLC
methods in samples collected from five different geographic zones of India. Copyright # 2001 John Wiley &
Sons, Ltd.
Keywords: HPLC; HPTLC; marker compounds; Cissus quadrangularis.

INTRODUCTION

The roots, young shoots and stem of the climber Cissus

quadrangularis Linn. provide an important Ayurvedic
single crude plant drug used in the treatment of fractured
bones, dyspepsia, indigestion and asthma in the Indian
System of Medicine (Chopra et al., 1986). This plant is
found throughout the hotter parts of India and Srilanka
(Anonymous, 1950). Earlier, we had reported the
presence of two new unsymmetric tetracyclic triterpe-
noids, namely, onocer-7-ene-3a, 21b-diol (1) and onocer-
7-ene-3b, 21a-diol (2) alongwith d-amyrin (3) and d-
amyrone (4) (Bhutani et al., 1984) in this plant.
Subsequently, another unsymmetric tetracyclic triterpe-
noid, 7-oxoonocer-8-ene-3b, 21a-diol (5) has been
reported (Gupta and Verma, 1990). Apart from these,
seven alicyclic lipid constituents were also reported
(Gupta and Verma, 1991).
Because of our renewed interest in the development of
Ayurvedic Pharmacopoeial standards, a standardisation
study on this plant was conducted and to the best of our
knowledge this is reported for the first time. It was marcs obtained from hexane extraction. Compound 6,
observed that compounds 1, 3 and 4 were present in non- being a product of a specific enzyme system of catechol
polar (hexane) fractions derived from all randomly oxidases (Kandaswami and Vaidyanathan, 1973) is an
collected samples from different geographic zones of interesting marker constituent for this plant. The contents
India, and these compounds could be considered as of 1, 3 and 4 were quantified through HPTLC, and an
marker constituents. In addition a new constituent, HPLC quantitation method was developed for 6 as a part
namely 3,3',4,4'-tetrahydroxybiphenyl (6), was detected of this study.
and isolated for the first time from this plant. This
compound was found to be present in all of the
sequentially prepared methanolic extracts of the plant
EXPERIMENTAL

* Correspondence to: K. K. Bhutani, Department of Natural Products,
National Institute of Pharmaceutical Education and Research (NIPER), Sector
67, S.A.S. Nagar (Mohali), Near Chandigarh 160062, India.
E-mail: niper@chd.nic.in
Contract/grant sponsor: Department of Indian System of Medicine, Ministry
of Health and Family Welfare (Government of India).
Plant material, extraction and isolation. The whole
plant materials (samples A–E, respectively) were col-
lected from different wild locations in the central, south,
north, east and west geographic zones of India by the
Regional Research Centres of CCRAS located at Jhansi,

Received 30 September 1999

Copyright # 2001 John Wiley & Sons, Ltd. Revised 7 April 2000
Accepted 1 August 2000

92 M. MEHTA ET AL.
Figure 1. Outline scheme of the isolation procedure for onocer-7-ene-3a, 21b-diol (1), onocer-7-ene-3b, 21a-diol (2) d-amyrin (3)
and d-amyrone (4).
Trivenderum, Patiala, Guwahti and Junagarh, and were
pooled zone-wise. The identities of the collections were
confirmed by conducting macro- and microscopic studies
in our laboratory, and authenticated by comparison with
standard reference samples. The voucher samples are
maintained in the Department of Natural Products,
NIPER, India.
For preparation of the extracts, the roots and aerial
parts were powdered and each powdered sample (100 g)
was exhaustively extracted with hexane (750 mL) in a
soxhlet apparatus. The hexane extracts were evaporated
(in vacuo) to dryness and amounts of 5.29, 5.69, 3.78,
3.19 and 3.52 g were obtained from the central, south,
north, east and west geographic zone samples, respec-
tively. A portion of each extract was preserved separately
for quantitation purposes whilst the remaining portions
were combined for over silica gel (60–120 mesh)
phytochemical studies. The combined dried extract
(10 g) was subjected to MPLC eluted with hexane,
hexane: ethyl acetate mixtures, and pure ethyl acetate in
increasing order of polarity. A total of 64 fractions were
collected and those fractions showing identical TLC
patterns were combined (Fig. 1). The bulked fractions
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F003, F004 and F005 were further processed as they were
comparatively higher in yield and less complex in
mixture. Fraction F004 (600 mg), on storage in hexane
at room temperature, deposited a crystalline material
which was separated and recrystallised from n-hexane to
yield white crystals of d-amyrin (3; 108 mg).
Fraction F003 on TLC showed the presence of one
major compound, and was re-chromatographed over
silica gel, eluting with pure hexane and hexane: ethyl
acetate mixtures. Sub-fraction F012 (Fig. 1) showed a
single spot on TLC and, on storing in ethyl acetate
overnight at room temperature, yielded a crystalline
compound (80 mg) which was later identified as d-
amyrone (4).
Fraction F005 was also further subjected to column
chromatography to yield a total of 39 subfractions (Fig.
1); two of these subfractions, F017 (500 mg) and F018
(109 mg), exhibited single spots on TLC and were further
recrystallised to yield onocer-7-ene-3a, 21b-diol (1;
300 mg) and onocer-7-ene-3b, 21a-diol (2; 60 mg),
respectively. All four isolated and purified compounds
(1, 2, 3 and 4) were characterized by comparison of TLC,
melting point and IR with authenticated samples, and by
Phytochem. Anal. 12: 91–95 (2001)

STANDARDISATION OF CISSUS QUADRANGULARIS
co-chromatography and mixed melting point with
authentic standards (Bhutani et al., 1984).
The plant marcs, after preparing hexane extracts, were
further subjected to exhaustive extraction with methanol
(750 mL) in a soxhlet apparatus. The methanol extracts
were evaporated to dryness under reduced pressure and
amounts of 8.86, 7.53, 12.52, 10.48 and 10.82 g were
obtained from central, south, north, east and west
geographic zone samples, respectively. Again, a part of
each extract was preserved separately and the rest of the
material was combined for isolation purpose. The
combined methanolic extract (10 g) was fractionated by
MPLC over silica gel (60–120 mesh); six broad fractions
were obtained using hexane, and hexane and chloroform
mixtures as eluent. Fraction F001 (900 mg) was subjected
to further column chromatography, eluting with hexane:
chloroform (95:5), to yield needle-shaped crystals from
ethyl acetate in minor amounts (20 mg). The compound
was characterized as 3, 3', 4, 4' - tetrahydroxybiphenyl (6)
on the basis of spectral data (MS, IR, NMR) and
comparison with reported values in the literature (Joulie
et al., 1994), and with the fragmentation pattern of 6 as
given in the mass library of Shimadzu GCMS Model QP
5000.
HPTLC conditions. A Camag (Muttenz, Switzerland)
HPTLC system equipped with a Linomat IV sample
applicator was used to analyse the sample on aluminium-
backed TLC plates (20  20 cm) precoated with silica gel
60F254 (Merck, Darmstadt, Germany). For developing
the plates, a Camag twin trough development chamber
was used in which the vertical development of the plate
was performed using hexane:ethyl acetate (6:1) as the
mobile phase. Visualisation of the plates was performed
by treatment with 10% methanolic sulphuric acid for 30 s
followed by heating in an oven at 100°C for 10 min.
Detection was carried out at 366nm using a Camag TLC
scanner 3 and associated integration software (Cats 4.03
version).
HPLC conditions. An HPLC system supplied by Waters
Corporation (USA) comprising delivery pump model 600
with four channel online degasser and a Rheodyne
sample injector (20 mL loop) was used. The column was a
Novapak C18 (250  4.6 mm i.d.) held at 27°C. Isocratic
elution was carried out with acetonitrile:water (1:1) at a
flow-rate of 0.5 mL/min; detection was at 233 nm using a
Waters PDA detector (model 996). Millennium V 2.10
software was used for integration and calibration.
Calibration curves. The calibration curves were ob-
tained using six data points (0.5–5 mg) for compound 1,
using seven data points (0.25–5 mg) for compounds 3 and
4, and using four data points (0.0625–0.25 mg) for
compound 6.
Estimation of marker constituents in the plant. Each
of the dried hexane extracts (20 mg) and methanol
extracts (20 mg) were dissolved in 1 mL of chloroform or
methanol, respectively. Aliquots (5 mL) of chloroform
solutions were subjected to HPTLC, and aliquots (20 mL)
of methanol solutions were injected into the HPLC. The
HPTLC plates were developed to a distance of 10 cm
from the point of application, dried, subjected to the
derivatisation procedure and scanned. HPLC analyses
were continued for 20 min since the retention time of 6
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93
was 14.4 min. The contents of marker constituents were
estimated by using pure reference compounds (1, 3, 4 and
6) as external standards. Compound 2 was found not to be
present in most of the extracts and was thus excluded for
estimation purposes. The content of marker constituents
was calculated by linear regression, and mean percen-
tages were calculated from three separate experiments.
Assay validation. The precision of the experiments was
expressed by coefficients of variation. The R f values were
determined and the precision for each of the standard
marker constituents (1, 3 and 4) was evaluated in
triplicate using 1, 2 and 3 mg quantities. The efficiency
of the HPTLC assay procedure was determined by
separately adding individual marker constituent (0.5 mg)
to the plant material (2 g) and subjecting the samples to
the optimised extraction procedure (described above).
The recovery of the individual marker constituent was
then calculated by a densitometric method. The detection
limit for each individual marker constituent in the assay
procedure was also determined. The efficiency of HPLC
assay was determined by estimating the recovery of 6
from a sample of plant material (5 g) to which 1 mg of
exogenous 6 had been added prior to defatting. The
content of marker compound 6 was calculated by
comparison with an unspiked control sample.
RESULTS AND DISCUSSION
Consistent quality for products of herbal origin can only
be assured if the starting plant materials are defined in a
rigorous and detailed manner. Characterisation of a
herbal drug is, therefore, essential to allow specifications
to be established which are both comprehensive and
relevant. In the present study, we have tried to define
multiple characterising compounds, generally referred to
as marker constituents, for the identity and authentication
of Cissus quadrangularis Linn. with a modified chemo-
profiling approach. In this approach, the sampling of
crude drugs was randomly performed to include the
obvious inherent variations such as temperature, humid-
ity, soil etc., available in five geographic zones of the
Indian sub continent. Whilst collections were not
controlled with respect to age of material and other
environmental factors, the botanical identity of each of
the samples was established with total certainty in our
laboratory. For convenience, the chemical constituents
were partitioned into non-polar (hexane), moderately-
polar (methanol) and polar (aqueous) extracts using
successive exhaustive extractions. Qualitative TLC
fingerprints of the hexane and methanolic extracts were
established, and a general gross similarity with regard to
the presence of the constituents was observed.
Compounds 1, 3 and 4 were found to be present in the
hexane extracts of all collections, whereas compound 2,
although isolated from a combined extract, was absent in
most of the samples. Therefore, onocer-7-ene-3a, 21b-diol
(1), d-amyrin (3) and d-amyrone (4) could serve as non-
polar marker constituents for this plant. An HPTLC
method for the standardisation of these compounds has
been found to be suitable. TLC chromatograms of these
reference standards and of the hexane extracts prepared
from different zone-wise collections are shown in Plate 1.
All experiments were performed in triplicate and results
Phytochem. Anal. 12: 91–95 (2001)
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94 M. MEHTA ET AL.

Table 1. HPTLC calibration plot data and precision measurements of marker constituents

Calibration plot dataa Precision measurementsa

Mean area
Amount Amount of (coef®cient of Mean Rf (coef®cient
Marker constituent (mg) Mean area SD standard (mg) variation) of variation)

Onocer-7-ene-3a, 0.50 2250 128.34 1 4234 (3.19) 0.25 (2.28)

21b-diol (1) 1.00 4234 135.11
2.00 5669 155.81 2 5669 (2.74) 0.24 (2.37)
3.00 6673 225.96
4.00 7819 382.06 3 6673 (3.38) 0.25 (0)
5.00 9082 355.68
d-amyrin (3) 0.25 1931 72.75 1 3813 (4.28) 0.39 (2.56)
0.50 2707 129.06
1.00 3811 163.19 2 5486 (4.22) 0.39 (2.99)
2.00 5436 229.22
3.00 6561 172.29 3 6561 (2.62) 0.41 (2.44)
4.00 7857 258.39
5.00 9131 402.05
d-amyrone (4) 0.25 1228 79.00 1 3445 (2.29) 0.82 (1.22)
0.50 2209 104.50
1.00 3445 79.05 2 4281 (2.47) 0.80 (0.72)
2.00 4281 105.88
3.00 5886 227.18 3 5886 (3.86) 0.81 (0.71)
4.00 6883 348.62
5.00 8572 317.96
a
n = 3.

are expressed as mean  SD; good linearities of the
calibration curves were obtained for compounds 1, 3 and
4, and their correlation coefficients (r) were found to be
0.991, 0.993 and 0.979, respectively. The calibration plot
data and precision measurements obtained for there
compounds are given in Table 1. The regression equations
used for quantitative determination of 1, 3 and 4 were
y = 1380x ‡ 2381, y = 1462x ‡ 2058 and y = 1425x ‡
1435, respectively. The coefficient of variation for these
three compounds was found to be <4.28 in area counts
and <2.98 for R f values. These values indicated that the
assay method was fairly reproducible. Further, the
recovery percentages for 1, 3 and 4 were found to be
97.2, 96.0 and 97.5% in the plant samples and their zone-
wise content varied in the range of 38.8  10ÿ3–
71.6  10ÿ3, 28.1  10ÿ3–109.7  10ÿ3 and 40.2  10ÿ3
ÿ143.7  10ÿ3, respectively (see Table 2).
One minor constituent was found to be present in all of
the defatted methanolic extracts available. This com-
pound was targetted for isolation from the pooled
methanolic extracts and was characterized as 3,3',4,4'-
tetrahydroxybiphenyl (6). Incidently, this compound has
been detected and isolated for the first time from this
plant. Being highly sensitive to UV detection, compound
6 could serve as moderately polar marker constituent of
this plant. HPLC chromatograms of this reference
standard and of all of the methanolic extracts are shown
in Plate 1 and were used for quantitation purposes. All
experiments were performed in triplicate and results are
expressed as mean  SD. A good linearity of the
calibration curve was obtained for marker compound 6,
and the correlation coefficient (r) was found to be 0.999.
The regression equation used for the quantification of 6 in
the plant material was y = 12143089x ÿ 726.354. The
mean coefficient of variation for the peak areas was
<3.70, and for the mean retention time was <1.15. The
mean recovery of the reference standard 6 was found to
be 98.62% (SD = 1.140) in the plant samples, and its
zone-wise content varied in the range of 0.11  10ÿ3–
4.87  10ÿ3 (Table 2). All of these results suggest that
this HPLC method is fairly reproducible for this new
marker constituent of the plant.

Table 2. Percentage (w/w) of marker compounds 1, 3, 4 and 6 in crude drug samplesa of Cissus quadrangularis

Percentage (w/w) mean  SD (10ÿ3)

Sample code 1 3 4 6

A (central zone) 71.6  2.4 105.4  3.29 110.7  3.95 0.29  0.0054
B (south zone) 42.3  2.1 109.7  4.87 143.7  71.3 0.11  0.0053
C (north zone) 44.0  1.9 39.5  1.71 59.0  1.71 4.87  0.11
D (east zone) 38.8  1.9 28.1  1.82 56.3  2.79 3.62  0.08
E (west zone) 44.2  1.9 31.6  1.80 40.2  0.79 3.91  0.17
a
Identi®ed by geographical origin (see Experimental section).

Copyright # 2001 John Wiley & Sons, Ltd. Phytochem. Anal. 12: 91–95 (2001)
10991565, 2001, 2, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/pca.569 by UNIFAL - Universidade Federal de Alfenas, Wiley Online Library on [25/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Phytochem. Anal. 12 (2001)

Plate 1. Showing the HPTLC and HPLC chromatogram of marker compounds 1, 3, 4 and
6 together with hexane and methanolic extracts of Cissus quadrangularis (A±E)
identi®ed by geographical origin (see the Experimental Section).

Copyright # 2001 John Wiley & Sons, Ltd.


STANDARDISATION OF CISSUS QUADRANGULARIS
In conclusion, four marker constituents for C. quad-
rangularis have been determined and defined by employ-
ing a randomized collection approach; the possibility of
finding more marker constituents for this plant remains
open.
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Copyright # 2001 John Wiley & Sons, Ltd.
10991565, 2001, 2, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/pca.569 by UNIFAL - Universidade Federal de Alfenas, Wiley Online Library on [25/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
95
Acknowledgements
The authors are greatful to the Department of Indian System of
Medicine, Ministry of Health and Family Welfare (Government of
India) for financial support, and to the Director of NIPER, Dr C. L.
Kaul, for interest in this work. Thanks are due to K. Prasanna
(JTA) and Vipin Sharma (Project Assistant) for their technical
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Phytochem. Anal. 12: 91–95 (2001)