Research Article

Antiosteoporotic activity of phytoestrogen-rich fraction separated

from ethanol extract of aerial parts of Cissus quadrangularis in
ovariectomized rats
Urmila M. Aswar, V. Mohan1, Subhash L. Bodhankar2

ABSTRACT
Objective: Cissus quadrangularis L. (C. quadrangularis L.) (Vitaceae) has been reported
in Ayurveda for its antiosteoporotic activity. The study separated the phytoestrogen-rich
fraction (IND-HE) from aerial parts of C. quadrangularis L. and evaluated its effect on
osteoporosis caused by ovariectomy in rats.
Department of Pharmacology, Materials and Methods: IND-HE was separated from the ethanol extract of C. quadrangularis.
STES’S Sinhgad Institute of Ovariectomized female Wistar rats were divided into four groups (n = 6). Group 1: Control
Pharmacy, Narhe, 1Indus Biotech (distilled water), Group II: IND-HE (75 mg/kg p.o.), Group III: IND-HE (100 mg/kg p.o.) were
Private Limited, Kondhwa, treated once daily for 8 weeks and Group IV: standard estradiol group, received estrogen
2
Department of Pharmacology, (1 mg/kg, s.c. bi-weekly). The effects on body weight were determined. DEXA (Dual energy-
BVP’s Poona College of Pharmacy, emission X-ray absorptimatory analysis) of whole body bone and femur was carried out.
Erandwane, Pune, Maharashtra, Blood was removed and analyzed for biochemical parameters. After sacrificing the animals,
India.
biomechanical study of right tibia and histopathology of pelvic bone was carried out.
Results: IND-HE showed presence of phytoestrogen-rich fraction. IND-HE (75 and 100 mg/kg)
Received: 04-05-2011
Revised: 03-01-2012 and estrogen treatment showed statistically significant increase in bone thickness, bone
Accepted: 28-02-2012 density and bone hardness. IND-HE (75 and 100 mg/kg) and estrogen treatment significantly
increased serum estradiol. IND-HE (100 mg/kg) (P<0.05) and estrogen treatment increased
Correspondence to: serum vitamin D3 and serum calcium compared to control. Alkaline phosphatase was
E-mail: sbodhind@rediffmail.com significantly reduced by IND-HE (100 mg/kg p.o.) and estrogen treatment. Histopathology
and DEXA results indicated that IND-HE (75 and 100 mg/kg) prevented bone loss.
Discussion and Conclusion: These findings confirm that phytoestrogen-rich fraction
(IND-HE) possess good antiosteoporotic activity.

KEY WORDS: Antiosteoporosis, estrogenic activity, phytoestrogen-rich fraction,

ovariectomized rats, serum alcium

Introduction Traditional therapies for postmenopausal osteoporosis

Osteoporosis is characterized by a reduction in bone density
and strength to the extent that fractures occur after minimal
trauma. It is well known that in human females, estrogen
deficiency caused by ovariectomy as well as menopause leads
to acceleration of bone resorption and rapid bone loss, resulting
in development of osteoporosis.
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DOI: 10.4103/0253-7613.96310
have emphasized agents that inhibit bone resorption such as
estrogen and calcitonin.[1] Although the most effective method
to reduce the rate of postmenopausal bone loss is estrogen
replacement therapy, it may be accompanied by side effects.
It is recommended only for woman who are at high risk of
osteoporosis and have no contraindications for estrogen.[2]
Plants containing phytoestrogen and triterpenoids have
been used in traditional system of medicine for the treatment
of osteoporosis. Cissus quadrangularis L. (Vitaceae), a climbing
shrub, characterized by a thick quadrangular fleshy stem, is an
edible plant found in hotter parts of India, Sri Lanka, Malaya,
Java and West Africa. Commonly known as the “bone setter,”
the plant is referred to as “Asthisamdhani” in Sanskrit and
“Hadjod” in Hindi because of its ability to join bones. The plant
has been documented in Ayurveda for its medicinal uses in gout,

Indian Journal of Pharmacology | June 2012 | Vol 44 | Issue 3 345

 Aswar, et al.: Anti-osteoporotic activity of IND-HE of Cissus quadrangularis L.
syphilis, venereal disease, piles, leucorrhea, as an aphrodisiac
and in the Siddha system of medicine for the treatment of piles,
diarrhea and dysentery and in diseases of kapham (Kapha
represents water and earth element in body).[3] Khan et al.[4]
reported the multifarious medicinal claims of C. quadrangularis
by the Gond tribals of Raisen district, India. The stem juice is
used to treat scurvy and irregular menstruation, the plant juice
is used in otorrhea and epistaxis. The root is used specifically
for bone fracture.[5] The root is reported as most useful for the
fractures of bones, with the same effects as plaster externally.
The extract of the plant has been reported to possess fracture
healing property.[6,7]
The phytochemical analysis of the plant showed the
presence of vitamin C, b-carotene, two symmetric tetracyclic
triterpenoids, b-sitosterol, a-amyrin, a-amyrone and three
stilbene derivatives and quandragularins A, B, C. In addition to
vitamin C, it also contains a high amount of carotene A, anabolic
steroidal substance and calcium.[8] The phytoestrogen steroid
isolated has been shown to influence early regeneration and
quick mineralization of bone.[9,10]
The ethanol extract of the plant in the doses of 500
and 750 mg/kg, p.o. has shown antiosteoporotic activity
in ovariectomized rats. [3] The recent study showed the
prominent antiosteoporotic effect of petroleum ether extract
of C. quadrangularis L.[11] Previous study carried out in our
laboratory showed improvement in sexual behavior. Also we
observed increase in weight of rat uterus treated with IND-
HE as compared to control ovariectomized rats indicating
phytoestrogen like activity in IND-HE.[12] Thus the objective of
the study was to evaluate the fraction (rich in phytoestrogen)
in ovariectomized rats for its antiosteoporotic activity.
Materials and Methods
Preparation and standardization of phytoestrogen-rich
fraction (IND-HE)
Collection and authentication: The plant was collected from
district of Madurai, Coimbatore, and Tanjore in India in the
month of March to April. The plant material was authenticated
by Dr. P.G. Diwakar, Taxonomist, Botonical survey of India at
Pune, Maharashtra (India).
The aerial parts of Cissus quandragularis were cut into
small pieces and washed thoroughly in running water to free
it from the adhering soil. This material was dried under shade
(30°C, 45% humidity). The dry plant material was powdered
and passed through the sieve of mesh no. 40. The powder was
extracted then with ethanol (70%) at room temperature for
8 h. in a circulation bed extractor and filtered through filter
press (Kothari equipments, India) so as to remove suspended
foreign matters. This filtrate was concentrated under vacuum
at 45°C to obtain a paste, which was then redissolved in 0.01 M
sodium acetate buffer and filtered The clear liquid was loaded
into a counter current column (tubular) and extracted with
N-butanol. The butanol layer was separated; the solvent was
evaporated to obtain dry powder. This powder was dissolved
in demineralized water and passed through polymer adsorbent
column (Amberlite XAD-7, Rohm and Haas, USA). The column
was washed thoroughly with 2% w/v sodium chloride solution
followed by demineralized water and eluted in a gradient
346 Indian Journal of Pharmacology | June 2012 | Vol 44 | Issue 3
manner with ethanol: water mixture and the fractions were
collected. The elutants were characterized by comparing
with various standards such as friedelin, vetaxin, isovitaxin,
orientin, caempherol and quercitin (Sigma Aldrich) using
High Performance Liquid Chromatography (HPLC). Among
the standards, the peak of the elutant matched with peak
of friedelin. The fractions containing maximum quantity of
phytoestrogens were concentrated under vacuum at 40°C and
the powder was recrystallized in alcohol to get yellow crystals
(yield 2.5% w/v).
HPLC Details: Column: Kromosil reverse phase C-18
(250 × 4.6 mm with 5μ particle size). Mobile phase Gradient
was A) water and B) acetone according to following profile: at
0 min, (75% A and 25% B), at 20 min (65% A and 35% B). Flow
rate: 1 ml/min. Detector: UV (205 nm).[12]
Chemicals
Estradiol benzoate (Himedia Laboratories Mumbai,
India), Ketamine hydrochloride (Aneket, Neon laboratories,
India), Xylazine (Xylaxin, Indian immunologicals Ltd., India),
anesthetic ether (TKM Pharma, India), friedelin (SIGMA, USA)
were purchased. All the chemicals were of analytical grade and
solvents were of HPLC grade.
Animals
Female Wistar rats of weighing 120 g ± 5 g and adult mice
of either sex weighing 20 ± 5 g were purchased from National
Toxicology Centre, Pune, India and used for the study. They were
maintained at a temperature of 25 ± 1°C and relative humidity
of 45-55% under 12 h light: dark cycle. The animals had free
access to food pellets (Nav Maharashtra Chakan Oil Mills Ltd.,
Pune, Maharashtra, India) and water. The experimental protocol
was approved by the Institutional Animal Ethics Committee
(IAEC) of Poona College of Pharmacy, Pune, Maharashtra, India,
constituted under Committee for the Purpose of Control and
Supervision of Experiment on Animals (CPCSEA). The protocol
approval no. is CPCSEA/76/07.
Acute oral toxicity of phytoestrogen-rich fraction
Healthy adult Swiss mice of either sex weighing between 20
and 25 g were used for acute oral toxicity study. The study was
carried out according to OECD (Organization for Economic Co-
operation and Development 2001) guideline number AOT-425.
The mice were observed for 2 h for behavioral, neurological
and autonomic profiles and for any lethality or death for the
next 48 h.
Ovariectomy of the rats[13]
Three days after arrival all the rats were randomly subjected
to ovariectomy. For ovariectomy, the animals were anesthetized
with an intraperitoneal injection of 5% ketamine hydrochloride
(80 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). Bilateral dorsal
incisions were made on the back, both the ovaries were
identified. The ovarian blood vessels were clamped and the
ovaries were excised. The muscle layer was tied and skin
incision was sutured. After the surgery, the rats had free access
to feed and water.
Treatment schedule
After recovery the rats were divided into four groups of
six rats each. Group 1 received distilled water, Group II was
given IND-HE (75 mg/kg p.o.), Group III was given IND-HE
 Aswar, et al.: Anti-osteoporotic activity of IND-HE of Cissus quadrangularis L.

(100 mg/kg p.o.) and Group IV was a standard estrogen group, Results
received estradiol benzoate (1 mg/kg in olive oil suspension,
Oral administration of IND-HE (2000 mg/kg) did not cause
s.c bi-weekly). The treatment period was 8 weeks. Following
mortality or any signs of clinical abnormality in both the groups.
parameters were studied.
At necropsy, no gross pathological changes were observed in the
Body weight measurement
target organs. The LD50 of IND-HE was more than 2000 mg/kg
Body weight of all the animals was recorded daily.
body weight.
Serum estrogen estimation The final product (standardized 2.5% friedelin) from
On the last day of treatment the blood was withdrawn by C. quadrangularis was labeled as IND-HE. The high-performance
retroorbital plexus. Blood was centrifuged to separate the liquid chromatogram [Figure 1] of IND-HE showed the presence
serum. The serum estrogen (E2) was analyzed by Clia: immulite, of friedelin, as major peak at 4.2 min.
fully automated immunoassay analyzer, USA.
Serum estrogen measurement
Hydroxycholecalciferol (Vitamin D3) estimation The serum estradiol (pg/ml) in control group, IND-HE
The serum for vitamin 25 hydroxycholecalciferol was (75 mg/kg), IND-HE (100 mg/kg) and estrogen group was
estimated by radioimmuno assay (BARC, INDIA/DPC, USA/ 15.83 ± 1.8, 46.67 ± 6.5, 52.67 ± 0.9 and 105.9 ± 5.3,
DSL, USA). respectively. The results showed significant increase in serum
Serum alkaline phosphatase estradiol with estrogen (P<0.01); IND-HE (75 mg/kg) (P<0.05)
The serum alkaline phosphatase was measured by an Auto- as well as IND-HE (100 mg/kg) (P<0.05) as compared to control
analyzer Hitachi, model No-912 using kits (Roche, USA) [by pnpp group [Figure 2a]. The rise in serum estradiol was 568.9, 232.7
(P-Nitrophenylphosphate)-kinetic method]. and 194.8% in estrogen group, IND-HE (100 mg/kg) and IND-HE
Serum calcium (75 mg/kg) groups, respectively.
The serum calcium was estimated by Cresolpthalein (Roche) Vitamin D3 estimation
method using an auto-analyzer Hitachi, model no-912. The Significant increase in serum vitamin D3 (ng/ml) was
protocol provided by the manufacturer was followed for each observed in IND-HE (100 mg/kg) that is 49.83 ± 6.19 (P<0.05)
assay. and estrogen group which was 66.83 ± 2.63 (P<0.01) as
Measurement of thickness, density and breaking strength compared to control group (30.0 ± 2.58) [Figure 2b]. The rise
of femur bone in vitamin D3 was 65 and 121% in estrogen group and IND-HE
After removing the blood for biochemical analyses the (100 mg/kg), respectively, compared to control group.
animals were sacrificed. The right femur was removed and was Serum alkaline phosphatase
freed of soft tissue using small scissors, tweezers and cotton
The serum alkaline phosphatase (U/Lt) activity was
gauge. The bone was dried overnight in the oven and bone
significantly reduced (P<0.01) when ovariectomized animals
marrow was carefully removed. The thickness of femur was
were treated with IND-HE (75 mg/kg, p.o.) −140 ± 12.6,
measured with a vernier caliper. Bone was weighed. Bone volume
IND-HE (75 mg/kg, p.o.) – 94.0 ± 12.13 and estrogen (1 mg/
was measured by using plethysmometer (model No.7140, UGO
kg s.c. biweekly) – 78.50 ± 6.95 as compared to control group
Basile, Italy) and bone density was calculated (mass/volume).
−159.7 ± 6.0. The percentage reduction was 12.3, 41.0, and
The breaking strength of left femur bone was estimated using
50.4% in IND-HE (75), IND-HE (100) and estrogen group,
hardness tester (Pharma Test PTB, Incorp., India).
respectively [Figure 2c].
DEXA of whole body and femur
Serum calcium
Dual energy-emission X-ray absorptimatory analysis of
There was significant increase in serum calcium in
whole body bone, femur was carried out using p-DEXA saber,
IND-HE (75 mg/kg) – 9.72 ± 0.10 mg/dl, IND-HE (100 mg/
ORTHOMETRIX.INC.
kg) – 9.93 ± 0.09 mg/dl and estrogen treated group –
Histopathological study 9.79 ± 0.007 mg/dl (P<0.01) as compared to control group
The pelvic bone was also removed and was stored for 24 h – 8.38 ± 0.14 mg/dl [Figure 2d].
in 10% formalin. The bone specimen was dehydrated by placing
it 3 times in xylene (1 h each) and later in alcohol 70, 90 and
Figure 1: HPLC tracing of IND-HE (standardized to friedelin)
100% strength, respectively, each for 2 h. The infiltration and
impregnation was carried out by treating with paraffin wax
twice, each time for on 1 h. Paraffin wax was used to prepare
paraffin L moulds. Specimens were cut into sections of 3-5-µm
thickness and were stained with haematoxylin and eosin. The
specimen was mounted on slide by use of distrene phthalate
xylene (D.P.X).
Statistical analysis
Data for each parameter was analyzed by one-way ANOVA
followed by Bonferroni post hoc test using Graph Pad, Prism
software, version 4.03, USA. The percentage increase was
calculated using the formula (test reading – control reading/
control reading) × 100.

Indian Journal of Pharmacology | June 2012 | Vol 44 | Issue 3 347

 Aswar, et al.: Anti-osteoporotic activity of IND-HE of Cissus quadrangularis L.
Body weight
The body weight of animals on the last day of treatment did
not change significantly in all groups as compared to control
group. The difference in increase in body weight was control
– 89.67 ± 9.2 g, IND-HE (75 mg/kg) – 93.8 ± 7.7 g, IND-HE
(100 mg/kg) −105 ± 8.6 g, estrogen (1 mg/kg) −107 ± 14.41 g
[Figure 3a].
Measurement of thickness, density and breaking strength of
femur bone
In animals decreased bone thickness, bone density and
bone hardness was observed as compared to estrogen treated
animals. The results showed significant increase in bone
thickness in estrogen (1 mg/kg) group −3.41 ± 0.21 mm
(P<0.01), IND-HE (100 mg/kg) −3.34 ± 0.12 mm (P<0.01), as
well as in IND-HE (75 mg/kg) group (2.67 ± 0.05 mm) (P<0.05)
as compared to control group −1.85 ± 0.12 mm [Figure 3b]. The
rise in bone thickness was 84, 80.8 and 54.6% in estrogen group,
IND-HE (100 mg/kg) and IND-HE (75 mg/kg), respectively.
There was significant increase in bone density in estrogen
Figure 2: Data represented are mean number of observations ± S.E.M.
of (a) serum estradiol (b) vit D3, (c) alkaline phosphatase activity, (d)
serum calcium content in ovariectomized female rats (n = 6) analyzed
by one way ANOVA followed by Bonferroni’s post hoc test. *P<0.05,
**P<0.01 as compared with control group
Figure 3: Data represented are mean number of observations ± S.E.M.
of difference of (a) rise in body weight, (b) bone thickness, (c) bone
density, (d) breaking strength in ovariectomized female rats (n = 6)
analyzed by one way ANOVA followed by Bonferroni’s post hoc test.
*P<0.05, ** P<0.01 as compared with control group.
348 Indian Journal of Pharmacology | June 2012 | Vol 44 | Issue 3
group −1.95 ± 0.18 g/cm 3 (P<0.01) as well as IND-HE
(100 mg/ kg) −1.32 ± 0.12 g/cm3 (P<0.05) but not in IND-
HE (75 mg/kg) −0.96 ± 0.03 g/cm3 [Figure 3c]. The rise in
bone density was 141, 52 and 20% in estrogen group, IND-HE
(100 mg/kg), IND-HE (75 mg/kg), respectively, as compared
to control group.
The breaking strength was significantly increased in estrogen
(1 mg/kg) −1.13 ± 0.03 kg/cm2 and IND-HE (100 mg/ kg)
−1.96 ± 0.17 kg/cm2 (P<0.05) but not in IND-HE (75 mg/
kg) group as compared to control (1.132 ± 0.037 kg/ cm2)
[Figure 3d]. The increase in breaking strength was 79.6, 73.4
and 59.2% in estrogen group, IND-HE (100 mg/kg) and IND-HE
(75 mg/kg), respectively, as compared to control group.
DEXA of whole body and femur and spinal cord
The results showed significant increase in bone mineral
density (BMD) of whole body bone and femur in IND-HE
(100 mg/ kg) and estrogen group (P<0.05, P<0.01) and
nonsignificant increase in BMD is observed in IND-HE (75 mg/ kg).
Bone mineral content of whole body and femur is significantly
increased in all treatment groups (P<0.01) [Table 1].
Histopathology of tibia bone
A scoring scale was designed to assess osteoporosis. The
scoring was 1: <25% bone showing osteopenia, 1+: 25-50%
bone showing osteopenia, 2+: 50-75% bone showing osteopenia
3+: >75% bone showing osteopenia.
The score were Control – 3+, IND-HE (75 mg/kg) – 2+, IND-
HE (100 mg/kg) – 1+, and estrogen (1 mg/kg) – 1. The results
thus indicated marked reduction in osteopenia in estrogen
(1 mg/kg) and IND-HE (100 mg/kg) treated group [Figure 4].
Discussion
Ovariectomy due to sex hormone deficiency in animals leads
to decrease in bone thickness, bone density and bone hardness.
Accelerated loss of bone occurs in women following menopause.
Both share many similar characteristics. These include
increased rate of bone turnover with resorption exceeding
formation and initial rapid phase of bone loss followed by a
much slower phase; greater loss of cancellous than cortical
Figure 4: Effect of estrogen (1 mg/kg), IND-HE (100 mg/kg) and IND-
HE (75 mg/kg) on histology of bone (200X). The score were (a) Control
– 3+, (b) IND-HE (75 mg/kg) – 2+, (c) IND-HE (100 mg/kg) – 1+ and
(d) estrogen (1 mg/kg) – 1
a b
c d
 Aswar, et al.: Anti-osteoporotic activity of IND-HE of Cissus quadrangularis L.
Table 1:
DEXA of estrogen (1 mg/kg), IND-HE (100 mg/kg) and IND-HE (75 mg/kg) treated rats
Parameter Control
BMD g/cm2 (whole body) 0.07332 ± 0.01
BMC (whole body) 2.512 ± 0.12
BMD g/cm2 (femur) 0.08062 ± 0.01
BMC (femur) 0.1075 ± 0.01
*P<0.05 and **P<0.01 considered statistically significant using one –way ANOVA followed by Bonferroni post hoc test
bone and decreased intestinal absorption of calcium.[14]
The estrogenic effect of ethanol extract of C. quadrangularis
has previously been reported in a number of in vivo models. [6–8]
Local and systemic administration of C. quadrangularis alcohol
extract in experimental fractures in dog and rat models
appeared to hasten all the healing phases.[8] Radiological
evidence of complete union and histological evidence of denser
bony trabeculae were reported in the animal group treated with
C. quadrangularis.[6] The treatment also restored fractured
bone mechanically by normalizing the tensile strength. The
antiosteoporotic effect of ethanol extract of this plant also
indicated the same consequence.[3] Treatment with ethanolic
extract of C. quadrangularis showed increase in alkaline
phosphatase activity in the murine osteoblasts cell lines
(MC). The observation suggested that the extract stimulated
osteoblastic activity and enhanced mineralization in MC.[15]
Results obtained in present study indicated that ovariectomy
in animals decreased bone thickness, bone density and bone
hardness compared to estrogen-treated animals [Figure 3] which
are in accordance with earlier report.[16] The results obtained in
case of bone thickness and hardness of rats treated with IND-HE
were similar to the rats treated with estrogen. The bone density
results of IND-HE and estrogen groups were not similar. The
possible reason for this observation may be incomplete healing of
the bone (partial prevention of osteoporosis) which is also evident
by histopathology of pelvic bone [Figure 4]. The histology of pelvic
bone showed increase in intratrabecular distance indicating
ovariectomy-induced osteoporosis in control ovariectomized rats.
This distance was partially normalized by IND-HE treatment while
prominently by standard estrogen treatment group.
Ovariectomy causes gain in body weight, [17] we found
nonsignificant increase in body weight of animals treated with
either estrogen or IND-HE. The nonsignificant increase in body
weight of the animals treated with IND-HE (100 mg/kg p.o.) and
estrogen might be due to increase in sodium and water retention
which is in accordance with the study done by Stachenfeld.[18]
IND-HE may not have potent estrogenic activity as the serum
estradiol was not elevated significantly compared to estrogen
treated (1 mg/kg s.c.) group. However, mild estrogenic activity
may be apparent.
Friedelin, a pentacyclic triterpene (a phytoestrogen),[19] which
we claim to be rich in IND-HE. Friedelin from different plant
sources has been reported for diseases like arthritis and gout.[20]
One of the factors responsible for the progression of bone loss
due to declined levels of gonadal hormones is inflammation.[21]
Bone and bone marrow cells produce cytokines which act locally
to regulate osteoclast function. Normally, their production is
regulated by estrogen and cytokine mediated action of steroid on
IND-HE (100) IND-HE (75) Estradiol (1)
0.1142 ± 0.01* 0.0949 ± 0.01 0.1289 ± 0.01**
3.737 ± 0.24** 3.687 ± 0.12** 4.479 ± 0.23**
0.1149 ± 0.00* 0.1075 ± 0.01 0.1869 ± 0.02**
0.2525 ± 0.01** 0.2458 ± 0.01** 0.3376 ± 0.02**
the skeleton. Therefore, hormonal depletion during menopause
affect cytokine levels causing accelerated bone loss.[22]
In an earlier study another phytoestrogen, genistein has
been investigated in post-menopausal women. The authors have
hypothesized the conversion of this phytoestrogen to estrogen
in the body.[23] In corollary to this hypothesis the possibility of
increase in serum estrogen [Figure 2a] observed in IND-HE
(75 and 100) treated rats appears to be due to phytoestrogens
present in IND-HE getting converted into estrogen in the body.
Menopause is associated with increased renal excretion of
calcium (Ca++) and decreased intestinal calcium absorption.[24]
Active form of vitamin D3, also known as calcitriol enhances
absorption of Ca++ and phosphate from intestine. This is brought
by increasing synthesis of a carrier protein for Ca++ called
calcium-binding protein or calcibindin. Evidence suggests that the
activation of vitamin D3 receptor promotes endocytotic capture of
Ca++ and its transport across duodenal mucosal cells in vesicular
form.[25] IND-HE (75 and 100 mg/kg) treatment also increased
the level of vitamin D3 [Figure 2b] thereby, increasing calcium
absorption from the intestine which is evident from increase in
serum calcium of IND-HE-treated rats. The possible explanation
for these findings could be phytosteroids present in IND-HE might
be the precursor of vitamin D3 as shown by similar studies.[26]
Thus increased vitamin D3 is responsible for increased absorption
of calcium from intestine which is involved in mineralization of
bone. This is apparent from the results obtained from DEXA
[Table 1]. IND-HE in both the doses showed increase in bone
mineral content of whole body bone as well as femur.
Alkaline phosphatase elevation in serum occurs in diseases
of bone, liver and pregnancy.[27] Reduction in serum alkaline
phosphatase by IND-HE correlated with histopathology of IND-
HE in preventing osteoporosis.
Other than nutrients, C. quadrangularis also contains
saponins. Saponins have been reported to affect the
permeability of the small intestinal mucosal cells due to its
strong surface–active properties and thus have an effect on
active nutrient transport. Rich calcium content as well as
presence of saponins in IND-HE further appears to contribute
to the absorption of IND-HE in blood.
Thus IND-HE (75 and 100 mg/kg p.o. for 2 months) and
standard estrogen (1 mg/kg s.c.) decreased the progression
of the bone loss in ovariectomized rats. The increase in serum
estrogen may be preventing the bone loss caused by ovariectomy.
Conclusions
The study confirms the antiosteoporotic activity of C.
quadrangularis and also phytoestrogen-rich fraction (IND-HE)
of C. quadrangularis increased blood calcium, vitamin D3, serum
Indian Journal of Pharmacology | June 2012 | Vol 44 | Issue 3 349
 Aswar, et al.: Anti-osteoporotic activity of IND-HE of Cissus quadrangularis L.
estrogen, bone mineral density and bone mineral content in
ovariectomized animals. The antiosteoporotic activity of IND-HE
is due to combined contribution from vitamin D3, calcium and
estrogen. Thus potency of IND-HE in preventing osteoporosis is
ranked as standard estrogen (1 mg/kg, s.c., biweekly) > IND-HE
(100 mg/kg. p.o.) > IND-HE (75 mg/kg. p.o.) based on this study.
Acknowledgment
The author would like to profusely thank Dr. Mohan Wani, NCCS,
Pune, Dr. S. S. Kadam, Dean, Bharati Vidyapeeth University, Pune and Dr.
K.R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth
University, Pune for providing necessary facilities to carry out the study.
References
1. Canalis E, McCarthy T, Centrella M. Growth factors and the regulation of bone
remodeling. J Clin Invest 1988;81:277-81.
2. Aloia JF, Cohn SH, Vaswani A, Yeh JK, Yuen K, Ellis K. Risk factors for
postmenopausal osteoporosis. Am J Med 1985;78:95-100.
3. Shirwaikar A, Khan S, Malini S. Antiosteoporotic effect of ethanol extract of Cissus
quadrangularis Linn. On ovariectomized rat. J Ethnopharmacol 2003;89:245-50.
4. Khan SS, Singh MP, Chaghtai SA. Ethnomedicobotany of Cissus quadrangularis
Linn. Orient J Chem 1991;7:170-2.
5. Kumbhojkar M, Kulkarni D, Upadhye A. Ethnobotany of Cissus quadrangularis {L}.
from India. Netherlands: Barkhuis; 1991.
6. Udupa KN, Prasad G. Biomechanical and Calcium-45 Studies on the Effect of
Cissus quadrangularis in Fracture Repair. Indian J Med Res 1964;52:480-7.
7. Udupa KN, Prasad GC. Further Studies on the Effect of Cissus Quadrangularis
in Accelerating Fracture Healing. Indian J Med Res 1964;52:26-35.
8. Deka D, Lahon L, Saikia J, Mukit A. Effect of Cissus quadrangularis in accelerating
healing process of experimentally fractured radius-ulna of dog, a preliminary study.
Indian J Pharmacol 1994;26:44-5.
9. Prasad GC, Udupa KN. Pathways and site of action of a phytogenic steroid from
Cissus quadrangularis. J Res Indian Med 1972;4:132.
10. Udupa KN, Gupta LP. The effect of growth hormone and thyroxine in healing of
fractures. Indian J Med Res 1965;53:623-8.
11. Potu BK, Rao MS, Nampurath GK, Chamallamudi MR, Prasad K, Nayak SR,
et al. Evidence-based assessment of antiosteoporotic activity of petroleum-ether
extract of Cissus quadrangularis Linn. on ovariectomy-induced osteoporosis. Ups
J Med Sci 2009;114:140-8.
12. Aswar UM, Bhaskaran S, Mohan V, Bodhankar SL. Estrogenic activity of friedelin-
rich fraction (IND-HE) separated from Cissus quadrangularis and its effect on
female sexual function. Pharmacognosy Res 2010;2:138.
350 Indian Journal of Pharmacology | June 2012 | Vol 44 | Issue 3
13. Wang FS, Ko JY, Lin CL, Wu HL, Ke HJ, Tai PJ. Knocking down dickkopf-1
alleviates estrogen deficiency induction of bone loss. A histomorphological study
in ovariectomized rats. Bone 2007;40:485-92.
14. Kalu DN, Liu CC, Salerno E, Hollis B, Echon R, Ray M. Skeletal response of
ovariectomized rats to low and high doses of 17 beta-estradiol. Bone Miner
1991;14:175-87.
15. Parisuthiman D, Singhatanadgit W, Dechatiwongse T, Koontongkaew S. Cissus
quadrangularis extract enhances biomineralization through up-regulation of
MAPK-dependent alkaline phosphatase activity in osteoblasts. In vitro Cell Dev
Biol Anim 2009;45:194-200.
16. Kalu DN. The ovariectomized rat model of postmenopausal bone loss. Bone
Miner 1991;15:175-91.
17. Chu SC, Chou YC, Liu JY, Chen CH, Shyu JC, Chou FP. Fluctuation of serum
leptin level in rats after ovariectomy and the influence of estrogen supplement.
Life Sci 1999;64:2299-306.
18. Stachenfeld NS, Dipietro L, Palter SF, Nadel ER. Estrogen influences osmotic
secretion of AVP and body water balance in postmenopausal women. Am J
Physiol Regul Integr Comp Physiol 1998;274: R187.
19. Gottlieb HE, Ramaiah PA, Lavie D. 13C NMR signal assignment of friedelin and
3 hydroxyfriedelan 2 one. Magn Reson Chem 1985;23:616-20.
20. Mahajan B, Taneja S, Sethi V, Dhar K. Two triterpenoids from Boswellia serrata
gum resin. Phytochemistry 1995;39:453-5.
21. Weitzmann MN, Pacifici R. Estrogen deficiency and bone loss: An inflammatory
tale. J Clin Invest 2006;116:1186.
22. Troen BR. Molecular mechanisms underlying osteoclast formation and activation.
Exp Gerontol 2003;38:605-14.
23. Marini H, Minutoli L, Polito F, Bitto A, Altavilla D, Atteritano M, et al. Effects of
the phytoestrogen genistein on bone metabolism in osteopenic postmenopausal
women. Ann Intern Med 2007;146:839.
24. O’Loughlin PD, Morris HA. Oestrogen deficiency impairs intestinal calcium
absorption in the rat. J Physiol 1998;511:313-22.
25. Levine MA. Normal Mineral Homeostasis. Vitam D and rickets. Endocr Dev
2003;6:14-33.
26. Lechner D, Bajna E, Adlercreutz H, Cross HS. Genistein and 17 – estradiol, but
not equol, regulate vitamin D synthesis in human colon and breast cancer cells.
Anticancer Res 2006;26:2597.
27. Hill P, Sammons H. An interpretation of the elevation of serum alkaline
phosphatase in disease. J Clin Pathol 1967;20:654.
Cite this article as: Aswar UM, Mohan V, Bodhankar SL. Antiosteoporotic
activity of phytoestrogen-rich fraction separated from ethanol extract of aerial
parts of Cissus quadrangularis in ovariectomized rats. Indian J Pharmacol
2012;44:345-50.
Source of Support: Nil. Conflict of Interest: None declared.
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