Antiosteoporotic activity of phytoestrogen-rich fraction separated from ethanol extract of aerial parts of Cissus quadrangularis in ovariectomized rats.

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.
MATERIALS AND METHODS: IND-HE was separated from the ethanol extract of C. quadrangularis. Ovariectomized female Wistar rats were divided into four groups (n = 6). Group 1: Control (distilled water), Group II: IND-HE (75 mg/kg p.o.), Group III: IND-HE (100 mg/kg p.o.) were treated once daily for 8 weeks and Group IV: standard estradiol group, received estrogen (1 mg/kg, s.c. bi-weekly). The effects on body weight were determined. DEXA (Dual energy-emission X-ray absorptimatory analysis) of whole body bone and femur was carried out. Blood was removed and analyzed for biochemical parameters. After sacrificing the animals, 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) and estrogen treatment showed statistically significant increase in bone thickness, bone 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 serum vitamin D3 and serum calcium compared to control. Alkaline phosphatase was 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.

Introduction

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.

Traditional therapies for postmenopausal osteoporosis 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, 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.[67]

The phytochemical analysis of the plant showed the presence of vitamin C, β-carotene, two symmetric tetracyclic triterpenoids, β-sitosterol, α-amyrin, α-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.[910]

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 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 (100 mg/kg p.o.) and Group IV was a standard estrogen group, received estradiol benzoate (1 mg/kg in olive oil suspension, s.c bi-weekly). The treatment period was 8 weeks. Following parameters were studied.

Body weight measurement

Body weight of all the animals was recorded daily.

Serum estrogen estimation

On the last day of treatment the blood was withdrawn by retroorbital plexus. Blood was centrifuged to separate the serum. The serum estrogen (E2) was analyzed by Clia: immulite, fully automated immunoassay analyzer, USA.

Hydroxycholecalciferol (Vitamin D3) estimation

The serum for vitamin 25 hydroxycholecalciferol was estimated by radioimmuno assay (BARC, INDIA/DPC, USA/DSL, USA).

Serum alkaline phosphatase

The serum alkaline phosphatase was measured by an Auto-analyzer Hitachi, model No-912 using kits (Roche, USA) [by pnpp (P-Nitrophenylphosphate)-kinetic method].

Serum calcium

The serum calcium was estimated by Cresolpthalein (Roche) method using an auto-analyzer Hitachi, model no-912. The protocol provided by the manufacturer was followed for each assay.

Measurement of thickness, density and breaking strength of femur bone

After removing the blood for biochemical analyses the animals were sacrificed. The right femur was removed and was freed of soft tissue using small scissors, tweezers and cotton gauge. The bone was dried overnight in the oven and bone marrow was carefully removed. The thickness of femur was measured with a vernier caliper. Bone was weighed. Bone volume was measured by using plethysmometer (model No.7140, UGO Basile, Italy) and bone density was calculated (mass/volume). The breaking strength of left femur bone was estimated using hardness tester (Pharma Test PTB, Incorp., India).

DEXA of whole body and femur

Dual energy-emission X-ray absorptimatory analysis of whole body bone, femur was carried out using p-DEXA saber, ORTHOMETRIX.INC.

Histopathological study

The pelvic bone was also removed and was stored for 24 h 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 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.

Results

Oral administration of IND-HE (2000 mg/kg) did not cause mortality or any signs of clinical abnormality in both the groups. At necropsy, no gross pathological changes were observed in the target organs. The LD50 of IND-HE was more than 2000 mg/ kg body weight.

The final product (standardized 2.5% friedelin) from C. quadrangularis was labeled as IND-HE. The high-performance liquid chromatogram [Figure 1] of IND-HE showed the presence of friedelin, as major peak at 4.2 min.

Figure 1

HPLC tracing of IND-HE (standardized to friedelin)

Serum estrogen measurement

The serum estradiol (pg/ml) in control group, IND-HE (75 mg/kg), IND-HE (100 mg/kg) and estrogen group was 15.83 ± 1.8, 46.67 ± 6.5, 52.67 ± 0.9 and 105.9 ± 5.3, respectively. The results showed significant increase in serum estradiol with estrogen (P<0.01); IND-HE (75 mg/kg) (P<0.05) as well as IND-HE (100 mg/kg) (P<0.05) as compared to control group [Figure 2a]. The rise in serum estradiol was 568.9, 232.7 and 194.8% in estrogen group, IND-HE (100 mg/kg) and IND-HE (75 mg/kg) groups, respectively.

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 hoctest. *P<0.05, **P<0.01 as compared with control group

Vitamin D3 estimation

Significant increase in serum vitamin D3 (ng/ml) was observed in IND-HE (100 mg/kg) that is 49.83 ± 6.19 (P<0.05) and estrogen group which was 66.83 ± 2.63 (P<0.01) as compared to control group (30.0 ± 2.58) [Figure 2b]. The rise in vitamin D3 was 65 and 121% in estrogen group and IND-HE (100 mg/kg), respectively, compared to control group.

The serum alkaline phosphatase (U/Lt) activity was significantly reduced (P<0.01) when ovariectomized animals were treated with IND-HE (75 mg/kg, p.o.) – 140 ± 12.6, IND-HE (75 mg/kg, p.o.) – 94.0 ± 12.13 and estrogen (1 mg/kg s.c. biweekly) – 78.50 ± 6.95 as compared to control group – 159.7 ± 6.0. The percentage reduction was 12.3, 41.0, and 50.4% in IND-HE (75), IND-HE (100) and estrogen group, respectively [Figure 2c].

There was significant increase in serum calcium in IND-HE (75 mg/kg) – 9.72 ± 0.10 mg/dl, IND-HE (100 mg/kg) – 9.93 ± 0.09 mg/dl and estrogen treated group – 9.79 ± 0.007 mg/dl (P<0.01) as compared to control group – 8.38 ± 0.14 mg/dl [Figure 2d].

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].

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 hoctest. *P<0.05, **P<0.01 as compared with control group.

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 group −1.95 ± 0.18 g/cm3 (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].

Table 1

DEXA of estrogen (1 mg/kg), IND-HE (100 mg/kg) and IND-HE (75 mg/kg) treated rats

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].

Figure 4

Effect of estrogen (1 mg/kg), IND-HE (100 mg/kg) and INDHE (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

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 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 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 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.