Activity of Wedelia calendulacea Less. in post-menopausal osteoporosis.
AbstractWedelia calendulacea Less., a perennial herb containing isoflavanoids, is used in liver disorders, uterine hemorrhage and menorrhagia. Osteoporosis in women occurs mainly due to estrogen deficiency following menopause. Studies indicate that isoflavones are estrogenic enough to promote bone formation. Our study was aimed to investigate the antiosteoporotic effect of the ethanol extract of W. calendulacea in the ovariectomized rat model of osteoporosis, at two different dose levels of 500 and 750 mg/kg/body wt. day. The findings, assessed on the basis of biomechanical and biochemical parameters, showed that the ethanol extract of the plant had a definite protective effect. This was further supported by the histopathological studies. Phytochemical investigation revealed the presence of isoflavones and wedelolactone, which are known to act as phytoestrogens and may be responsible for the antiosteoporotic activity.
© 2005 Elsevier GmbH. All rights reserved.
Keywords: Wedelia calendulace; Isoflavonoids; Rat model; Post-menopausal osteoporosis
Introduction
Osteoporosis, a silent epidemic has become a major health hazard in recent years, afflicting over 2000 million people worldwide (Meryl, 1997). According to the WHO, osteoporosis is "a disease characterized by low bone mass and microarchictectural deterioration of bone tissues, leading to enhanced bone fragility and consequent increase in fracture risk that results in fractures with minimal trauma" (Prabha Shankar, 2002). Bone formation and resorption is a balanced and continuous process, an imbalance between which results in osteoporosis. In osteoporosis there is an increased number of fractures, which are slow in healing. In most countries, the incidence of osteoporosis is about 2-4 times higher in women than in men. A sharp decrease in ovarian estrogen production is the predominant cause of rapid, hormone-related bone loss during the first decade after menopause (Gruber et al., 1984). Menopause, aging and hereditary factors, inadequate calcium intake and absorption, lack of exercise, prolonged steroid administration, excessive alcohol intake, and cigarette smoking are the major risk factors that predispose osteoporosis (Prabha Shankar, 2002).
The ovariectomized rat is a convenient and reliable model for studying the efficacy of pharmaceutical agents in post menopausal osteoporosis (Mitra et al., 2001; Bahram et al., 1996). The pharmacological agents used to manage osteoporosis act by decreasing the rate of bone resorption, thereby slowing the rate of bone loss, or by promoting bone formation. Many synthetic agents such as calcium, calcitonin, hormones, bisphosphonates and selective estrogen receptor modulators (SERMs) such as Raloxifene and Droloxifene have been developed to treat osteoporosis, but are associated with side effects such as hypercalcemia, hypercalciuria, increased risk of endometrial and breast cancer, breast tenderness, menstruation, thromboembolic events, vaginal bleeding, hot flashes, dyspepsia and GI ulcers (Genant et al., 1989; Bennet et al., 1984; Canalis et al., 1988).
To overcome the wide range of side effects produced by these synthetic drugs, there is an increasing demand for 'green medicines' which are thought to be healthier and safer for the treatment of osteoporosis. The phytoestrogens, which are known to bind to the estrogenic receptor sites of the cell and trigger the components and processes of estrogenic activity have a promising role in the treatment of osteoporosis (Adams., 1989). The isoflavonoids are among the most active phytoestrogens in the flavonoid class. Ipriflavone, a synthetic flavonoid derivative (Agnusdei et al., 1989) was found to be effective in preserving bone mass in several models of experimental osteoporosis (Benvenuti et al., 1991). The isoflavones found in soybeans, such as genistein, were found to prevent bone loss in the ovariectomized rat model of osteoporosis (Bahram et al., 1996; Blair, 1996; Fanti et al., 1998). Genistein was also found to suppress osteclastic function, in vitro and in vivo (Blair, 1996).
Wedelia calendulacea (Less.) or W. chinensis (Anonymous, 1964), known also as pila bhangra in Hindi and manjal karisalankanni in Tamil, is a perennial herb with bright yellow flowers and a light, camphor-like odor. Found in Uttar Pradesh, Assam, Arunachal Pradesh and Tamil Nadu, the plant is used traditionally as a cholagogue and deobstruent in hepatic enlargement and jaundice. A decoction of the herb is used in uterine hemorrhage and menorrhagia. As the leaves contain, as its principal constituent, isoflavanoids and wedelolactone, which is analogous in structure to the clover estrogen coumestrol (Anonymous, 1964), the present study is an attempt to evaluate the plant W. calendulacea for its potential antiosteoporotic activity.
Materials and methods
Plant material
The aerial parts of W. calendulacea Less. were collected from Udupi district, Karnataka, India, in the month of April 2002 and authenticated by Dr. Gopalkrishna Bhat, Prof. and Head, Department of Botany, Poorna Prajna College, Udupi, India. A voucher specimen (No. PP513) has been deposited at the Department of Pharmacognosy, College Of Pharmaceutical Sciences, Manipal, India.
Phytochemical screening
The coarse powder of the aerial parts of W. calendulacea (50 g) was subjected to successive extraction with different solvents in increasing order of polarity from petroleum ether (60-80°C), to benzene, acetone, alcohol and finally to chloroform: water (Kokate, 1991; Harborne, 1984). The dry extracts were subjected to various chemical tests to detect the presence of different phytoconstituents (Rashmi et al., 2002).
Preparation of ethanol extract
The shade-dried, powdered aerial parts (5 kg) were extracted exhaustively with 95% ethanol using a Soxhlet apparatus. The total ethanol extract was concentrated in vacuo to a syrupy consistency (yield 500 g). Half of the total ethanol extract was used for the preparation of the ether extract. The aqueous suspension of the ethanol extract (500 ml) was extracted exhaustively with hexane [500 ml x 5] followed by ether [500 ml x 5]. The ether fraction was washed with distilled water (5 ml), dried over anhydrous sodium sulfate and concentrated in vacuo to a semisolid consistency (yield 4 g). This was then loaded on to a silica gel column and was eluted with 100% chloroform followed by graded mixtures of methanol in chloroform. TLC of the methanol: chloroform (20:80) eluate showed a single blue fluorescent spot under UV. The eluates containing this spot on concentration yielded a yellowish-white colored compound (mp 330°C), which was recrystallized from methanol and designated as WC1.
hRf value: 58 [chloroform: methanol (70:30)]. UV [?.sub.max] 351, 304, 249 (MeOH), 390, 309, 288 (NaOH), 370, 310, 300 (Al[Cl.sub.3]), 445, 351, 304, 249 (Al[Cl.sub.3]/Hcl), 370, 304, 247 (NaoAc), 364, 307, 247 (NaoAc/[H.sub.3]B[O.sub.3]). IR ([cm.sup.-1]) KBr: 3303, (d-lactone), 1706 (carbonyl), 1620 (C = C), 1452 (C = C skeletal vibrations of aromatic ring), 1413 (aromatic C-H), 1332 (asymm strech), 1197, 1157 (C-O streching), 1072 (O-H bend). [.sup.1]H NMR (400 MHz DMSO): d 10.87 (1 H, s, 5-OH), 9.37 (2 H, s, 3' and 4'OH), 7.3 (1 H, s, 10-H), 7.2 (1 H, s, 13-H), 6.6 (1 H, s, 8-H), 6.4 (1 H, s, 6-H), 3.9 (3 H, s, 7-OC[H.sub.3]). [.sup.13]C-NMR (DMSO): d 162(C = O), 158(C-5), 157(C-9), 155(C-7), 148(C-4, C-6'), 145(C-4'), 144(C-3'), 113(C-5'), 104(C-10), 101(C-1'), 98(C-8), 96(C-6), 94(C-2), 93(C-3), 55(OC[H.sub.3]).
Ethics
The experimental protocol was subjected to the scrutin of the Institutional Animal Ethics Committee, and was cleared by same before beginning the experiment (No.IAEC/KMC/75/2001-2002).
Selection of animals
Healthy Wistar albino female rats weighing each between 150-170 g and within 90 days of age were selected for the study. They were fed with standard food pellets (Hindustan Lever, rat pellets) and water ad libitum. They were housed in polypropylene cages maintained under standard conditions (12 h light/dark cycle; 23 ± 2°C, 35-60% humidity).
Acute toxicity studies
Acute toxicity studies were conducted as per the OECD guidelines, ADOPTION; 425. Two groups of ten, healthy albino rats of either sex, fasted overnight, were selected randomly. One group of the rats was fed with aqueous suspension of ethanol extract at a limit test dose of 5000 mg/kg body wt. and the control group was fed with an equal volume of saline. The animals were observed at 0 min, 30 min, 1 h, 2 h, 4 h, 6 h and thereafter every day for 14 days. At the end of the 14th day the animals were sacrificed by ether anesthesia and dissected for examination of vital organs for pathological changes.
Antiosteoporotic study
Forty animals in five randomized groups (n = 6) were used for the study. The rats from all groups were ovariectomized except for the rats from group 1, which served as sham-operated control. Rats from group 2 received an equal volume of saline and served as ovariectomized control. Group 3 served as a standard and received Raloxifen (Dr. Reddy's Lab, Hyderabad, India) at a dose of 5.4 mg/kg body wt. Groups 4 and 5 received ethanol extracts of W. calendulacea (500 and 750 mg/kg body wt., respectively).
The extracts and the standard drug were administered orally, 15 days after ovariectomy, and the treatment was continued until the end of 90 days. Ovariectomy was performed by ligation and excision of the ovaries, after tracing them along the fallopian tubes through a small vertical incision on the lower abdomen under ketamine anesthesia (120 mg/kg body wt.) in aseptic conditions. In the sham operation, the ovaries were exposed as above and manipulated gently but not excised (Mitra et al., 2001).
Biochemical evaluation
After 90 days, the blood samples of rats were withdrawn from the tail vein and the serum samples were analyzed for serum alkaline phosphatase (ALP), tartarate resistant acid phosphatase (TRAP), and serum calcium, using the diagnostic reagent kit from Span Diagnostic Ltd., Surat, India.
Biomechanical evaluation
At the end of the study, animals were sacrificed by ether anesthesia. The freshly isolated femur and tibia and fourth lumbar vertebra were assessed for loading test of the femoral neck, three-point bending test (Peng et al., 1994) and compression test (Ogey et al., 2001), respectively using the tensile strength testing machine (Gayatri Machine Products, Allahabad). The readings were recorded in Newtons.
Histopathology
The histopathological studies were carried out on sections of decalcified left femur (fixed in 10% neutral buffered formalin) embedded in paraffin wax. Sections stained with hematoxylin and eosin were observed under microscope for the micro-architectural changes.
Statistical analysis
The data were analyzed using one-way ANOVA followed by Post Hoc Sheffe's Test using SPSS computer software version 7.5. Level of significance was fixed at 0.05.
Results
The preliminary phytochemical screening revealed the presence of flavonoids, saponins, phytosterols, gums and mucilage and carbohydrates. The comparison of UV, IR and H-NMR spectra of the compound WC 1 with literature data (Wagner and Fessler, 1986), showed that all the spectra of WC1 match with that of wedelolactone. The [.sup.13]C NMR spectral data further support the findings.
The acute toxicity studies showed that oral administration of the ethanol extract of W. calendulacea produced neither mortality nor signs of clinical abnormality in either group. At necropsy, no gross pathological observations could be made in the target organs. The L[D.sub.50] value of W. calendulacea was thus found to be more than 5000 mg/kg body wt.
The effect of ethanol extract of W. calendulacea on biomechanical parameters of osteoporosis in rats after 3 months are shown in Table 1. The biomechanical parameters, viz., load testing of the femoral head of the femur (Femur), three-point bending (Tibia), and compression test of vertebra (Vertebra) evaluated at the end of 3 months following ovariectomy (group 2) showed a significant reduction in biomechanical strength as compared to group 1. A significant increase in biomechanical strength was observed in treated groups 3, 4 and 5 as compared to group 2. The results obtained for groups 4 and 5 were similar.
The effect of the ethanol extract of W. calendulacea on biochemical parameters of osteoporosis in rats after 3 months is shown in Table 2. Compared to group 1, group 2 animals showed a significant increase in both ALP and TRAP at the end of 3 months; however, no changes were observed in the calcium levels. A significant increase was observed in the ALP of groups 3-5 as compared to group 2. Also, there was a tremendous decrease in the TRAP value of group 5 as compared to groups 2-4.
Demineralized sections of femur of group 1 stained with haematoxylin and eosin showed normal micro-architecture without any pathological change (Plate 1), while the sections of group 2 (Plate 2) revealed disruptive and lytic changes along with fibrocartilageneous matrix, leading to osteodystrophy. Few osteocytes and uneven and disruptive shaft with lytic thinning were also observed. Treated groups 3-5 revealed similar findings (Plates 3-5), characterized by fairly uniform thickness of the bone shaft and variably dense osteocytic areas. Osteoclastic activity was marginal and the trabecular components were predominantly of a dense character with fibrous and cartilagenous matter. An appreciable balance with fibrocartilagenous proliferation was observed with mineralization of the cartilagenous matter from within. The epiphysial cartilagenous layer, with downward extension into spikules, cancellous and shaft matrices showed progress towards ossification. The cortical portion was found to be fairly dense and uniform with mineralized cartilage in predominance.
Discussion
Osteoporosis associated with ovarian hormone deficiency following menopause (postmenopausal osteoporosis) is by far the most common cause of agelated bone loss (Bahram et al., 1996). The ovariectomized rat model of osteoporosis was taken as an experimental model for conducting the study (Bahram et al., 1996; Wronski et al., 1989). In adult rats, oophorectomy is followed by an increase in bone turnover associated with bone loss and a permanent deficit of bone mass at several skeletal sites (WHO Guidelines, 1998). In 3 month old ovariectomized rats, the phase of accelerated estrogen-dependent bone loss lasts 3-4 months (Wronski et al., 1989), a phenomenon comparable with the bone loss associated with newly estrogen-depleted women.
Following ovariectomy at the end of three months, a significant reduction was observed in biomechanical parameters such as compression strength of the femur, vertebra and the three-point bending test of tibia as compared to the sham group, thereby indicating a reduction in bone mechanical strength. The biochemical parameters correlated with the biomechanical study as characterized by an increase in ALP and TRAP. This indicates an increase in the osteoblastic and osteoclastic activity, respectively (Marcus, 1994), thereby showing that bone turnover, which takes place at a faster rate, may account for bone disorder. There was no significant change in the serum calcium levels viewing that the hemostatic mechanisms maintain the serum calcium levels even after ovariectomy. The histopathological examination of the femur also showed evidence of osteoclastic and osteolytic activity leading to osteodystrophy. All these changes showed that following ovariectomy, there is a decreased bone mineral density and increased bone fragility, indicating that estrogen deficiency causes osteoporosis.
Treatment for 3 months with an ethanol extract of W. calendulacea (both 500 and 750 mg/kg body wt.) revealed a significant increase in the compression strength of the femur, vertebra and the three-point bending strength of the tibia which was comparable to that seen with Raloxifen, SERM. While all the treated groups showed a significant increase in ALP, only the group treated with 750 mg/kg body wt. of extract showed a tremendous decrease in TRAP, thus showing an enhancement of osteoblastic activity and a reduction of osteoclastic activity. Histopathological examination of the femur of extract-treated groups 4 and 5 also revealed ossification, mineralization, calcified cartilagenous deposits and a marginal osteoclastic activity, all of which indicate marked restorative action and thereby suggest that the protective effect of the drug may be due to an increase in bone formation and a reduction in bone resorption.
Studies have shown that genistein, an isoflavone (phytoestrogen) from soya, offered a protective effect in osteoporosis by suppressing osteoclastic function and by decreasing resorption of bone in the ovariectomized rat model. (Fanti et al., 1998; Blair, 1996; Yamaguchi and Gao, 1998). Further reports have indicated that ipriflavone, a synthetic flavonoid derivative is effective in preserving bone mass in several models of experimental osteoporosis. The protective action of the ethanol extract of W. calendulacea against osteoporosis induced by ovariectomy may therefore be due to the isoflavones and wedelolactone present therein, which act as phytoestrogens.
To conclude, our studies have shown that the aerial parts of W. calendulacea possess a marked antiosteoporotic activity with minimal toxicity and could therefore have a promising role in the treatment of osteoporosis due to estrogen deficiency. Further studies on the principal component, wedelolactone, and its potential as an antiosteoporotic agent must be performed.
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Shirwaikar Annie (a,*), R.G. Prabhu (a), S. Malini (b)
(a) Department of Pharmacognosy, College of Pharmaceutical Sciences, Manipal, India
(b) Department of Pharmacology, Kasturba Medical College, Manipal, India
Received 9 April 2003; accepted 8 January 2004
*Corresponding author. Tel.: 08252 71201 x 22793; fax: 08252 70062, 70061.
E-mail addresses: annie.shirwaikar@cops.manipal.edu, annieshirwaikar@yahoo.com (S. Annie).
Table 1. Effect of ethanolic extract of W. calendulacea on biomechanical parameters of osteoporosis in rats after 3 months (n = 6/group) Biomechanical parameters Dose in mg/kg body (Newtons) Groups n = 6/group wt. Of rat orally Compression Femur Sham Equal volume of 65.33 ± 5.62 saline Ovariectomized (Ovx) Equal volume of 32.33 ± 1.40 (a) 3 months saline Raloxifen 5.4 55.33 ± 4.77 (b) Wedelia calendulacea 500 70.66 ± 3.81 (b) extract Wedelia calendulacea 750 70.00 ± 1.71 (b) extract Biomechanical parameters (Newtons) Three point bending Groups n = 6/group Tibia Compression vertebra Sham 40.00 ± 1.03 171.66 ± 11.37 Ovariectomized (Ovx) 16.66 ± 1.60 (a) 98.33 ± 4.27 (a) 3 months Raloxifen 74.66 ± 2.51 (b) 174.66 ± 15.34 (b) Wedelia calendulacea 62.33 ± 6.07 (b) 167.33 ± 17.75 (b) extract Wedelia calendulacea 61.33 ± 1.97 (b) 202.00 ± 17.18 (b) extract (a) p < 0.05 vs. Sham. (b) p < 0.05 vs. Ovx 3 months. Table 2. Effect of ethanolic extract of W. calendulacea on biochemical parameters of osteoporosis in rats after 3 months (n = 6/group) Dose in mg/kg body wt. of rat orally Biochemical parameters Groups n = 6/group ALP (KA units) Sham Equal volume of saline 15.37 ± 0.96 Ovariectomized (Ovx) Equal volume of saline 31.77 ± 1.66 (a) 3 months Raloxifen 5.4 38.13 ± 4.03 Wedelia calendulacea 500 50.63 ± 4.07 (b) extract Wedelia calendulacea 750 36.67 ± 3.91 extract Biochemical parameters Groups n = 6/group TRAP (KA Units) Calcium (mg/dl) Sham 1.9 ± 0.14 10.15 ± 0.32 Ovariectomized (Ovx) 5.35 ± 0.42 (a) 10.31 ± 0.36 3 months Raloxifen 18.96 ± 1.19 (b) 9.06 ± 0.19 (b) Wedelia calendulacea 13.48 ± 1.25 (b) 9.63 ± 0.33 (b) extract Wedelia calendulacea 3.64 ± 0.56 9.72 ± 0.13 extract (a) p < 0.05 vs. Sham. (b) p < 0.05 vs. Ovx 3 months.
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| Author: | Annie, Shirwaikar; Prabhu, R.G.; Malini, S. |
|---|---|
| Publication: | Phytomedicine: International Journal of Phytotherapy & Phytopharmacology |
| Article Type: | Clinical report |
| Geographic Code: | 9INDI |
| Date: | Jan 1, 2006 |
| Words: | 3527 |
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