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Journal of Chinese Integrative Medicine ›› 2011, Vol. 9 ›› Issue (10): 1125-1132.doi: 10.3736/jcim20111014

• Original Experimental Research • Previous Articles     Next Articles

Anti-osteoporotic activity of methanolic extract of an Indian herbal formula NR/CAL/06 in ovariectomized rats

Praveen Srikanta1, Shivaprasad H. Nagarajappa2, Gollapalle L. Viswanatha1(), Mukund Handral1, Rajesh Subbanna1, Rangappa Srinath1, Ganesh Hiremath1#br#   

  1. 1. Department of Pharmacology, People’s Education Society (PES) College of Pharmacy, Bangalore 560050, India
    2. Natural Remedies Pvt. Ltd, Veerasandra Industrial Area, Bangalore 560100, India
  • Received:2011-06-24 Accepted:2011-08-05 Online:2011-10-20 Published:2011-10-15
  • Contact: L. Viswanatha Gollapalle

Objective: The present study was aimed to evaluate the anti-osteoporotic activity of methanolic extract of NR/CAL/06 in ovariectomized (OVX) rats.

Methods: Bilateral ovariectomy was performed in female Sprague-Drawley rats under aseptic conditions and the rats were divided into five groups (n=10). Two different doses of methanolic extract of NR/CAL/06 (200 and 400 mg/kg, per oral (p.o.)) were evaluated for anti-osteoporotic activity and raloxifene (5.4 mg/kg, p.o.) was used as a reference standard. Treatment was given for 90 d. Anti-osteoporotic potential of NR/CAL/06 was evaluated based on various parameters, namely, body weight, organ weight, bone weight, bone mineral content, bone strength, calcium and phosphorus in serum and urine, and serum alkaline phosphatase (ALP). External diameter, length and thickness of the femur bone were measured by using scanning electron microscopy (SEM).

Results: The bilateral ovariectomy in rats resulted in decreased bone strength, bone mineral content and bone weight. The SEM images showed porous, perforated and disintegrated femur bone architecture and decreases in bone length and thickness in OVX rats. These changes were associated with elevated serum levels of calcium, phosphorus and ALP. Increases in body weight and adipose weight and a decrease in uterine weight were also observed and the changes were highly significant when compared with the sham-control group. Treatment with methanolic extract of NR/CAL/06 (200 and 400 mg/kg, p.o.) for 90 d dose-dependently restored the ovariectomy-induced alterations in bone weight, bone mineral content, bone strength, serum calcium, phosphorus and ALP, body weight and adipose weight nearly to normal levels. Furthermore, the SEM images of the femur bones of NR/CAL/06 (200 and 400 mg/kg, p.o.)-treated rats showed reduced pore formation and improved bone compactness compared with the OVX-control group.

Conclusion: These findings suggest that the methanolic extract of NR/CAL/06 possesses significant anti-osteoporotic activity and may be useful in the treatment of osteoporosis.

Key words: osteoporosis, ovariectomy, plant extracts, medicine, Ayurvedic, microscopy, electron, scanning, rats


Group n Body weight (g) Increase in body
weight (%)
Day 0 Day 45 Day 90
Sham-control 10 198.20±3.43 209.10±6.87 232.60±6.71 17.47
OVX-control 10 213.10±9.26 233.70±8.18 272.00±9.77** 28.12**
Raloxifene (5.4 mg/kg) 10 209.60±13.19 222.29±14.42 244.15±14.45△△ 16.82△△
NR/CAL/06 (200 mg/kg) 10 216.87±38.63 229.90±41.90 258.24±44.67 19.56△△
NR/CAL/06 (400 mg/kg) 10 208.80±5.38 226.45±4.77 242.33±5.39 16.50△△


Group n Adipose mass
Uterine weight
Femur weight
Femur length
Femur thickness
Sham-control 10 2.642±0.775 0.383±0.014 0.713±0.021 35.028±0.760 3.907±0.120
OVX-control 10 3.857±0.747** 0.173±0.043** 0.690±0.178* 35.597±9.007 3.792±0.949*
Raloxifene (5.4 mg/kg) 10 1.907±0.394△△ 0.223±0.035△△ 0.808±0.166△△ 35.853±7.310 3.905±0.797
NR/CAL/06 (200 mg/kg) 10 3.107±0.608 0.220±0.051△△ 0.810±0.168△△ 35.797±6.926 3.862±0.752
NR/CAL/06 (400 mg/kg) 10 2.703±0.394△△ 0.297±0.095△△ 0.818±0.170△△ 36.210±7.562 3.933±0.823


Group n Calcium (mg/dL) Phosphorus (mg/dL)
Day 0 Day 45 Day 90 Day 0 Day 45 Day 90
Sham-control 10 10.08±0.19 10.13±0.13 10.09±0.09 3.04±0.34 2.70±0.17 3.05±0.18
OVX-control 10 10.09±0.17 11.08±0.05** 11.21±0.07** 3.95±0.37 4.08±0.16 5.97±0.10**
Raloxifene (5.4 mg/kg) 10 9.98±0.11 10.25±0.05△△ 10.23±0.07△△ 3.02±0.08 2.47±0.14 2.62±0.08△△
NR/CAL/06 (200 mg/kg) 10 10.16±0.14 10.25±0.09△ 10.15±0.07△△ 4.18± 0.51 2.87±0.39 2.52±0.43△△
NR/CAL/06 (400 mg/kg) 10 10.29±0.13 10.18±0.07△ 10.21±0.08△△ 3.02±0.27 2.94±0.07 3.19±0.12△△
Group n ALP (Iu/L)
Day 0 Day 45 Day 90
Sham-control 10 110.9±14.0 119.2±14.6 128.4±14.0
OVX-control 10 111.9±17.2 262.8±23.7** 318.9±39.6**
Raloxifene (5.4 mg/kg) 10 119.7±11.3 165.3±25.3 155.6±27.2△△
NR/CAL/06 (200 mg/kg) 10 121.4±18.0 158.0±25.0 149.9±24.4△△
NR/CAL/06 (400 mg/kg) 10 113.5±07.9 148.2±24.3 147.9±29.8△△


Group n Urine calcium
Urine phosphorus
Bone mineral content
Ash weight (g) Ash calcium
(mg/g Ash)
Ash phosphorus
(mg/g Ash)
Sham-control 10 4.860±0.534 9.860±0.634 0.650±0.034 11.6±0.12 6.50±0.54
OVX-control 10 8.190±0.374** 11.200±0.774* 0.580±0.054** 10.0±0.14** 6.56±0.56
Raloxifene (5.4 mg/kg) 10 4.920±0.088△△ 7.840±0.688 0.670±0.059△△ 10.8±0.13△△ 6.56±0.56
NR/CAL/06 (200 mg/kg) 10 6.350±0.513△△ 8.750±0.813 0.600±0.055 11.3±0.11△△ 6.47±0.60
NR/CAL/06 (400 mg/kg) 10 6.160±0.274 8.610±0.674 0.620±0.053 11.9±0.10△△ 6.53±0.53


Group n Force (N)
Sham-control 10 112.800±4.885
OVX-control 10 78.540±2.974**
Raloxifene (5.4 mg/kg) 10 168.000±5.948△△
NR/CAL/06 (200 mg/kg) 10 74.270±2.182
NR/CAL/06 (400 mg/kg) 10 99.170±2.148△△

Figure 1

Bone architecture of rats in different groups observed under a scanning electron microscopeMagnifications are 1 000×. Arrow indicates the pore formation and reduced compactness. The OVX-control group showed porous perforated and disintegrated bone architecture compared with the sham-control group. NR/CAL/06 (200 mg/kg) treatment showed partial restoration; NR/CAL/06 (400 mg/kg)- and raloxifene (5.4 mg/kg)-treated groups showed complete restoration of osteoporosis bone changes to a compact firm.A: Sham-control; B: OVX-control; C: NR/CAL/06 (200 mg/kg); D NR/CAL/06 (400 mg/kg); E: Raloxifene (5.4 mg/kg). OVX: ovariectomized."

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