With a longitudinally designed study, we tested whether an acetone soluble fraction (ASF) from the stem bark of
Bone mass after menopause is critically dependent on the bone quantity attained during young adulthood and the rate at which bone is lost afterwards. The highest bone amount, peak bone mass, has a vital role in bone health. It is suggested that one standard deviation increase in peak bone mass could cut down the risk of fracture by as much as 50%, since individuals who accrue a high peak bone mass may preserve a higher BMD throughout their lives [
Peak bone mass is a combination of multiple factors, with genetic influence being central [
Accordingly, the present study used a longitudinal design in which female rats at weaning were treated with either vehicle or ASF by gavage for 12 weeks, followed by concurrent termination of treatment and induction of estrogen deficiency by OVx. Since ASF has been reported to induce new bone formation and prevent OVx-induced bone loss at 100 mg/kg dose [
E2, calcein, and tetracycline were purchased from Sigma-Aldrich (St. Louis, MO, USA). Osteocalcin (midportion) and fragments of type 1 collagen (CTx) ELISA kits were purchased from Immunodiagnostic Systems Ltd. (Tyne and Wear, UK). Total antioxidant status (TAS) Kit was purchased from Randox Laboratories Ltd. (Crumlin Co., Antrim, UK). The stem bark of
The study was conducted in compliance with the standards mentioned by Institutional Animal Ethical Committee (IAEC) at Central Drug Research Institute; the CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals) registration number of the IAEC is 34/1999. Female Sprague-Dawley rats were obtained from the National Laboratory Animal Centre, CSIR-CDRI. All rats were housed in a room maintained at 25°C in 12 : 12 hour light/dark cycles. Standard laboratory rodent chow diet devoid of soy protein and water was provided ad libitum.
Figure
Experimental design. Three-week-old (weaned) female SD rats were given either vehicle (daily/p.o.) or ASF (100 mg/kg/d/p.o.) for 12 weeks (baseline). At baseline, BMD and microarchitectural parameters were recorded and serum TAS was measured. Rats were then either sham operated or OVx and were given either vehicle or E2 (10
Subsequently, all the rats were ovariectomized (OVx). ASF treated rats after OVx and concurrent treatment withdrawal (vehicle replaced ASF treatment) referred as ASF withdrawal (ASFW;
After an additional 12 weeks (endpoint) all groups were killed (Figure
For determination of new bone formation
Bone length was determined from the rendered 3D images in CTAn software by drawing scale bar to individual bone image, for femur total length was defined as distance from greater trochanter to the edge of the femoral condyles while total length of tibia was from medial condyle to medial malleolus. The growth plate was isolated from the surrounding bone tissue in the micro-CT images by manual segmentation of 2D slices of sagittal images. The segmented sections were then reconstructed to render 3D images. From these images, growth plate height was measured using data viewer software. To analyze trabecular region, ROI was drawn at a total of 100 slices in the region of secondary spongiosa situated 1.5 mm away from the distal border of growth plate excluding all primary spongiosa and cortical bone. For cortical bone analysis, 350 serial image slides were discarded from growth plate to exclude the trabecular region, and 200 consecutive image slides were selected and quantification was done using CTAn software. Various trabecular parameters (3D) and cortical parameters (2D) were analyzed by following previously published protocols [
Bone mechanical strength was examined by 3-point bending strength of femur middiaphysis and compressive strength of the fifth lumbar vertebrae using Bone Strength Tester Model TK-252C as we reported earlier [
Methods followed our previously published protocol with some modifications [
Measurement of total antioxidant status (TAS) in serum sample (baseline) was carried out using ABTS+ (2,2′-azidodiethylbenzothiazolin sulphonate) radical formation kinetics (Randox Laboratories Ltd.). The presence of antioxidants in serum suppressed the bluish-green staining of the ABTS + cation, which was proportional to the antioxidant concentration. Kinetics was measured at 600 nm.
On the basis of our previously published protocols [
Data are expressed as mean ± SEM. The data obtained in experiments with multiple treatments were subjected to one-way ANOVA followed by post hoc Newman-Keuls multiple comparison test of significance using GraphPad Prism 5 software. An unpaired form of Student’s
ASF (100 mg/kg/day dose) treatment to weaned female rats for 12 weeks was well tolerated and resulted in no significant change in body weight when compared to the vehicle group (control). Plasma TAS was 57% higher in the ASF group compared to the control. ASF group had
Effect on body weight, antioxidant status, bone mass, and microarchitecture in growing female rats after 12 weeks of treatment (baseline).
Parameters | Vehicle |
ASF |
---|---|---|
Body weight (g) | 201 ± 4.58 | 216.1 ± 9.68 |
Total antioxidant status (mmol/L) | 1.44 ± 0.05 | 2.27 ± 0.07b |
Bone length | ||
Femur (cm) | 2.43 ± 0.02 | 2.57 ± 0.02b |
Tibia (cm) | 2.69 ± 0.03 | 2.91 ± 0.06c |
Growth plate height | ||
Femur ( |
241.77 ± 2.23 | 264.9 ± 3.81a |
Tibia ( |
148.07 ± 2.73 | 161.97 ± 3.94c |
BMD (g HA/cm3) | ||
Femur diaphysis | 1.02 ± 0.007 | 1.071 ± 0.004a |
Femur metaphysis | 0.23 ± 0.007 | 0.298 ± 0.01b |
Tibia diaphysis | 1.01 ± 0.01 | 1.067 ± 0.01c |
Proximal tibia metaphysis | 0.19 ± 0.01 | 0.257 ± 0.01b |
Micro-CT measurements at femur cortical bone | ||
B.Ar (mm2) | 4.28 ± 0.09 | 4.68 ± 0.11c |
Ct.Th (mm) | 0.21 ± 0.01 | 0.26 ± 0.05c |
T.Ar (mm2) | 9.88 ± 0.35 | 10.22 ± 0.55 |
Ma.Ar (mm2) | 6.02 ± 0.39 | 5.53 ± 0.45 |
MMI (mm4) | 10.21 ± 0.5 | 11.76 ± 0.58c |
Micro-CT measurements at femur cancellous bone | ||
BV/TV (%) | 15.8 ± 0.49 | 20.8 ± 0.61a |
Tb.N (1/mm) | 1.74 ± 0.05 | 2.252 ± 0.08a |
Tb.Th (mm) | 0.08 ± 0.002 | 0.089 ± 0.002 |
Conn.D (1/mm3) | 157.37 ± 11.83 | 229.8 ± 14.81a |
Tb.sp (mm) | 0.48 ± 0.01 | 0.33 ± 0.03b |
Tb.pf (1/mm) | 3.45 ± 0.13 | 1.415 ± 0.11a |
SMI | 1.96 ± 0.05 | 1.68 ± 0.02b |
DA | 1.91 ± 0.01 | 1.892 ± 0.02 |
Micro-CT measurements at tibial cancellous bone | ||
BV/TV (%) | 9.6 ± 0.81 | 15.3 ± 1.33b |
Tb.N (1/mm) | 1.28 ± 0.04 | 1.87 ± 0.15b |
Tb.Th (mm) | 0.08 ± 0.001 | 0.08 ± 0.001 |
Conn.D (1/mm3) | 103.66 ± 12.72 | 166.36 ± 13.6b |
Tb.sp (mm) | 0.51 ± 0.03 | 0.32 ± 0.02b |
Tb.pf (1/mm) | 17.75 ± 1.78 | 12.56 ± 1.09c |
SMI | 2.26 ± 0.05 | 1.89 ± 0.02a |
DA | 2.11 ± 0.02 | 2.16 ± 0.08 |
Values represent mean ± SEM;
BMD: bone mineral density, B.Ar: bone area, Ct.Th.: cortical thickness, T.Ar: periosteal area, MMI: mean polar moment of inertia, Ma.Ar: marrow area, BV/TV: percent bone volume, Tb.N: trabecular number, Tb.Th: trabecular thickness, Conn.D: connection density, Tb.sp: trabecular separation, Tb.pf: trabecular pattern factor, SMI: structure model index, DA: degree of anisotropy.
The effect of ASF treatment on bone mass and microarchitectural parameters in cancellous and cortical bone are described in Table
At femur metaphysis, ASF treatment resulted in significant increase in bone volume fraction (BV/TV), trabecular number (Tb.N), and connectivity density (Conn.D) compared to control, while trabecular separation (Tb.sp), trabecular pattern factor (Tb.pf), and structure model index (SMI) were reduced. Trabecular thickness (Tb.Th) and degree of anisotropy (DA) were not different between the groups. Tibial data at metaphysis also showed substantial gain in BV/TV, Tb.N, and Conn.D and fall in Tb.sp, Tb.pf, and SMI in ASF treated group when compared to control.
The results of
Effect of OVx and concurrent ASF withdrawal (ASFW) on appendicular bones. (a) BMD at femur diaphysis and metaphysis, (b) BMD at tibia diaphysis and metaphysis, (c) BV/TV at femur metaphysis, and (d) BV/TV at proximal tibia metaphysis. Mean percent changes from baseline were calculated and shown (
Figure
Table
Effect of 8-week treatment withdrawal and ovariectomy on trabecular bone microarchitecture among various groups.
Parameters | OVx + vehicle |
OVx + vehicle |
OVx + E2 |
---|---|---|---|
Femoral cancellous bone | |||
BV/TV | −48.7 ± 0.6*** | −17.6 ± 0.3 |
−20.2 ± 0.6 |
Tb.N | −50.9 ± 5.28*** | −20.8 ± 2.1 |
−34.08 ± 2.7 |
Tb.Th | −5.6 ± 1.05 | −2.5 ± 1.99 | −1.8 ± 0.85 |
Conn.D | −60.8 ± 7.35*** | −40.3 ± 5.53** | −50.08 ± 3.03** |
Tb.sp | 135.4 ± 10.57*** | 81.8 ± 8.8 |
118.3 ± 10.8 |
Tb.pf | 338.8 ± 17.8*** | 49.4 ± 4.4 |
120.3 ± 3.8 |
SMI | 11.5 ± 1.42* | 2.32 ± 0.22p | 1.9 ± 0. |
DA | 1.5 ± 0.05 | 2.7 ± 0.17r | 1.9 ± 0.43 |
Tibial cancellous bone | |||
BV/TV | −51.7 ± 5.37*** | −21.5 ± 2.0 |
−25.8 ± 3.9 |
Tb.N | −65.5 ± 2.45*** | −20.8 ± 3.6 |
−17.4 ± 1.0 |
Tb.Th | −2.4 ± 0.01 | −2.2 ± 0.013p | −5.1 ± 0.0 |
Conn.D | −71.1 ± 2.57*** | −50.3 ± 2.94**,p | −42.3 ± 4.0 |
Tb.sp | 192.5 ± 10.61*** | 48.5 ± 4. |
92.4 ± 7.1 |
Tb.pf | 73.7 ± 4.83*** | 10.2 ± 3.29p | 28.9 ± 5.2 |
SMI | 21.6 ± 2.57* | 1.7 ± 0.73p | 2.4 ± 0.9p |
DA | 4.04 ± 1.01 | 3.2 ± 0.86 | 5.4 ± 1.42 |
All parameters expressed in terms of mean percentage change from treatment termination (baseline). Values are mean ± SEM from 15 rats/group. ***
p
One-way ANOVA was followed for intergroup statistics.
BV/TV: percent bone volume, Tb.N: trabecular number, Tb.Th: trabecular thickness, Conn.D: connection density, Tb.sp: trabecular separation, Tb.pf: trabecular pattern factor, SMI: structure model index, DA: degree of anisotropy.
At the endpoint, body weight of OVx rats treated with vehicle for 12 weeks weighed 30% more than the sham (
Serum OCN and urinary CTx were increased by 132% and 145%, respectively, in the OVx + veh group compared with sham (both
Micro-CT was performed on excised bones to compare various parameters between the groups at the endpoint. When BMD measurement of femur metaphysis, tibia proximal metaphysis, and L5 vertebra was compared with the sham, it showed remarkable fall in all OVx groups. OVx + E2 group had significantly higher BMD at all three sites compared to OVx + veh group. On the other hand, ASFW group had maintained the gain attained during treatment to certain extent, such that it has significantly higher BMD when compared to OVx + veh, while BMD was not different between OVx + E2 and ASFW group.
Femoral trabecular data showed that compared with the sham, all OVx groups resulted in the deterioration of the trabecular parameters represented by reduced BV/TV, Tb.N and Conn.D and increased Tb.sp, Tb.pf, and SMI (Table
Effect on body weight, bone biochemical markers, trabecular bone mass, microarchitecture, and strength following ovariectomy and treatment withdrawal at the endpoint.
Parameters | Sham + vehicle |
OVx + vehicle |
OVx + vehicle |
OVx + E2 |
---|---|---|---|---|
Body weight (gm) | 232.3 ± 8.89 | 303.1 ± 8.27p | 297.4 ± 7.93p | 276.7 ± 13.05r |
Bone biochemical markers | ||||
Serum OCN (ng/mL) | 105.49 ± 8.76p | 244.96 ± 6.76 | 203.93 ± 9.84q,a | 97.27 ± 9.99p,e |
Urinary CTx (ng/mL) | 46.75 ± 11.4p | 113.69 ± 4.66 | 76.6 ± 3.23p,b | 66.37 ± 3.24p,c |
BMD (g HA/cm3) | ||||
Femur metaphysis | 0.234 ± 0.01q | 0.166 ± 0.001 | 0.198 ± 0.007r | 0.185 ± 0.01r |
Proximal tibia metaphysis | 0.248 ± 0.01p | 0.118 ± 0.006 | 0.17 ± 0.006q,a | 0.171 ± 0.01q,a |
Vertebral cancellous bone | 0.31 ± 0.014p | 0.219 ± 0.006 | 0.264 ± 0.007q,b | 0.265 ± 0.009q,b |
Micro-CT measurements at femur cancellous bone | ||||
BV/TV (%) | 24.8 ± 1.87p | 12.07 ± 0.12 | 17.12 ± 0.38p,a | 14.12 ± 0.56r,a,f |
Tb.N (1/mm) | 2.67 ± 0.27p | 1.27 ± 0.01 | 1.79 ± 0.06r,a | 1.71 ± 0.05r,a |
Tb.Th (mm) | 0.09 ± 0.0004 | 0.09 ± 0.001 | 0.09 ± 0.001 | 0.09 ± 0.002 |
Conn.D (1/mm3) | 158.66 ± 11.79p | 58.48 ± 3.6 | 82.37 ± 3.76r,a | 72.15 ± 4.78a |
Tb.sp (mm) | 0.3 ± 0.03p | 0.73 ± 0.04 | 0.65 ± 0.01a | 0.59 ± 0.02r,a |
Tb.pf (1/mm) | 1.42 ± 0.51p | 6.33 ± 0.3 | 2.44 ± 0.27p | 3.39 ± 0.4p,c |
SMI | 1.48 ± 0.07q | 1.7 ± 0.03 | 1.43 ± 0.02q | 1.5 ± 0.04q |
DA | 1.8 ± 0.03 | 1.82 ± 0.06 | 1.8 ± 0.06 | 1.69 ± 0.02 |
Micro-CT measurements at tibial cancellous bone | ||||
BV/TV (%) | 20.35 ± 1.64p | 8.8 ± 0.65 | 13.38 ± 0.64r,a | 13.67 ± 1.06r,a |
Tb.N (1/mm) | 2.32 ± 0.17p | 0.98 ± 0.07 | 1.36 ± 0.06r,a | 1.45 ± 0.09r,a |
Tb.Th (mm) | 0.08 ± 0.001 | 0.08 ± 0.001 | 0.09 ± 0.001 | 0.09 ± 0.001 |
Conn.D (1/mm3) | 137 ± 10.12p | 33.9 ± 2.12 | 55.52 ± 6.32r,a | 59.42 ± 4.15r,a |
Tb.sp (mm) | 0.23 ± 0.01p | 0.69 ± 0.04 | 0.5 ± 0.02p,a | 0.46 ± 0.01p,a |
Tb.pf (1/mm) | 6.41 ± 0.35q | 12.23 ± 0.78 | 8.19 ± 0.48q | 8.29 ± 0.48q |
SMI | 1.74 ± 0.03p | 2.05 ± 0.05 | 1.84 ± 0.02q | 1.83 ± 0.05q |
DA | 2.16 ± 0.04 | 2.18 ± 0.05 | 2.006 ± 0.03 | 2.01 ± 0.03 |
Micro-CT measurements at vertebral cancellous bone | ||||
BV/TV (%) | 29.96 ± 1.85p | 15.06 ± 1.36 | 21.06 ± 0.63r,b | 22.24 ± 3.25r,c |
Tb.N (1/mm) | 2.003 ± 0.1q | 1.28 ± 0.14 | 1.61 ± 0.07 | 1.62 ± 0.19 |
Tb.Th (mm) | 0.13 ± 0.005p | 0.102 ± 0.001 | 0.12 ± 0.001p,c | 0.11 ± 0.002p,b |
Conn.D (1/mm3) | 64.99 ± 4.23p | 17.77 ± 1.56 | 26.35 ± 2.16r,a | 28.83 ± 1.12r,a |
Tb.sp (mm) | 0.38 ± 0.009q | 0.49 ± 0.02 | 0.41 ± 0.01r | 0.41 ± 0.02r |
Tb.pf (1/mm) | 0.86 ± 0.76r | 6.37 ± 0.82 | 4.16 ± 0.48 | 4.49 ± 1.22 |
SMI | 0.86 ± 0.11p | 1.44 ± 0.04 | 0.96 ± 0.09p | 1.19 ± 0.01r,b,g |
DA | 2.09 ± 0.07p | 3.57 ± 0.09 | 2.62 ± 0.11p,b | 2.7 ± 0.15p,b |
Vertebral bone strength | ||||
Ultimate load (N) | 203.33 ± 16.99p | 92 ± 4.32 | 172 ± 17.65p | 143.33 ± 4.71q,b |
Energy (mJ) | 1009.05 ± 104.5q | 544.27 ± 83.01 | 936.88 ± 43.47q | 1031.44 ± 112.01q |
Stiffness (N/mm) | 268.03 ± 41.77p | 66.57 ± 13.67 | 206.86 ± 18.93q | 252.86 ± 40.6q |
Values represent mean ± SEM;
BMD: bone mineral density, BV/TV: percent bone volume, Tb.N: trabecular number, Tb.Th: trabecular thickness, Conn.D: connection density, Tb.sp: trabecular separation, Tb.pf: trabecular pattern factor, SMI: structure model index, DA: degree of anisotropy.
Similar deterioration in trabecular profile in the OVx groups at the tibia metaphysis was observed. ASFW group or OVx + E2 group resulted in significantly increased BV/TV due to increase in Tb.N and also increased Conn.D, but decreased Tb.sp, Tb.pf, and SMI when compared to OVx + veh group. None of these parameters were different between OVx + E2 and ASFW groups.
L5 vertebra exhibited enormous trabecular deterioration in all three groups 12 weeks following OVx (Table
We next studied the effect of various treatments on vertebral strength by compression test. Compared to the sham group, L5 of OVx + veh group showed significant reduction in ultimate load, energy to failure, and maximum stiffness. All parameters between the sham and ASFW groups were comparable whereas those between the OVx + E2 and sham, energy to failure and maximum stiffness were not different but ultimate load was higher in the sham group.
Dynamic histology of femur diaphysis was compared between the groups (Table
Effect on bone formation indices, microarchitecture, and strength at femur diaphysis following ovariectomy and treatment withdrawal at the endpoint.
Parameters | Sham + vehicle |
OVx + vehicle |
OVx + vehicle |
OVx + E2 |
---|---|---|---|---|
Bone formation indices at femur mid-diaphysis | ||||
pMS/BS (%) | 96.96 ± 0.5p | 85.92 ± 2.72 | 95.43 ± 0.46q | 89.87 ± 1.86c,g |
pMAR ( |
0.43 ± 0.01p | 0.2 ± 0.01 | 0.34 ± 0.01p,a | 0.23 ± 0.001a,e |
pBFR /BS ( |
3.79 ± 0.05p | 1.87 ± 0.08 | 3.13 ± 0.13p,b | 2.05 ± 0.16a,e |
Micro-CT measurements at femur mid-diaphysis | ||||
B.Ar (mm2) | 4.7 ± 0.14q | 3.79 ± 0.06 | 4.53 ± 0.06q | 4.62 ± 0.21q |
Ct.Th (mm) | 0.49 ± 0.01r | 0.35 ± 0.02 | 0.47 ± 0.02r | 0.4 ± 0.03 |
T.Ar (mm2) | 8.16 ± 0.05 | 8.19 ± 0.21 | 8.13 ± 0.1 | 8.25 ± 0.6 |
Ma.Ar (mm2) | 3.37 ± 0.06r | 4.66 ± 0.26 | 3.45 ± 0.13q | 3.77 ± 0.32r |
MMI (mm4) | 7.33 ± 0.61r | 10.2 ± 0.69 | 7.63 ± 0.37r | 8.41 ± 0.65 |
Bone strength at femur mid-diaphysis | ||||
Ultimate load (N) | 142.66 ± 10.5p | 101.33 ± 4.27 | 161.66 ± 5.49p | 149.75 ± 1.88q |
Energy (mJ) | 70.66 ± 2.45q | 42.65 ± 3.85 | 82.91 ± 6.52q | 65.241 ± 1.16r |
Stiffness (N/mm) | 247.62 ± 13.39p | 133.58 ± 20.91 | 252.475 ± 13.12p | 248.161 ± 22.41q |
Values represent mean ± SEM;
pMS/BS: periosteal mineralizing surface per bone surface, pMAR: periosteal mineral apposition rate, pBFR/BS: periosteal bone formation rate/bone surface. B.Ar: bone area, Ct.Th.: cortical thickness, T.Ar: periosteal area, MMI: mean polar moment of inertia, Ma.Ar: medullary area.
2D-micro-CT measurements at the site of femur mid-diaphysis (Table
A three-point bending test was used to assess bone strength at the femoral midshaft (Table
There is a continuing debate, whether the attainment of peak bone mass or bone loss is the most crucial factor when it comes to the mechanism of developing osteoporosis [
Increased consumption of fruits and vegetables, soy-based diets, dietary supplementation with antioxidant vitamins (C and E), and phytopreparations rich in polyphenolic antioxidants are considered beneficial for health in general. With respect to skeleton, antioxidants not only counteract the increased production of osteoclasts due to reactive oxygen species (ROS) action on the precursor cells [
In rats, a skeletal growth spurt for the first 5 weeks is followed by a sluggish phase giving way to skeletal maturity, which is attained by 11.5–13 weeks [
The effect of ASF treatment on connectivity measures and trabecular geometry was better than the placebo group. This gain in cortical bone and improvement in microarchitectural measures are related to the increased rate of bone elongation. The extent of changes in various parameters between femur and tibia by ASF treatment was largely comparable. Increase in Tb.N might have arisen from stimulation of new trabeculae from the growth plate. Conn.D reflects trabecular bone connectivity, which is a structural property of cancellous bone that affects cortical bone strength and make the bone less fracture prone [
Trabecular bone is readily lost due to E2 deficiency that characterizes postmenopausal bone loss [
Twelve weeks following OVx (endpoint), femur and tibia of OVx control rats showed a significant reduction in trabecular BMD, bone volume, and Tb.N, as well as an increase in Tb.sp, a series of changes that resulted in a decreased connectivity among trabeculae and led to increased Tb.pf value. Apart from poor connectivity, geometric measure showed worsening impact in OVx due to higher SMI (an important strength surrogate) compared with the sham. Trabecular deterioration at L5 in OVx control was more severe than the appendicular sites as each of the connectivity and geometric measures was negatively affected. The endpoint micro-CT measurement in ASFW group in all cancellous bones was, by and large, comparable to E2 group, suggesting that ASF pretreatment afforded trabecular preservation after OVx equivalent to that of E2 supplementation. Furthermore, better architectural indices in the trabecular rich vertebra in the ASFW group translated to greater resistance to compressive failure in the lumbar vertebra, thereby implying that ASF supplementation during the period of peak bone gain could effectively reduce the risk of osteoporotic compressive fracture in postmenopausal women.
Better cortical parameters at baseline appear to have contributed in better cortical thickness and cortical bone area in the femur of ASFW group compared to OVx + veh. In fact, the cortical parameters in ASFW were comparable to the sham group. Interestingly, MMI in the diaphysis was increased after OVx, which was similar to a previous report [
E2 deficiency is characterized by high turnover bone loss and OCN and CTx serve as surrogates for monitoring the efficacy of treatment of osteoporosis in both clinical and preclinical studies [
In conclusion, our studies in the preclinical setting in female rats provide evidence that ASF supplementation during the skeletal growth and maturity could enhance peak bone mass and bestow greater bone conserving efficacy after OVx despite treatment withdrawal. ASF supplementation to young girls for an extended period may provide an effective preventive strategy for decreasing the risk of developing osteoporosis and fragility fracture after menopause.
K. Srivastava and K. Khan contributed equally to this work.
All the authors declare that they have no conflict of interests.
Authors acknowledge the generous grant support from CSIR (ASTHI programme). Research fellowship grants from the Council of Scientific and Industrial Research (K. Srivastava, A. M. Tyagi, D. K. Yadav), Department of Biotechnology (K. Kainat), and the Indian Council of Medical Research (MPK), Government of India, are also acknowledged.