Evaluation of Antiosteoporotic potential of Sesbania grandiflora Linn. aqueous fraction in Ovariectomised Rats

 

S. Gupta1, A. M. Shaikh2, B. Mohanty3, P. Chaudhari4, P. B. Parab5, K. G. Apte6

1,6APT Research Foundation, Pune, 411041

2Faculty of Health and Biomedical Sciences, Symbiosis International University, Pune, 412115

3AAEMF’S Delight College of Pharmacy, Pune, 412216, India.

4,5Small Animal Imaging Facility, Advanced Centre for Treatment, Research and Education in Cancer,

Tata Memorial Centre, Navi Mumbai, 410210.

*Corresponding Author E-mail: shrutiguptashruti70@gmail.com

 

ABSTRACT:

Sesbania grandiflora (SG), an Indian herbal drug has been traditionally employed to treat or prevent female related hormonal disorders and mitigating symptoms of menopausal conditions. The present study assessed the antiosteoporotic potential of the aqueous extract of Sesbania grandiflora on bone metabolism in ovariectomised (OVX) rat model and its safety in the uterus. Thirty Sprague-Dawley 6 months old female rats were randomly divided into 5 groups: Sham operated group and four OVX subgroups (n=6), that were undergone bilateral ovariectomy. OVX rats were further subdivided into vehicle, raloxifene 5.4mg/kg/day, Sesbania grandiflora aqueous leaf extracts–250mg/kg/day and 500mg/kg/day respectively. The pharmacological effects of the extract were evaluated against osteoporosis by body weight, uterus wet weight, serum and urine biochemical parameters, bone mineral density, biomechanical strength, trabecular microarchitecture, histomorphology and uterus immunohistochemistry. Daily oral administration of aqueous leaf extract significantly assuages the symptoms of ovariectomy as shown by decreased levels of serum ALP, TRAP, hydroxyproline and urinary calcium in the treatment groups. Moreover, improved femur parameters were seen as increased bone strength, BMD, trabecular bone mass and microarchitecture similar to raloxifene. Histopathological data also showed significant restorative progression with increased ossification and mineralization of trabecular bone, without uterine hypertrophy. The results suggests that Sesbania grandiflora had a remarkable antiosteoporotic activity and may be a promising candidate for treatment of postmenopausal osteoporosis induced by estrogen deficiency in a natural way through herbal resources.

 

KEYWORDS: Sesbania grandiflora, Osteoporosis, Ovariectomized rats, Bone mineral density, Micro-CT.

 

 

 

INTRODUCTION:

Osteoporosis is a systemic skeletal disorder, characterized by low bone mass and microarchitectural deterioration of bone tissues leading to enhanced bone fragility and fracture risk1. Postmenopausal osteoporosis majorly affects elderly people and women within 10-15 years after menopause. Various biological, endocrinological, genetic, nutritional and environmental factors predisposes osteoporosis in both male and  female 2.

 

Fortunately, the available treatments for osteoporosis such as hormone replacement therapy, bisphosphonates, selective estrogen receptor modulators (raloxifen and droloxifen), strontium ranelate, denosumab, calcitonin, synthetic parathyroid hormone and other anabolic therapies, reduces the fracture rates, but each exhibits its own potential adverse effects leading to atraumatic fracture3.

 

Hence, natural extracts and its constituents derived from medicinal plants are urgently required as alternative approach for the prevention and treatment of osteoporosis4. Phytoestrogens such as isoflavones (genistein, daidzein, glycitein, equol and biochanin A), lignins (enterolactone, enterodiol), flavonoids (quercetin, kaempferol) and coumestans, share structural and functional similarities with naturally occurring or synthetic estrogens5. Moreover, their estrogenic activity is mediated via estrogen receptor binding, hence preventing postmenopausal osteoporosis and cardiovascular risks, by improving the defensive system6.

 

Sesbania grandiflora (L.) Pers. (Leguminosae), is an Indian medicinal plant, also known as ‘sesbania’, ‘agathi’, ‘humming bird tree’. It has also been listed as Ayurvedic drug in Indian Materia Medica7. Various pharmacological studies affirm that S. grandiflora possess phytoestrogens namely quercetin and kaempferol8 and are also rich source of catechin, epicatechin, luteolin, myricetin, naringenin, beta-carotene, grandiflorol, leucocyanidin, neoxanthin and oleanolic acid. The various parts of the plant have been classically used in gynecological applications for leucorrhoea, amenorrhea, anemia, emaciation, milk stimulation after child birth7,9 and gonorrhea in males. Also, the plant has been used for various disorders as: night blindness, antiulcer, headache, swellings, anemia, bronchitis, pains, liver disorders, laxative, analgesic, fever, astringent, and tumors. The pharmacological properties, viz. anticancer, antiurolithiatic, hepatoprotective, anxiolytic, anticonvulsive, cardioprotective, antiinflammatory, hypotensive, depressant, diuretic, hypoglycemic and hemolytic are scientifically proven10–17 The plant product is variedly used in traditional medicine and modern formulation to assuage leucorrhoea (Keva Leucorrhoea Care, Keva Industries, Bangalore) and fever relief (Ratnagiri rasa; Zandu Ayurveda, Mumbai; Imis Pharma, Andhra Pradesh).

 

Howbeit, S. grandiflora has never been investigated as alternative preventive medicine for osteoporosis. The present study, systematically researched the effects of aqueous leaf extract of S. grandiflora (AQSG) in an ovariectomy-induced osteoporosis model. We hypothesized that S. grandiflora may beneficially prevent bone loss caused by estrogen deficiency. Our data suggest that AQSG treatment is safe, inhibits bone deterioration in OVX rat model likely via new bone formation, suggesting its role as a protective agent for mediating bone diseases.

 

MATERIALS AND METHODS:

Chemicals Reagents and Diagnostic kits:

All chemicals and reagents of analytical grade were purchased from Sigma–Aldrich (St Louis, MO, USA). Phosphorous, calcium, tartrate resistance acid phosphatase and alkaline phosphatase kits were purchased from Coral Biosystems, India. Raloxifene, ketamine and xylazine were obtained from CIPLA Ltd. (Goa, India), Themis Medicare Ltd. (Haridwar, India) and Indian Immunologicals Ltd. (Hyderabad, India).

 

Extract Preparation:

Sesbania grandiflora leaves were procured from Bhugaon, Pune, Maharashtra. Identified, authenticated and submitted at Botanical Survey of India, Pune, Maharashtra through voucher specimen (BSI/WRC/Cert./2014, ATP15). Fresh, shade dried and powdered leaves (1kg) were extracted with distilled water (4.5 l) in Soxhlet apparatus for 24 hrs at 50-60°C. Extract was concentrated under rotary evaporator and stored in air tight container at -20°C until further use. Daily fresh solution of extract was made and antiosteoporotic studies were carried out in ovariectomized rats18.

 

Identification of constituents:

HPLC analysis was carried out on Dionex Ultimate 3000 liquid chromatograph (Germany) comprised of a LPG 3400 SD pump, diode array detector (DAD 3000) with 5 cm flow cell, a manual sample injection valve equipped with a 20µl loop and Chromeleon (c) Dionex Version 7.2.5.9377 system manager as data processor. The separation was performed by reversed-phase AcclaimTM 120 C18 column (5μm particle size, 250 x 4.6mm) at wavelength 272nm19.

 

In vivo studies:

The study was initiated after obtaining approval (Research Project No.43/1415) from Institutional Animal Ethical committee (IAEC, National Toxicology Centre, Pune; Reg.No. 40/ CPCSEA/1999) and as per the guidance of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. Thirty Sprague-Dawley 6 months old female rats (220±20g) were ovariectomized bilaterally and left for 2 months to develop osteopenic condition. For treatments, ovariectomised rats were randomly divided into four groups (n=6) as follows, for 90 days oral treatment: Sham operated+vehicle (Distilled water), Ovariectomised (OVX)+vehicle (Distilled water), OVX+RLX (Raloxifene 5.4mg/kg/day), OVX+AQSG 250mg/kg/day and OVX+AQSG 500 mg/kg/day. All animals were housed under standard conditions of 12 h/12 h light/dark illumination cycles at ambient temperature (22±2°C) and controlled relative humidity (50-60%) with free access to standard diet and water ad libitum throughout the experimental period. At the end of 12-week, animals were anesthetized with ketamine HCl (50mg/kg) and xylazine (25mg/kg) intraperitoneally and bone mineral density (BMD) was measured by dual energy X-ray absorptiometry (DEXA). The animals were sacrificed and blood was withdrawn from cardiac puncture, collected and centrifuged at 1900xg for 10 minute. Serum and urine was kept at -70°C for biochemical analysis. The rats were autopsied to collect bones for biomechanical testing and structural analysis.

Serum and urine biochemistry:

Serum calcium (S-Ca), Alkaline phosphatase (S-ALP), Urinary calcium (U-Ca) and Urinary phosphorous (U-P) were estimated by semi automatic analyzer (Pathozyme Smart-7, India) using diagnostic kits (Coral Biosystems, India). Tartrate resistant acid phosphatase (S-TRAP) was measured by using diagnostic kit from Labcare Diagnostic Pvt. Ltd., Gujarat, India. Urinary (U-HOP) and serum hydroxyproline (S-HOP) was measured as per modified Neuman and Logan method (Neuman and Logan, 1950). Serum estradiol (E2) levels were determined with Enzyme-linked Fluorescent (ELFA) method using kit (Mini Vidas BioMerieux, France), according to manufacturer’s instruction.

 

Bone mineral density:

Bone mineral density (BMD, g/cm2) of total femora were measured using Lunar iDXA (DEXA, GE Healthcare, USA). BMD was calculated using the BMC of the measured area with software (enCORE Version 16, GE healthcare, Madison, WI, USA) using the small animal scan mode 20.

 

Micro-CT analysis:

Trabecular bone microarchitecture of the right distal femoral metaphysis were analyzed using high-resolution micro-CT (Tri-Foil imaging, CA, USA) and bone histomorphometric analysis were performed using MicroView version ABA 2.4 Software. The scanning system was set in 1x1 bining FLY mode, Voltage- 60Kev, Current-175µA, Exposure-1700ms, focal spot 32 µm, magnification 4x4 with FOV 29.59mm, Frames-3, Number of projections-1024 and total acquisition time of 37 minutes. The region of interest (ROI) was selected as the region 20 to 25 slices away from the growth plate at the proximal end of the femur. 3D images were obtained for visualization and display. Bone morphometric parameters include bone mineral content (BMC), tissue mineral content (TMC), tissue mineral density (TMD), bone volume (BV), relative bone volume (BV/TV), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th) and connectivity density (Conn.D). The scan analysis was blindly conducted by the operator without any knowledge of the treatments associated with the groups 21.

 

Biomechanical evaluation:

The dried right femora were weighed using digital balance and the length was measured with vernier caliper from proximal tip of femur head to the distal tip of medial candyle. Bones were assessed for their biomechanical strength by using Universal Tensile Testing Machine, Veekay Testlabs, Model No.UTMG410B, Mumbai, India) at a speed of 2 mm/min. Three point bending of femur, femoral neck loading test and lumbar compression test of 4th lumbar vertebra were measured by method of Shirwaikar et al., 200322.

Histomorphometric and Immunohistochemical Analysis:

For histopathological evaluations, left femur was fixed in 10% formalin and decalcified by immersion in 10% ethylenediaminetetra-acetic acid (EDTA) for 10 days. Distal femur was sectioned (5-μm thickness) longitudinally on a rotary microtome (Leica RM22, Germany), processed for hematoxylin and eosin staining and examined under microscope for microarchitectural changes. Immunohistochemical (IHC) evaluation of estrogen receptor (ER) expression on uteri was performed with ER monoclonal antibody (Lab Vision Corporation, Fremont, USA) according to the manufacturer’s instructions. The cells were examined qualitatively under light microscopy (Nikon H550S Eclipse Ci-L, Japan) with NIS Elements Imaging Software version 4.

 

Statistical analysis:

All data were expressed as mean±SEM and analyzed using one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s test to compare all treatment groups with normal and OVX control. Group means were considered to be significantly different at 5% level of significance, 𝑃 <0.05. All statistical analyses were performed using the “GraphPad Prism V-5.03”.

 

RESULTS:

Identification of Phytoconstituents:

HPLC analysis revealed the presence of gallic acid, catechin and quercetin in the aqueous extract of Sesbania grandiflora as presented in Fig. 1a and 1b. Visible peaks in the test sample was confirmed with retention time against those of the standards: gallic acid (5.059), catechin (9.179) and quercetin (24.833) respectively.

 

Effect of S. grandiflora on body weight and uterine weight:

The initial and final body weights of all the animals in each group are shown in Table 1. Although no significant difference was observed in the initial mean bodyweight, however, at the end of the study, mean weight of OVX (estrogen-deficient) rats increased significantly than sham (p < 0.001) by (~37%), relative to initial body weight. Administration of AQSG at all doses significantly inhibited the increased body weight by ~30% and ~26% respectively (p<0.05), compared to the OVX group where as raloxifene group showed (p<0.05) ~20% inhibition after 90 days. As expected, ovariectomy resulted in significant (p<0.001) reduction in uterus weight in OVX group, by ~86.5% compared with sham group, indicating the uterus atrophy by estrogen deficiency. Daily oral administration of 250 mg/kg and 500 mg/kg of S. grandiflora aqueous extract showed neither much increase nor decrease in uterine weight compared to OVX group, suggesting that AQSG did not have any uterotrophic effect. Raloxifene also shown significant prevention in uterine weight loss compared to the OVX group (p<0.05).

 

 

Table 1. Effect of aqueous extract of S. grandiflora on body and uterus weight

Parameters

Body weight (g)

Uterus weight (g)

Groups

Initial

Final

Difference

Sham

223.0±2.5

251.3±8.4

29.5±7.3

0.63±0.109

OVX

226.3±5.3

308.2±7.5***

82.5±7.1**

0.085±0.007***

RLX

229.6±4.8

274.5±9.5#

47.8±11.1#

0.133±0.018#

AQSG 250

222.9±4.5

289.2±5.6

70.0±9.9*

0.105±0.007***

AQSG 500

222.3±5.3

278.9±6.2#

62.8±9.7

0.109±0.012***

 

Body weight difference and Uteri wet weight changes in the OVX model of osteoporosis. OVX rats received no treatment (OVX), RLX (5.4mg/kg/day), and AQSG (250 and 500 mg/kg/day) for 12 weeks. The results were expressed as mean ±S.E.M, n=6 in each group. Data were analyzed by one way analysis of variance followed by Dunnett’s t-test. *p< 0.05, **p< 0.01, ***p< 0.001 versus Sham group. #p< 0.05, versus OVX group.

 

 

Effect of S. grandiflora on biochemical parameters:

Effects of the AQSG on serum and urine biochemical parameters are indicated in Table 2. As expected, ovariectomy significantly elevated the levels of S-ALP, U-Ca, S-TRAP, U-HOP and S-HOP excretion (p<0.001, p<0.05) compared to sham group. Treatment with RLX and AQSG (250, 500 mg/kg) significantly (p<0.05) decreased the urine calcium, hydroxyproline and serum TRAP (bone resorption marker) levels without any effect on U-P and S-Ca. Serum ALP (bone formation markers) were significantly elevated in OVX rats (p<0.001) which were reversed (p<0.05) by AQSG 250 and 500mg/kg treatments by ~23% and ~41% compared to OVX. Further, serum estradiol (pg/ml) levels were notably increased in RLX, AQSG (250, 500 mg/kg) groups compared with OVX group (p<0.05), which indicates attenuation of high bone turnover and resorption. Based on the results, it was concluded that AQSG reduced the bone loss associated with estrogen deficiency.

 

Table 2. Effect of aqueous extract of S. grandiflora on biochemical parameters

Parameters

Sham

OVX

RLX

AQSG 250

AQSG 500

S-ALP(U/L)

121.5±20.2

369.8±54.7***

319.3±20.5

284.8±36.8

216.8±48.0#

S-TRAP(IU/L)

2.1±0.31

5.1±0.28***

2.6±0.32##

2.2±0.33##

3.2±0.98#

S-Ca (mg/dl)

6.7±0.21

6.9±0.29

6.1±0.23#

6.6±0.14

6.1±0.22

S-HOP(ug/ul)

0.95±0.09

1.34±0.11**

0.68±0.05###

0.89±0.05###

0.73±0.04###

Estradiol (pg/ml)

3.68±0.12

2.56±0.26*

4.62±0.46#

4.56±0.72#

3.98±0.71#

U-Ca(mg/dl)

2.65±0.27

3.33±0.12*

2.09±0.08###

2.05±0.16###

2.67±0.09##

U-P(mg/L)

3.39±0.09

3.36±0.02

3.41±0.05

3.40±0.02

3.42±0.02

U-HOP(ug/ul)

0.51±0.029

0.83±0.11**

0.31±0.03###

0.33±0.03###

0.22±0.02###

All data are expressed as mean ±S.E.M. n=6 in each group. Results were analyzed by one way analysis of variance followed by Dunnett’s t-test. *p< 0.05, **p< 0.01, ***p< 0.001 versus Sham group. #p< 0.05, ##p< 0.01, ### p< 0.001 versus OVX group.

Effect of aqueous extract of Sesbania grandiflora on femur parameters

As mentioned in Table 3, the femur ash, ash percentage and ash calcium content were significantly reduced (p<0.05) upto 12%, 9% and 18% in OVX rats as compared to sham group, which was significantly improved (p<0.05) after treatment with RLX, AQSG 250 and 500mg/kg. No significant difference was observed in femur length among all groups. Ovariectomy resulted in significant reduction of femur weight by ~30% that was improved with RLX, AQSG 250 and 500mg/kg treatment by ~24%, ~35% and ~38% compared to OVX group.

 

Table 3. Effect of aqueous extract of S. grandiflora on ash content of femoral bone

Parameters

Ash weight (g)

Ash (%)

Calcium (mg/cm3)

Femur Length (mm)

Femur weight (g)

Sham

499.0±25.5

57.5±0.9

123.6±8.1

35.4±0.4

1.026±0.12

OVX

441.5±9.4*

52.5±1.1*

101.9±2.9**

34.2±0.8

0.718±0.05***

RLX

460.4±4.0

55.8±0.2

114.6±1.4#

34.5±0.4

0.89±0.02##

AQSG 250

491.9±1.5##

55.8±0.3

115.8±1.2##

34.1±0.3

0.97±0.01###

AQSG 500

522.3±7.5###

58.6±1.0#

121.1±3.2##

33.7±0.1

0.99±0.03###

All data are expressed as mean ±S.E.M. n=6 in each group. Results were analyzed by one way analysis of variance followed by Dunnett’s t-test. *p< 0.05, **p< 0.01, ***p< 0.001 versus Sham group. #p< 0.05, ##p< 0.01, ### p< 0.001 versus OVX group.

 

 

Effect of aqueous extract of Sesbania grandiflora on biomechanical parameters:

As observed in Table 4, the mechanical strength of femur shaft was decreased significantly (p<0.05), whereas femur neck loading and lumbar vertebra was decreased non-significantly in OVX group as compared with sham. Although an increasing trend of improvement was observed after 90 days of treatment with AQSG 500mg/kg dose in mechanical strength of femur and vertebra (p<0.05), while femoral neck loading test showed non-significant increase. Similarly, significant (p<0.05, p<0.001) improvement was observed in femoral neck loading and lumber vertebra compression test with raloxifene treatment as compared with OVX control.

 

Table 4. Effect of aqueous extract of S. grandiflora on biomechanical parameters

Parameters

Three point bending of Femur (N)

Femoral neck load (N)

Compression of 4th lumber vertebra (N)

Sham

107.7±6.7

159.2±9.6

76.8±3.9

OVX

77.3±1.1*

132.4±5.8

62.8±5.8

RLX

89.2±5.1

182.7±6.3#

97.2±5.6###

AQSG 250

95.4±5.8

171.8±14.1

103.8±6.7###

AQSG 500

118.7±11.4#

170.9±8.4

82.4±3.8#

All data are expressed as mean ±S.E.M. n=6 in each group. Results were analyzed by one way analysis of variance followed by Dunnett’s t-test. *p< 0.05 versus Sham group. #p< 0.05, ### p< 0.001 versus OVX group.

 

Bone mineral density:

BMD were considered to be the golden standards to evaluate the incidence of osteoporosis. The total femoral bone mineral density (BMD) of OVX rats declined to 0.105 ±0.001 g/cm2 from 0.115 ±0.005 g/cm2 compared to Sham rats (p<0.05) (Fig 2). This suggested that after 12 weeks of ovariectomy BMD was decreased by 8.6%. However, treatment with AQSG 250 and 500mg/kg/day notably enhanced the BMD by 4.63% and 5.9% (p < 0.05), respectively, as compared to OVX group. Similarly, raloxifene treated rats also showed elevated BMD by 8.45% (p < 0.05) compared to OVX rats.

 

All data are expressed as mean ±S.E.M. n=6 in each group. Results were analyzed by one way analysis of variance followed by Dunnett’s t-test. *p< 0.05 versus Sham group. #p< 0.05, ##p< 0.01 versus OVX group.    

 

Micro-CT analysis:

The results of distal femur Micro CT scan were quantitatively evaluated as BV/TV, BMC, TMC, TMD, BV, Tb.Th and Conn.D in Table 5. Gross observation of femur morphometric parameters exhibited deterioration in bone microarchitecture by estrogen deficiency at the distal femur metaphyses in the OVX group. These results were evidenced by significant decrease in trabecular BV/TV, BMC, TMC, TMD and BV (p<0.05), reduction in Tb.Th and Conn.D and increased Tb.Sp. These parameters were further improved by raloxifene, AQSG 250 and AQSG 500mg/kg treatments. From the data, it can be suggested that AQSG have bone preventive action in the maintenance of bone mass and trabecular microarchitecture at skeleton sites (Fig 3).

 

 

 

Table 5. Effects of S. grandiflora on microarchitecture of right distal femoral metaphysis

Parameters

Sham

OVX

RLX

AQSG 250

AQSG 500

BV/TV

0.489±0.056

0.377±0.005*

0.408±0.003

0.422±0.022#

0.455±0.011##

BMC (mg)

1159.9±68.1

368.2±107.7**

651.6±84.9*

819.3±166.2

842.3±51.8#

TMC (mg)

1071.9±110.9

329.7±54.2***

611.2±99.1#

641.7±95.6#

730.8±12.2##

TMD (mg/cc)

790.3±7.2

357.2±21.2***

503.2±30.1#

627.2±16.9###

480.1±60.3#

Tb.Sp (mm)

2.25±0.19

2.94±0.35

2.47±0.49

2.67±0.16

2.46±0.17

Tb. Th (mm)

1.22±0.11

1.03±0.15

1.19±0.16

1.33±0.20

1.46±0.27

CD (1/mm3)

0.026±0.003

0.01±0.002***

0.019±0.003#

0.016±0.002*

0.016±0.001*

BV (mm^3)

1352.9±128.1

673.4±74.9*

1263.4±272.3

1035.3±174.4

1629.1±173.5##

 

All data are expressed as mean ±S.E.M. n=4 in each group. Results were analyzed by one way analysis of variance followed by Dunnett’s t-test. *p< 0.0,**p< 0.01, ***p< 0.001 versus Sham group. #p< 0.05,##p< 0.01, ### p< 0.001 versus OVX group. Bone mineral content (BMC), tissue mineral content (TMC), tissue mineral density (TMD), Bone volume fraction (BV/TV), Trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), Bone volume (BV) and connectivity density (CD).

 

 

Histological and Immunohistochemical examination:

Alternation in bone microarchitecture and new bone formation during 12 weeks of treatment was assessed by histological evaluation of left femur diaphysis (Fig 4). In OVX rats osteodystrophy was observed as trabecular disruption, lytic changes, trabecule thinning and intertrabecular spaces widening compared with sham group with normal trabecular microarchitecture and bone compactness. As observed from the photomicrographs, raloxifene and AQSG (250, 500mg/kg) treated groups showed significant restorative progression with increased ossification, mineralization, increased osteoblastic activity, compactness, reduced bone resorption and increased proliferation of trabecular fibrocartilaginous. The uterine sections showed degenerative changes in uterine mucosal epithelium, loss of uterine glands and increased atrophy in OVX rats. Moreover, reversed uterotrophic changes and restoration of uterine mucosal epithelium and glands were observed in RLX and AQSG treated group. From this data, it can be suggested that AQSG treatment had no uterine estrogenicity and antiestrogenicity. As examined in (Fig 5), the ER expression appeared as a yellow-brown cytoplasmic staining, in the endometrium, interstitial and smooth muscle cells in all groups. The ER labeling was decreased in OVX group compared with Sham group whereas a relative increase in the expression was observed after 12-weeks of treatment with RLX and AQSG (250, 500mg/kg).

 

DISCUSSION:

According to the World Health Organization (WHO), osteoporosis has been considered as a major global public health problem secondary to coronary heart disease affecting millions of people worldwide irrespective of racial or ethnic group of any age23,24. The International Osteoporosis Foundation estimated the worldwide annual cost burden of osteoporosis (for all ages) to be increased by USD 131.5 billion by 205025. The ovariectomised (OVX) rat model is widely accepted experimental model of postmenopausal osteoporosis. Ovariectomy resulted in increased body weight, enhanced bone turnover and marrow cavity of femoral diaphysis, repressed BMD and increased bone resorption on the endocortical surface26 and cancellous bone loss similar to human osteoporosis27. In the current study, AQSG effects were compared with raloxifene that act like hormone by modulating hormonal receptor with fewer side effects. From the previous studies estrogen exerts osteoprotective effects in maintaining bone structure and density28 by directly binding to estrogen receptors (ERα and ERβ) located on bone cells29. This binding leads to the activation of estrogen-receptor complex which further stimulate osteoblast differentiation via RANKL suppression30, and osteoprotegerin (OPG) upregulation, hence inhibiting osteoclastogenesis. Therefore, estrogen deficiency enhanced the osteoclastogenesis via RANKL activation and OPG downregulation leading to bone loss31.

 

From the previous studies, it is reported that estrogen deficient state may induced fat accumulation and metabolism via reduction in the leptin level, a hormone involved in regulating energy intake and appetite inhibition32,33. Leptin exert direct effect on bone metabolism by ameliorating osteoblastic differentiation, inhibiting osteoclastogenesis and reducing trabecular bone loss in ovariectomized rats31. Our data showed that AQSG and raloxifene treatment for 12 weeks significantly prevents increase in body weight as compared to OVX rats despite of similar food supply.

 

The effect of various drugs on bone remodeling is clinically evaluated by bone turnover biochemical markers. Various reported studies of postmenopausal women and laboratory animals have shown the association between ALP (osteoblast-specific bone formation marker) and TRAP levels (osteoclast-specific bone resorption marker) with microarchitecture34. As evident from our data, the levels of ALP, TRAP and HOP were significantly increased in OVX rats while urinary HOP was significantly upregulated as a marker for collagen degradation; under high TRAP levels released from activated osteoclast35. Bone sparing action of AQSG and raloxifene treatment in estrogen deficient rats showed significant reduction in ALP, TRAP and urinary HOP levels. Further, RLX and AQSG significantly reversed the urinary calcium excretion that was not observed in serum calcium and urine phosphorus levels in treatments groups, suggesting that AQSG does not interfere mineral homeostasis. The serum E2 level was markedly decreased in OVX rats as reported in previous studies36 (Table 2) which were restored by AQSG administration. These results surmised that AQSG ameliorated the OVX-induced bone loss by having an antiresorptive as well as osteogenic function in new bone formation.

 

Loss of bone mass is the leading cause jeopardizing bone integrity, reduced bone strength and increased fractures susceptibility34,37. In the present study, significant decrease in femur BMD was marked due to an increase in bone turnover in the OVX rats especially at skeletal sites containing cancellous bone compared with sham group. Further, oral administration of AQSG significantly improved the BMD and could prevent the progress of bone loss induced by ovariectomy (Fig.2). In accordance with this hypothesis, femur compressive strength and vertebral compression values were significantly improved in treatment groups compared to OVX rats, which were supported by increased calcium content in bone of treatment groups.

 

Although BMD act as vital determinant of bone strength, it does not measure the architectural changes occurring in trabecular bone38. Recently, Micro-CT has been extensively used as a new high resolution digital imaging technique with statistical software to provide detailed quantitative nondestructive analysis of 3D microscopic bone architecture39,40. The current study evaluated metaphyseal region near the growth plate of the distal femur, as it is the newly formed trabecular bone, apparently more sensitive to dietary factors affecting mineralization. Loss of bone volume fraction and organization in trabecular region was clearly seen in ovariectomized rats41. Daily supplementation of AQSG for 12 weeks markedly increased trabecular BV/TV, Conn.D, BMC, TMC, TMD, BV and Tb.Th, and decreased Tb.Sp (Table 5 and Fig. 3) indicating better geometry of trabecular network as compared with OVX group. These results suggested that microarchitectural changes of trabecular bone are more sensitive than BMD and to evaluate bone strength and fracture risk prediction, combination of microarchitectural properties of trabecular bone and BMD are required. Above findings were supported by histopathological data indicating marked restoration of bone loss by ossification, mineralization and calcified cartilagenous deposits with a marginal osteoclastic activity in AQSG and RLX groups. Data from this study suggest that AQSG effectively ameliorate estrogen-deficiency related-bone loss in the trabecular bone. Hence, the clinical administration of AQSG could efficaciously minimize the fracture risk in postmenopausal osteoporosis.

As shown in previous studies OVX resulted in the uterine atrophy. Usage of phytoestrogens as alternate therapy for postmenopausal osteoporosis has been widely accepted but a secondary concern is endometrial hyperplasia, or excessive cell growth in the uterus, which may occasionally lead to precancerous stage. In the present study, treatment with Raloxifene and AQSG 250 and 500 mg/kg, had increased uterus weight without causing hypertrophy as depicted by microscopic examination of uterus cells with no cell proliferation or hyperplasia. Consequently, an increased ER expression was observed in the rat endometrium after administration of AQSG and raloxifene. From the data, it may be proposed that AQSG is safe (non-uterotrophic) for use in postmenopausal osteoporosis and possess a similar potential like raloxifene with lower risk of endometrial carcinoma.

 

The present data of Sesbania grandiflora provide an insight for the possible mechanism(s) of its antiosteoporotic action. (1) Presence of phytoestrogens (quercetin and kaempferol) and other phytocompounds (saponins, catechin, myricetin, triterpenoids, etc) that may have antiosteoporotic role in bone protection by inhibition of tyrosine kinase, inhibition of DNA topoisomerase, antioxidant activity, inhibition of angiogenesis, stimulation of sex hormone binding globulin, inhibition of 5α reductase, 17β-OH-steroid-dehydrogenase and aromatase enzymes and also by enhancing IGF-I and OPG secretion from osteoblast via ER beta receptor 42. (2) Antioxidative potential of its polyphenolic compounds (quercetin, myricetin, catechin kaempferol) and tocopherols that actively participate in ROS scavenging 43. (3) Further, HPLC results demonstrated that aqueous extract of Sesbania grandiflora contented (+1)-catechin and quercetin, with known oestrogen-like effects. Presence of quercetin in S. grandiflora may have exerted antiosteoporotic effect by inhibiting osteoclastic differentiation induced by RANKL via a mechanism involving transcription factor NFkB and AP-1 44 and also inhibits bone loss without effect on the uterus 45. Additionally, as reported (+1)-catechin has a direct stimulatory effect on osteoblast growth in cultured osteoblastic MC3T3-E1 cells in vitro and also inhibits TNF-α induced apoptosis and inflammatory cytokines production 46.

 

CONCLUSION:

For the first time, this study reveals that daily administration of AQSG in estrogen deficient female rats for 12 weeks improved serum E2 levels, trabecular microarchitecture and preservation of bone mass and biomechanical quality. AQSG can be prepared conveniently, easily available and cost effective, suggesting it as a clinical advantage as an alternative medicine to oestrogen therapy for preventing postmenopausal osteoporosis. However, studies are required for the identification of the bioactive compounds associated with the AQSG bone-protective effects in vivo and delineate the molecular mechanism(s), underlying therapeutic approach of AQSG, as a candidate for preventing post menopausal osteoporosis.

 

ABBREVIATIONS:

BMD, bone mineral density (BMD); TRAP, tartrate-resistant acid phosphatase; OVX, ovariectomized; DEXA, dual energy X-ray absorptiometry; aqueous leaf extract of Sesbania grandiflora, AQSG; RLX, Raloxifene; BMC, bone mineral content; TMC, tissue mineral content; TMD, tissue mineral density; BV, bone volume; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; Conn.D, connectivity density.

 

CONFLICT OF INTEREST:

The authors assert no conflict of interest associated with this project.

 

ACKNOWLEDGEMENT:

The authors are grateful to Dr. C.S. Yajnik, KEM Hospital for providing DEXA and Dr. Mandavi Garge for constant motivation and support.

 

FUNDING:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

 

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Received on 16.08.2019         Modified on 13.09.2019

Accepted on 02.11.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(4):1804-1812.

DOI: 10.5958/0974-360X.2020.00325.X