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
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.
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 (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.
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.
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.
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.
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:
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.
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.
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.
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.
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.
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.
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).
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.
The authors assert no conflict of interest associated with this
project.
The authors are grateful to Dr. C.S.
Yajnik, KEM Hospital for providing DEXA and Dr. Mandavi Garge for
constant motivation and support.
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