Antiosteoporotic activity of lupeol on postmenopausal symptoms through promoting estrogenic activity and bone markers in ovariectomized rats

Abstract

Background

Menopause is caused by age-related follicular degeneration and is marked by menstrual cycle cessation with insufficiency of an ovarian hormone, particularly estrogen. Estrogen insufficiency causes vasomotor and urogenital symptoms, as well as an increased risk of osteoporosis. Lupeol (LP) is a triterpene compound found in aloe leaves, carrot root, mango pulp, melon seeds, cucumber, soybean, and other plants. The present study aims to assess the effect of LP on estrogen insufficiency in ovariectomy-induced postmenopausal osteoporosis in rats. Female rats were ovariectomized bilaterally and placed into five groups (n = 8) as ovariectomy control (OVX), standard (17β-Estradiol 50 µg/kg) group, and LP (25, 50, and 100 mg/kg) groups. Two additional groups Normal Control (NC) and Sham Control (SC) rats went through a sham operation procedure. The rats were given an oral dose for 90 days. The tail skin temperature (TST) was recorded every day, and vaginal cornification was assessed every fourth day. Serum hormones, biochemical parameters, bone physical, radiographic, and histological markers were measured on the 90th day of treatment.

Results

In LP-treated rats, there was a decrease in TST, induction of vaginal cornification, improved serum hormonal and lipid profile, improved bone physical properties, increased bone density, and enhanced cytological and histological alterations.

Conclusions

These findings imply that LP exhibited an antiosteoporotic activity against postmenopausal symptoms in ovariectomized rats, suggesting that it could be used therapeutically to treat menopausal problems.

Background

Postmenopausal osteoporosis (PMO) has become a serious medical concern, affecting globally around fifty percent of postmenopausal females, and is regarded to be a condition with one of the maximum frequencies in the elderly (Sambrook & Cooper, 2006). In India, the estimated osteoporosis populace will be 1613 million by 2050, with 315 million persons above 60 years of age, demonstrating a higher occurrence than the 26 million people in the year 2003 (Desai et al., 2017). During PMO, the trabecular bone structure and the crystalline features of mineral deposits change, resulting in fracture of cancellous bone-rich areas (Zhang et al., 2006a). In females, PMO is mostly caused by a decrease in estrogen, which causes a decrease in bone production and a rise in bone resorption action. Environmental risks, sitting lifestyle, early puberty, excessive stress, ovarian problems, amenorrhea, and hormonal changes all contribute to an increase in the occurrence of PMO (Jay & Stephen, 2004). Osteoporosis is caused by changes in the functions of osteoblasts and osteoclasts cells. In osteoporosis, stimulated osteoclasts lead to the generation of reactive oxygen species (ROS) such as superoxides, and a rise in blood malondialdehyde level. Such oxidative stressors also increase osteoporotic bone loss (Ahmed et al., 2022; Desai et al., 2017).

The present PMO therapy approach focuses on i) preventing bone resorption, ii) promoting bone development, and iii) both preventing bone resorption and promoting bone development. Raloxifene, teriparatide, hormone replacement therapy (HRT), bisphosphonates, vitamin D, calcitonin, strontium ranelate, and selective estrogen receptor modulators are among the medications used for this purpose (Satpathy et al., 2015). The mentioned treatments, however, have a greater risk-to-benefit proportion because they are linked to adverse effects such as hypercalcemia, vaginal bleeding, ovarian cancer, hot flushes, breast cancer, breast soreness, and heart attack (Tripathi, 2008).

As a result, searching for novel medications having a high benefit/low-risk ratio, low cost, and lesser side effects is desirable for osteoporosis treatment. Phyto-estrogenic medications, occasionally known as "dietary estrogens," are becoming more popular as an alternate therapy for PMO management. Isoflavones, flavonoids, lignins and their derivatives, coumestans, and stilbenoids are known as phytoestrogens as they bind to estrogen receptors and lead to a change in gene expression, and show their effect on hormones (Bedell et al., 2014). Phytochemicals with antiosteoporotic effects include berberine, quercetin, rutin, luteolin-7-O-D-glucopyranoside, luteolin, genistein, wedelolactone, epigallocatechin-3-gallate, afzelin, astragalin (Jia et al., 2012). Several mechanisms for the antiosteoporotic activity of phytoconstituents include: i) enhanced osteoblast mineralization, ii) prevention of bone resorption and osteoclast development, iii) decrease in bone-resorbing cytokines and oxidative stress, iv) increased bone formation parameters such as phosphorus, calcium, osteocalcin, and parathyroid hormone, v) enhancement of osteoblasts growth and alkaline phosphatase (ALP) activity, vi) interface with estrogen receptor α and/or β, vii) enhancement of bone biomechanical features, and viii) prevention of osteoclastic tartrate-resistant acid phosphatase (TRAP).

Lupeol (LP) (Fig. 1) is a triterpene compound found in aloe leaves, carrot root, mango pulp, melon seeds, cucumber, soyabean, and other plants (Duke, 1992). It has anticancer (Saleem, 2009), anti-inflammatory, and antioxidant activities (Geetha and Varalakshmi, 2001), hepatoprotective (Preetha et al., 2006), cardioprotective (Wal et al., 2011), antidiabetic (Lakshmi et al., 2014), antimicrobial (Wal et al., 2015). Antiosteoporotic activity has been reported for Bombax cieba extract. According to reports, LP is a prominent ingredient in the Bombax cieba extract (Chauhan et al., 2018). Echinops echinatus Roxb. is a traditional herb used to treat reproductive system diseases, and Aspidosperma nitidum, which is used to treat uterine and ovarian inflammation, contains LP as the main ingredient (Wal et al., 2011). According to many researchers, herbal medicines enriched with LP caused anti-inflammatory response in the complete Freund adjuvant-induced arthritis model in rats (Geetha and Varalakshmi, 2001). LP has also been shown in studies to bind to the estrogen receptor alpha (ER-α). LP, in particular, had an influence on cell proliferation in in vitro experiments that reported lupeol binding to ER-α. LP has estrogenic potential, according to in vitro research such as cell proliferation assays (Zingue et al., 2016). The effectiveness of LP in the treatment of PMO symptoms may be linked to its estrogenic activity. Even though previous research suggests that LP is effective in treating bone problems, a comprehensive study using appropriate in vivo model is needed. There is a lack of complete knowledge of LP effects on bone formation, its likely mechanism of action, and the search for lead molecules accountable for its bone-protective functions.

Fig. 1

Chemical structure of LP

Therefore, the goal of this research work is to better understand the role of LP in bone remodeling by comparing its influence on osteoblastic and osteoclastic activity in OVX-induced PMO in female rat models in comparison to 17β-estradiol (17β ED). To back up the findings, an array of biochemical, radiological, and histopathological markers was examined.

Methods

Chemicals

LP and 17β ED were purchased from Sigma-Aldrich Co. (USA). Rat estrogen, progesterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) kits were purchased from Genexbio Health Sciences Pvt. Ltd., Delhi, India. Total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), alkaline phosphatase (ALP), calcium (Coral Clinical Systems, India), and acid phosphatase (Accurex Biomedical Pvt. Ltd., India) kits were used.

Animals

Nine-week-old female Sprague Dawley rats (180–220 g) were procured from the National Institute of Biosciences, Pune. Animals were housed in polypropylene cages and maintained in an environment with a temperature of 25 ± 1°, relative humidity of 45–55%, and 12-h light: 12-h dark cycle. All the experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC) of Poona College of Pharmacy, Pune. The protocol approval number is CPCSEA/PCP/ PCL 10/2017–2018.

Experimental design

After a quarantine period of one week, rats were randomly separated into seven groups (n = 8) as Normal Control (NC), Sham Control (SC), Ovariectomy Control (OVX), Standard (17β-estradiol 50 µg/kg), OVX with a low dose of LP (25 mg/kg), OVX with a low dose of LP (50 mg/kg), and OVX with a low dose of LP (100 mg/kg). All the groups of animals except for NC and SC were subjected to bilateral ovariectomy. Animals of the SC group underwent sham operations. The animals were anaesthetized using thiopentone sodium (50 mg/kg i.p). A dorsolateral longitudinal incision of 1 cm was made to expose the abdominal cavity. Ovaries surrounded with fat were isolated. The fallopian tube was ligated with absorbable sutures and the ovary was removed. The muscle layer was sutured followed by the skin layer. The procedure was repeated on the other side. After surgery animals were administered antibiotics (amoxicillin 30 mg/kg) for 3 days to prevent infection. The animals of NC, SC, and OVX control were administered with 1% sodium carboxy methyl cellulose solution as a vehicle for 90 days. The other groups were orally administered with respective treatments for 90 days. Tail skin temperature (TST) was recorded for all the animals each day, and vaginal smears were prepared and analyzed every fourth day after treatment initiation. After 90 days of intervention, all rats were euthanized with thiopentone sodium, and a range of samples was acquired, including left/right femur, uterus, and vagina (Zhang et al., 2016).

Measurement of vaginal cornification assay

The various stages of the estrous cycle were identified by preparing the vaginal smears and observing them under a microscope. In the morning, samples of vaginal mucus were collected by flushing a few drops of saline solution into the rat’s vagina. The estrous phase of the rat was studied by seeing the saline and its cellular contents under the microscope and rated as per the following parameters (Satpathy et al., 2013).

0-Diestrous phase: Leukocytes with few epithelial cells.

1-Mixture of leukocytes and epithelial cells.

2-Proestrous phase: Nucleated or nucleated and cornified cells.

3-Estrous phase: Only cornified cells.

Only animals showing a score of 2 or 3 were considered to be positive for estrogenic activity.

Measurement of tail skin temperature

TST was measured every day at 8 am by using 8 channels PowerLab recording system (AD instruments). The probe was placed 2 cm from the base of the tail, and the temperature was recorded using LabChart software (Kobayashi et al., 2000).

Measurement of serum hormones

The blood sample was removed from the retro-orbital plexus under anesthesia on the last day of the study, and serum estrogen, progesterone, FSH, and LH were measured using ELISA kits (Biocodon Technologies, USA). Sandwich ELISA was carried out using Streptavidin-HRP enzyme and the intensity of color formed was measured using a microplate reader (LISAscan, Erba) in the form of change in optical density at 405 nm.

Measurement of serum lipid profile

Serum total cholesterol, triglyceride, HDL-C, and LDL-C levels were estimated using biochemistry kits (Coral Clinical systems, India) on day 90. The optical density was measured using an ELISA plate reader (LISAscan, Erba) at 505 nm.

Measurement of serum biochemistry markers of bone turnover

The blood samples were collected from the retro-orbital plexus, and serum samples were tested for ALP, TRAP, and calcium after 90 days of treatment. Serum ALP and calcium were estimated by using kits of Coral Clinical Systems, India. Serum TRAP level was measured by using autozyme kit of Accurex Biomedical, India.

Measurement of body weight and relative uterine weight

The body weight of each animal was measured weekly during the experimental period. Rats were weighed and killed by overdose of thiopentone sodium (100 mg/kg, i.p.) on the 90th day of study. The uterus was cut and isolated from fat and connective tissues and weighed. Relative uterine weight was calculated by using the following formula (Thanamool et al., 2013);

$$\text{Relative uterine Wt }(\text{\% RU})\left(g\right)=\frac{\text{Uterus Wt}. (g)}{\text{Body Wt}. (g)} \times 100$$

Measurement of femur bone physical parameters

The femur bones were isolated and weighed using an electronic balance. Femur length was measured from the proximal tip of the femur head to the distal tip of the medial condyle using digital vernier callipers. Bone volume was measured by the fluid displacement method (Halekunche et al., 2016). The thickness was measured at midshaft using digital vernier callipers (Bodhankar et al., 2012).

Radiographic imaging of femur bone

The femur bone was subjected to high-resolution X-ray imaging to reveal bone density changes. The scanning protocol used X-ray energy of 53 kVp and 6 mAs (Mahl et al., 2009).

Uterus and vaginal histopathology

The uterus and vagina were excised, and cut free of adherent fat tissues and fixed in 10% formalin. The formalin-fixed organs were embedded in paraffin, cut into slices (4 μm), and stained with hematoxylin–eosin (H&E) staining. H&E staining was carried out as described earlier by Huang et al. (2002). Uterus and vaginal histological changes were observed under a light microscope.

Statistical analysis

The data were expressed as Mean ± SEM. The data were analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni’s test for repeated parameters and one-way ANOVA followed by Dunnett’s multiple comparison test for last-day parameters using the GraphPad Prism demo 5.0. Values p < 0.05 were considered statistically significant.

Results

Effect of LP on vaginal cornification assay in ovariectomized rats

The appearance of the cornified cell was used as an indicator of estrogenic activity. The vaginal smear of OVX control rats did not show any vaginal cornification (Score 0). It was observed that treatment with 17β ED showed only cornified cells in the vaginal smear (Score 3). The treatment of rats with LP (25 mg/kg) exhibited the presence of nucleated as well as cornified cells (Score 2). However, all cells were cornified in the rats administered with a mid and high dose of LP (50 and 100 mg/kg) (Score 3) (Fig. 2 and Table 1).

Fig. 2

Effect of LP on vaginal cornification in OVX rats a vaginal smears of ovariectomized rats observed on Day 1, b vaginal smears of ovariectomized rats observed on Day 90 A: NC (2), B: SC (3), C: OVX Control (0), D:17β ED (50 µg/kg) (3), E: LP (25 mg/kg) (2), F: LP (50 mg/kg) (2), G: LP (100 mg/kg) (3). Values in bracket indicate vaginal cornification score. NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

Table 1 Percentage of number of animals showing positive cornification within each group

Effect of LP on tail skin temperature in ovariectomized rats

The TST was found to be considerably (p < 0.001) increased in OVX control rats as compared to SC group rats from day 1 to day 70. Treatment of rats with 17β ED exhibited a significant (p < 0.001) decrease in TST from day 14 to day 70 as compared to OVX control rats, whereas treatment of rats with LP (25 and 50) decreased the TST significantly (p < 0.001) from day 28 to day 70 as compared to OVX control rats. Treatment of rats with a high dose of LP (100 mg/kg) exhibited a significant (p < 0.01) decrease in TST from day 14 to day 70 as compared to OVX control rats (Fig. 3).

Fig. 3

Effect of LP on tail skin temperature in OVX rats Data expressed as Mean ± SEM. n = 8. Data were analyzed by two-way ANOVA followed by Bonferroni’s test. ^###p < 0.001 when compared to SC control, ^**p < 0.01, ^***p < 0.001 when compared to OVX control. NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

Effect of LP on serum hormones in ovariectomized rats

The level of serum estrogen and progesterone hormones was found to be considerably (p < 0.001) lowered in OVX control rats as matched to SC group rats. Rats treated with 17β-estradiol (50 µg/kg) showed a significant (p < 0.001) increase in the serum estrogen level as compared to OVX control rats. However, treatment of rats with 17β-estradiol (50 µg/kg) did not significantly change serum progesterone levels as compared to OVX control rats. The treatment of rats with a mid and high dose of LP (50 and 100 mg/kg) displayed a significant (p < 0.01 and p < 0.001) increase in the serum estrogen as compared to OVX control rats. Means the low dose of LP (25) is not having significant blood estrogenic activity when compared with OVX rats. The serum progesterone level was found to be not significantly changed in rats treated with LP (25, 50 and 100 mg/kg) as compared to OVX control rats (Fig. 4).

Fig. 4

Effect of LP on serum hormones in OVX rats. Data expressed as Mean ± SEM. n = 8; data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. ^##p < 0.01, ^###p < 0.001 when compared to SC control. ^*p < 0.05, ^**p < 0.01 ^***p < 0.001 when compared to OVX control rats FSH: Follicle-Stimulating Hormone; LH: Luteinizing Hormone. NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

The level of serum FSH and LH hormones was observed to be considerably (p < 0.01) increased in OVX control rats as matched to SC group rats. The rats treated with 17β-estradiol (50 µg/kg) showed a significant (p < 0.001) decrement in the FSH and LH level in serum as compared to OVX control rats. The serum FSH was found to be significantly (p < 0.05 and p < 0.01) decreased in LP (50 mg/kg and 100 mg/kg) as compared to OVX control rats. However, treatment of rats with LP (25 mg/kg) did not show any significant change in serum FSH level as compared to OVX control rats. The rats treated with LP (25, 50, and 100 mg/kg) exhibited significant (p < 0.05, p < 0.01, and p < 0.001) decrement in serum LH levels as compared to OVX control rats (Fig. 4).

Effect of LP on serum lipid profile in ovariectomized rats

The serum level of TG, TC, and LDL-C was observed to be considerably (p < 0.001) increased in OVX control rats as compared to SC rats. The rats treated with 17β-estradiol (50 µg/kg) showed a significant (p < 0.001) increase in the TG level in serum as compared to OVX control rats, whereas LP (25, 50, and 100 mg/kg) treatment to rats has exhibited a significant (p < 0.01 and p < 0.001) reduction in TG level as compared to the OVX control group. Treatment of rats with 17β ED caused a significant (p < 0.001) reduction in TC and LDL-C levels as compared to OVX control rats. Furthermore, treatment of rats with LP (25, 50, and 100 mg/kg) was found to significantly (p < 0.001) reduce the TC and LDL-C level as compared to OVX control rats.

The serum level of HDL-C was observed to be considerably (p < 0.01) reduced in OVX control rats as compared to SC rats. The serum HDL-C was found to be improved considerably (p < 0.01) following treatment with 17β ED as compared to OVX control rats, whereas treatment of rats with LP (25, 50, and 100 mg/kg) was found to increase the serum HDL-C levels significantly (p < 0.05, p < 0.01 and p < 0.001) as compared to OVX control rats (Fig. 5).

Fig. 5

Effect of LP on serum lipid profile in OVX rats. Data expressed as Mean ± SEM. n = 8; data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. ^##p < 0.01, ^###p < 0.001 when compared to SC control. ^*p < 0.05, ^**p < 0.01 ^***p < 0.001 when compared to OVX control. TG: Triglycerides; TC: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol. NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

Effect of LP on serum ALP, TRAP, and calcium in ovariectomized rats

The serum ALP and TRAP levels were observed to be considerably (p < 0.001) increased in OVX control rats as compared to SC rats. The rats treated with 17β-estradiol (50 µg/kg) showed a significant (p < 0.001) decrease in the serum ALP and TRAP level as compared to OVX control rats, whereas the rats treated with LP (25, 50, and 100 mg/kg) displayed significant decrease in serum ALP and TRAP levels as compared to the OVX control group. However, no significant change was observed in serum calcium levels of any of the tested groups (Fig. 6).

Fig. 6

Effect of LP serum biochemical markers in OVX rats. Data were expressed as Mean ± SEM. n = 8; data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. ^###p < 0.001 when compared to SC; *p < 0.05, **p < 0.01 ***p < 0.001 when compared to OVX rats. NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol, ALP: Alkaline phosphatase, TRAP: Tartarate-resistant acid phosphatase

Effect of LP on body weight in ovariectomized rats

The body weight of the OVX control group was found to be significantly (p < 0.01) increased as compared to the SC group rats on days 56, 70, and 84. Treatment of rats with 17β-estradiol caused no significant change in body weight as compared to the OVX control group on any of the tested days. Treatment of rats with LP (50 mg/kg and 100 mg/kg) exhibited significant (p < 0.05, p < 0.01 and p < 0.001) decrease in the body weight on day 42, 56, 70, and 84. However, treatment of rats with LP (25 mg/kg) caused a significant (p < 0.001) decrease in body weight as compared to OVX rats on day 84 (Fig. 7).

Fig. 7

Effect of LP on body weight (g) in OVX rats. Data expressed as Mean ± SEM. n = 8; data were analyzed by two-way ANOVA followed by Bonferroni’s test. ^##p < 0.01 when compared to SC control, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 when compared to OVX control. NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

Effect of LP on relative uterine weight in ovariectomized rats

The relative uterine weight was observed to be considerably decreased (p < 0.001) in OVX control rats as related to SC rats. The treatment of rats with 17β ED significantly (p < 0.001) improved the relative uterine weight as compared to OVX control rats. However, the treatment of rats with LP (25, 50, and 100 mg/kg) caused a significant (p < 0.01 and p < 0.001) rise in relative uterine weight as compared to OVX control rats (Fig. 8 and 9).

Fig. 8

Effect of LP on ovariectomy-induced changes in relative uterine weight of rats A: NC; B: SC; C: OVA; D: 17β Estradiol; E: LP (25 mg); F: LP (50 mg); G: (100 mg). NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

Fig. 9

Effect of LP on relative uterine weight in OVX rats. Data were expressed as Mean ± SEM. Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. ^###p < 0.001 when compared to SC, ^**p < 0.01, ^***p < 0.001 when compared to OVX control. NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

Effect of LP on bone physical parameters in ovariectomized rats

The femur length, thickness, weight, volume, and density were observed to be considerably (p < 0.001) reduced in OVX control rats as compared to SC rats. The rats treated with 17β-estradiol (50 µg/kg) showed a significant (p < 0.001) increase in the femur length, thickness, weight, volume, and density as compared to OVX control rats, whereas rats treated with LP (50 and 100 mg/kg) displayed markedly significant (p < 0.05, p < 0.01, and p < 0.001) increment in femur length, thickness, weight, volume, and density as compared to the OVX control group. Treatment of rats with LP (25 mg/kg) did not show any significant change in femur length, thickness, weight, volume, and density (Table 2).

Table 2 Effect of LP on bone physical parameters in OVX rats

Effect of LP on radiographic imaging of femur bone in ovariectomized rats

The bone density was found to be decreased in OVX control rats as compared to NC and SC groups, whereas treatment of rats with 17β ED and LP (50 and 100 mg/kg) was found to increase the femur bone density (Fig. 10).

Fig. 10

Effect of LP on X-ray images of femur bones in OVX rats X-ray images of isolated femur bone isolated on day 90. Intensity of the image is proportional to the density of femur bone. A: NC, B: SC, C: OVX control, D: 17β estradiol (50 µg/kg), E: LP (25 mg/kg), F: LP (50 mg/kg), G: LP (100 mg/kg). NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

Effect of LP on vaginal and uterine histopathology in ovariectomized rats

Multiple layers of stratified squamous epithelial cells lining the vaginal wall were found to be present in NC and SC control rats, whereas the lining was found to be absent in the OVX control group. Treatment of rats with 17β ED and lupeol (25, 50, and 100 mg/kg) exhibited multiple layers of stratified squamous epithelial cells lining the vaginal wall (Fig. 11). Under the microscope, marked atrophy of the rat uterus to about half its normal size was observed in OVX rats. The uterine atrophic changes indicated by decreased grooves in the uterine lining and absence of glands were observed in the OVX control group as compared to SC group rats. However, treatment of rats with 17β ED and LP (25, 50, and 100 mg/kg) displayed reversal of the vaginal atrophy accomplished by the proliferation of uterine tissue (Fig. 12).

Fig. 11

Effect of LP on vaginal histology in OVX rats. Sections of vagina stained with hematoxylin–eosin (H&E) and observed under 40X objective. Arrows show layers of stratified squamous epithelial cells. A: NC, B: SC, C: OVX control, D: 17β estradiol (50 µg/kg), E: LP (25 mg/kg), F: LP (50 mg/kg), G: LP (100 mg/kg). NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

Fig. 12

Effect of LP on uterine histology in OVX rats Sections of uterus stained with hematoxylin–eosin (H & E) and observed under 40X objective. Arrows show uterine glands. A: NC, B: SC, C: OVX control, D: 17β estradiol (50 µg/kg), E: LP (25 mg/kg), F: LP (50 mg/kg), G: LP (100 mg/kg). NC: Normal Control, SC: Sham Control, OVX: Ovariectomy Control, 17-βED: 17β-Estradiol, LP: Lupeol

Discussion

Osteoporosis is an age-related condition characterized by enlarged bone brittleness, reduced bone mass, and a high frequency of bone fractures as a result. The majority of female osteoporosis cases are caused by postmenopausal deficiency of estrogen hormone (Kanis, 1994). Female rodents with ovariectomies have osteoporotic bone morphologies similar to that of postmenopausal osteoporosisin women. As a result, estrogen therapy reduces estrogen deficiency-induced bone loss in OVX rats and human females after menopause (Moriya et al., 1998). OVX-induced osteoporosis in rats has been frequently used as a PMO model in humans. Despite the pharmacological and clinical importance of hormone replacement therapy as a widely established therapeutic method for osteoporosis, substantial long-term adverse effects have been described (LaCroix & Burke, 1997). Hence, identifying other viable therapeutic alternatives with superior safety profiles became critical.

Menopause is an inevitable phenomenon as a part of the normal physiology of a woman. It occurs mainly due to ovarian failure resulting in termination of the menstrual cycle and a deficit of ovarian hormones like estrogen and progesterone (Shifren et al., 2014). Estrogen exerts physiological effects by acting through estrogen receptors. Estrogen receptors are located in various tissues such as the urogenital tract, brain, bone, etc. Thus, estrogen deficiency results in diverse symptoms out of which only vasomotor symptoms subside after a few years, but the other symptoms persist and affect the quality of life of a woman (Dalal & Agarwal, 2015). Estrogen deficiency increases bone resorption resulting in bone loss and osteoporosis (Prestwood & Raisz, 2002). HRT is so far the only treatment that improves all the symptoms and provides protection against osteoporosis, but certain risk factors such as the increased risk of breast and endometrial cancer, increased risk of CVD, etc., outweigh the benefits of HRT (Santen et al., 2010). In rodent’s ovarian failure results in estropause. Although ovarian failure can be induced by the administration of ovotoxic compounds such as VCD, ovarian failure occurs gradually taking a longer duration for induction of osteoporosis (Merchenthaler et al., 2014). Surgical removal of ovaries results in rapid deficiency of ovarian hormones, changes in body weight, uterine and vaginal atrophy, elevated TST, changes in lipid profile, and induction of osteoporosis. This can be well correlated with human postmenopausal symptoms. In the present study bilateral ovariectomy was performed and cessation of the estrous cycle was confirmed by the consistent diestrus phase observed in vaginal smears of the animals which showed the presence of only leukocytes. This observation was consistent with previous studies (Zhang et al., 2020).

Previous reports showed that estrogen deficiency in adipose tissue caused by OVX significantly increased body weight and decreased uterine weight in rats (Høegh-Andersen et al., 2004; Zhao et al., 2011). Human and rodent adipocytes express estrogen receptors (ERα and ERβ) and estrogen deficiency induces visceral adiposity. In the present study, the body weight of rats was increased in OVX rats and treatment of rats with LP significantly reduced body weight. Treatment with 17β ED however did not reduce the body weight significantly. This can be attributed to ERβ-mediated effect of 17β ED resulting in adipocyte proliferation (Blüher, 2013).

A vaginal cytology assay is particularly used to determine the estrogenic activity of synthetic estrogens. Endogenous estrogen induces the estrous phase in rodents and brings about the cornification of the vaginal epithelium (Cordial et al., 2006). Postovariectomy, the vaginal smear consists of only leukocytes which are characteristic of the diestrus phase. The appearance of cornified cells in vaginal smears of OVX rats suggests the estrogen-like effects of the compound (Satpathy et al., 2013). Treatment with 17β ED showed induction of estrous in rats indicated by vaginal smear consisting of only cornified cells. In the current study presence of cornified cells in a vaginal smear of rats was observed even after treatment with a low dose of LP, but a full estrous developed gradually. This highlights the estrogenic potential of LP (Stroheker et al., 2003). The hot flush is characterized by a sensation of heat on the face, neck chest, etc., that persists for a few minutes. A hot flush is a characteristic vasomotor symptom which occurs because of the resetting of the thermoneutral zone in the hypothalamus resulting in the simulation of the heat dissipation mechanism with minimal changes in core temperature. Flushing is not observed in rodents, but a consistently elevated temperature can be observed in areas such as the ear, tail, abdomen, etc. Tail skin is the principal heat dissipation site in rodents. An increase in TST as a result of tail skin vasodilation following ovariectomy is comparable to a human hot flush (Kobayashi et al., 2000). In the present study LP and 17β ED treatment lowered TST as compared to elevated TST in OVX rats. This indicates that LP would decrease the incidence of a hot flush.

The female reproductive cycle is regulated by anterior pituitary hormones FSH and LH. The secretion of estrogen from developing follicles is stimulated by FSH and that from the corpus luteum is stimulated by LH. Estrogen reduces FSH and LH secretion by negative feedback (Mwampagatwa et al., 2013). Similar effects are observed in rodents after ovariectomy where decreased levels of serum estrogen increase serum FSH and LH in absence of negative feedback (Zhang et al., 2016). The increased LH levels are believed to be responsible for vasomotor symptoms. In the present study, lupeol treatment increased serum estrogen and consequently decreased FSH and LH. TC, TG, and LDL cholesterol are increased, while HDL cholesterol is decreased following ovariectomy in rats. Estrogen therapy is known to improve lipid profile, but it increases TG levels potentiating the risk of venous thromboembolism and other cardiovascular diseases (Zhang et al., 2016). A similar effect was observed in the current study confirming increased chances of development of atherosclerosis associated with treatment with 17β ED. Treatment with 17β ED reduced total cholesterol as well as LDL cholesterol and increased HDL cholesterol resulting in an improved HDL: LDL ratio, but the TG level was significantly increased. LP treatment, however, improved lipid profile and also lowered TG even at low doses, indicating a lower chance of atherosclerosis development.

ALP and TRAP activity are markers of osteoblast function in bones as well as bone production parameters (Shivakumar et al., 2012). Increased levels of serum ALP and TRAP thus indicate increased bone turnover (Halekunche et al., 2016). Estrogen lowers the blood levels of these indicators; hence, these markers are higher in the OVX group due to lower estrogen levels. In the present study, ALP and TRAP were increased following ovariectomy. LP was found to decrease ALP and TRAP which indicates its antiosteoporotic effect.

The principal role of estrogen is in the development of organs in the reproductive system. Atrophic changes in the uterus leading to reduced uterine weight are observed because of estrogen deficiency in OVX rats. Estrogen therapy has been known to induce the proliferation of uterine tissues (Salem, 2013). However excessive stimulation resulting in hyperproliferation of uterine tissue leading to endometrial cancer has been observed with estrogen therapy. In the current study uterine weights were increased following treatment with 17β ED and with LP, but no hyperproliferation was observed even after treatment with a high dose of lupeol for 90 days as evidenced by histopathology. This confirms the safety of LP on uterus tissue of rats. Osteoporosis is not a postmenopausal symptom, but the postmenopausal woman is at a much higher risk of developing osteoporosis. In postmenopausal osteoporosis microarchitectural damage to the bone tissue results in weakened, porous, fragile bones which are more prone to fractures (Prestwood & Raisz, 2002). PMO is mainly due to altered levels of estrogen. Estrogen by decreasing apoptosis as well as by other mechanisms decreases the process of bone remodeling. Estrogen deficiency associated with menopause thus promotes bone turnover. Estrogen deficiency impairs the process by promoting osteoclastogenesis causing increased bone resorption and inhibiting osteoblast formation which decreases bone formation causing a net loss of bone. As the healthy bones are usually compact and can withstand significant loads, therefore bone strength is employed as a criterion. In the current study, the length, thickness, weight, volume, and density of femur bone were reduced in OVX control rats, indicating degradation of bone. LP treatment was found to be effective in preventing bone loss as observed by higher femur bone length, thickness, weight, volume, and density as compared to OVX animals. A significant difference in bone physical parameters was observed in rats after treatment with LP. Though 17β ED treatment was better than lupeol in improving bone degeneration although LP could offer considerable protection against estrogen deficiency-induced bone loss. This was also confirmed by radiological images. Hence LP may induce bone formation without the adverse effects.

Calcium balance usually represents the degree of linkage of bone production and resorption processes because the bone is a metabolically active tissue with continuing turnover. Ninety-nine percent of the body’s calcium is present in the bones of the body, and calcium balance is determined by a variety of factors, including the quantity of calcium in the diet, the rate of intestinal calcium absorption, and calcium excretion. In the current investigation, we discovered a rise in bone calcium and an increase in serum estrogen levels in LP-treated rats. Our findings support previous observations that estrogen is essential for calcium absorption from the intestines (Arnaud & Sanchez, 1990). Microcomputed tomography studies highlight more clearly the structural deterioration of bone. The present investigation validates LP's estrogenic activity and its efficacy in osteoporosis prevention. Following LP therapy, histopathological examination showed stratification and cornification of the vaginal epithelium. Treatment with LP did not prevent uterine atrophy, although it did cause uterine gland proliferation. Treatment with LP did not result in hyperproliferation of uterine tissue. LP was discovered to exhibit estrogen-like actions, although without causing the adverse effect of estrogen.

Conclusions

The current study was designed to assess the effect of LP on estrogen deficiency and osteoporosis in Sprague Dawley rats. Treatment with LP caused a significant reduction in body weight, increase in uterine weight, lowering of TST, and induced cornification of the vaginal epithelium. LP treatment also enhanced serum hormone and serum lipid profile with enhanced bone physical and biochemistry parameters. Collectively, the results of this study concluded that lupeol is having estrogenic potential and is effective in the prevention of bone degeneration associated with osteoporosis. Lupeol thus can be regarded as a candidate for future studies to reveal more about estrogenic effects and antiosteoporotic activity. The knowledge regarding estrogenic potential can be helpful in pioneering future studies.