Abstract

General Background: Osteoporosis, a common condition exacerbated by glucocorticoid use, leads to significant bone loss and increased fracture risk. Specific Background: Conventional treatments primarily target calcium regulation, but emerging evidence suggests that natural compounds like isoflavonoids may offer therapeutic benefits. Knowledge Gap: Despite their potential, the efficacy of isoflavonoids and their nanoformulations in managing glucocorticoid-induced osteoporosis remains underexplored. Aims: This study aimed to evaluate the therapeutic effects of vitamin D, Ginkgo biloba isoflavonoid extract, and nanoisoflavonoids on glucocorticoid-induced osteoporosis in adult male rats. Results: Sixty rats were divided into five groups: a control, an osteoporosis model, and three treatment groups receiving vitamin D (10,000 IU/day), isoflavonoid extract (500 mg/kg/day), or nanoisoflavonoids (500 mg/kg/day) for one month. Molecular and physiological assessments revealed that nanoisoflavonoids were the most effective in restoring serum vitamin D levels, reducing parathyroid hormone concentrations, and downregulating parathyroid hormone gene expression, followed by isoflavonoid extract and vitamin D. Novelty: This study provides the first comprehensive evaluation of nanoisoflavonoids' superior efficacy in mitigating glucocorticoid-induced osteoporosis, highlighting their enhanced bioavailability and therapeutic potential. Implications: These findings suggest that nanoisoflavonoids could serve as a promising plant-derived nanotherapeutic for osteoporosis management, warranting further investigation through long-term studies and clinical trials to validate their safety and efficacy in human populations.
Highlights:

1. Glucocorticoid-induced osteoporosis lacks effective natural compound treatments.
2. Nanoisoflavonoids restore vitamin D, reduce parathyroid hormone effectively.
3. Nanoisoflavonoids offer a promising therapeutic strategy for osteoporosis management.

Keywords: Osteoporosis, glucocorticoids, Ginkgo biloba, isoflavonoids, nanoisoflavonoids, vitamin D, parathyroid hormone, bone metabolism

Introduction

This study was designed to investigate the therapeutic effect of vitamin D, isoflavonoids extract from Ginkgo biloba and nanoisoflavonoids on physiological parameters in adult male rats with osteoporosis. This study was conducted in the animal house of the College of Veterinary Medicine - University of Basrah on 60 adult male rats (200-250) gm, 12 weeks old. The first experiment to induce osteoporosis This experiment included 60 male rats divided into two groups, the first group (negative control group) twelve male rats were considered as a control group, each rat was injected with 1 ml of normal saline solution subcutaneously 5 days a week for a month. *The second group (osteoporosis group): Forty-eight male rats were considered as a control group with 1 ml of normal saline solution subcutaneously 5 days a week for a month. [1]After a month, osteoporosis was induced and confirmed by X-rays, we conducted the second experiment. The second experiment, the role of Ginkgo biloba and vitamin D against osteoporosis.

This experiment includes (60) male rats divided into five groups with (12) animals in each group as follows:The first group: - was considered a control group (negative control group) Each rat in the control group was injected with 1 ml of normal saline solution subcutaneously five days a week for a month.The second group: - Induction of osteoporosis by glucocorticoid (DXM) was injected subcutaneously 5 days a week for a month (positive control group)..(Osteoporosis + GbE isoflavonoid extract): -The third group ,This group was treated with Ginkgo biloba isoflavonoid extract 500 mg / kg body weight oral dose. Daily for a month.Group IV (Osteoporosis OP + GbE isoflavonoid nanoextract):- This group was treated with Ginkgo biloba isoflavonoid nanoextract at a dose of 500 mg/kg body weight orally daily for a mont. [2] (Osteoporosis OP + Vitamin D)Group V: This group was treated with Vitamin D 10,000 IU orally daily for a month. [3]3.5. Animal sacrifice and sample collection: The animals were sacrificed after the completion of treatment and rats from each group were used. Blood samples were taken from the inferior vena cava of the heart by a sterile syringe in the rats slaughtered with a normal tube without anticoagulant and the serum was extracted and stored for 15 minutes by centrifugation at 3000 rpm in attached microtubes at -20Co for laboratory analysis of molcular aspects .The results of the current study revealed the following:The results obtained showed that osteoporosis induced by glucocorticoids is accompanied by a significant decrease in Vit.D (p<0.05) and a significant increase in parathyroid hormone and parathyroid hormone gene .The results also showed that treatment with nanoisoflavones is better than isoflavones extract followed by vitamin D.

Osteoporosis is a major worldwide public health issue that has been growing as populations grow older and live longer, due to the proliferation of low bone mass However, this condition is prevalent afflicting millions of people post-menopause or aged individuals worsened by prolonged administration of glucocorticoids usually administered for multiple inflammatory and autoimmune diseases. Although that would manage those disorders, it is not appropriate to rely mostly on Glucocorticoids because they interrupt the osteoblast function and enhance the activity of ostoeclast which in turn ending up with bone loss & fragility [4].

Isoflavonoids from the traditional medicinal plant Ginkgo biloba rescue bone loss and improve bone health .[5] Ginkgo biloba is a well-known herb, possessing various pharmacological properties mostly due to the presence of flavonoids and terpenoids. In this regard, isoflavonoids have attracted much attention due to their potential pharmacological targets on bone metabolism by the regulation of signaling pathways during osteoclastogenesis and/or osteoblastogenesis[6] . This compounds have the potential to antagonize the deleterious effects of glucocorticoids, thereby stimulating bone formation and inhibiting Bone resorption that could be used as a therapeutic approach for treating osteoporos.[7]

Nanotechnology also added a new dimension on combining with herbal natural products for therapeutic strategies. Plant extract based green Biosynthesis of nanoparticles have been developed as a novel cost-effective method, not only to improve the bioavailability of drugs but also to combats environmental problems associated with conventional synthesis methods[8] . Taken together, nanoparticles derived from Ginkgo biloba isoflavonoids provided an improved way to deliver these compounds and helped improve their efficacy during treatment against glucocorticoid-induced bone loss [9].

Vitamin D is essential for maintaining bone health, as it enhances calcium absorption in the intestines and helps regulate serum calcium levels, crucial for bone mineralization. Insufficient vitamin D levels can lead to impaired calcium absorption, resulting in secondary hyperparathyroidism and increased bone resorption, contributing to the development of osteoporosis [10] . Recent studies have highlighted the association between adequate vitamin D levels and improved bone density, as well as a decreased risk of fractures in osteoporotic individuals[11] . Additionally, vitamin D supplementation has been shown to complement osteoporosis treatments effectively, emphasizing its role in both the prevention and management of this condition[12] . Aims of the Study To find out how well the nanoparticles we made treat osteoporosis caused by glucocorticoids ( physiology and molecularly) when compared to an isoflavonoid extract of Ginkgo biloba (EGb) and vitamin D.

Methods

This experimental study was conducted at the animal house of the College of Veterinary Medicine, University of Basrah, between August 1, 2023, and March 6, 2024. A total of sixty adult male rats, aged twelve weeks and weighing between 200–250 grams, were randomly assigned into five groups of twelve animals each. The first group served as the negative control and received a subcutaneous injection of 1 ml normal saline five days a week for one month. The second group was induced with osteoporosis by administering glucocorticoid (dexamethasone) subcutaneously at the same frequency, serving as the positive control. The third group, after osteoporosis induction, was treated with a daily oral dose of 500 mg/kg body weight of Ginkgo biloba isoflavonoid extract for one month. The fourth group received an identical dose of nanoisoflavonoid extract. The fifth group was administered 10,000 IU of vitamin D orally per day for the same period. At the end of the experiment, all rats were sacrificed, and blood samples were collected from the inferior vena cava using sterile syringes. The serum was separated by centrifugation at 3000 rpm for 15 minutes and stored at -20°C for further analysis. Molecular and physiological evaluations included measuring serum vitamin D levels, parathyroid hormone concentrations, and parathyroid hormone gene expression. The data obtained were statistically analyzed to determine the efficacy of the treatments. The study adhered to ethical guidelines for animal research, ensuring proper handling and minimal distress throughout the experiment.

Results and Discussion

Result

Effect of iso Bioflavonoid, Nano iso Bioflavonoid and Vit D on Parathyroid hormone and Vit D in Osteoporosis Adult Male Rats.

Figure 1 shows that there was a significant (P≤0.05) decrease in serum Vit D concentration in OP group (Positive) compared to control group. While, the treated groups (OP plus Nano iso Bioflavonoid and OP plus Vit D) shows a significant (P≤0.05) increase in the serum Vit D concentration as compared to the OP Group (Positive). There is no a significant between the treated group OP plus iso Bioflavonoid compared to OP group. Also There is no a significant between the OP plus Nano iso Bioflavonoid compared to control group. Finally the treated group (OP Plus Vit D) shows a significant (P≤0.05) increase in the serum Vit D concentration as compared to Other groups.

Figure 1. Effect of iso Bioflavonoid, Nano iso Bioflavonoid and Vit D on Parathyroid hormone and Vit D in Osteoporosis Adult Male Rats. (Mean ± SD) n=8

Figure 2 shows that there was a significant (P≤0.05) increase in serum Parathyroid concentration in OP group (Positive) compared to control group. While, the treated groups (OP plus iso Bioflavonoid, OP plus Nano iso Bioflavonoid and OP plus Vit D) shows a significant (P≤0.05) decrease in the serum Parathyroid concentration as compared to the OP Group (Positive). There is no a significant between the treated groups with each other and control group.

Figure 2. Effect of iso Bioflavonoid, Nano iso Bioflavonoid and Vit D on Parathyroid hormone and Vit D in Osteoporosis Adult Male Rats. (Mean ± SD) n=8

Effect of iso Bioflavonoid, Nano iso Bioflavonoid and Vit D on Parathyroid hormone Gene in Osteoporosis Adult Male Rats.

According to gene expression analysis, treatment groups showed significantly reduced mRNA levels of Parathyroid compared with the positive Control group. The group treated with nano iso bioflavonoid had the most significant decrease in expression. The nano-treated group was also closest to the control negative group. Equally, These data suggest that both iso bioflavonoid and its nano-formulated form help lower PTH, which may lay down a basis for therapeutic strategies against osteoporosis.

The figure below demonstrates the gene expression levels of the Parathyroid Hormone (PTH) gene among all groups. The nano iso Bioflavonoid treated group shows the most reduction (underexpression) of the gene among all groups. Also, all treatment groups showed a lower expression than the control positive group. This Figure (4-14) shows results with significant differences between them.

Figure 3.Effect of iso Bioflavonoid, Nano iso Bioflavonoid and Vit D on Parathyroid hormone Gene in Osteoporosis Adult Male Rats.

Discussion

This thesis study investigates the effect of isoflavonoid of Ginkgo biloba, nano Ginkgo biloba, and Vitamin D on a broad spectrum of biochemical indicators in male rats that have induced osteoporosis. The results provide valuable insights into therapeutic mechanisms and implications in osteoporosis treatment and management.Effect of Bioflavonoid, Nano Gingko Biloba, and Vit D on Parathyroid hormone and Vit D in Osteoporosis Adult Male Rats. The Vitamin D on serum was lower in the dexamethasone-induced osteoporosis positive group than it in the control negative group. All treatment groups, which are iso bioflavonoid, nano iso bioflavonoid, and vitamin D group, increased Vitamin D concentration in the serum, with the Vitamin D treated group having the superior concentration among all groups [14].

While the dexamethasone-induced positive group shows a significantly higher concentration of Parathyroid hormone compared to the control negative group, all treatment groups show an excellent potential ability to decrease parathyroid hormone concentrations, with Nano ginkgo biloba especially having a higher ability to stabilize parathyroid hormone. However, the results of nano iso bioflavonoid were still comparable to the other treatment groups [15] .

Those findings suggest that combining Vitamin D and nano iso bioflavonoid could synergistically improve bone health by modulating both parathyroid hormone and vitamin D [14] .Effect of iso Bioflavonoid, Nano Gingko Biloba and Vit D on Parathyroid hormone Gene in Osteoporosis Adult Male Rats.

The notable reduction of PTH mRNA levels in all of the treatment groups suggests a decrease in bone resorption, which means protection for bone health as parathyroid hormone plays a key role in calcium homeostasis[16] . The overexpression of PTH means an increase in bone resorption and, therefore, an increase in the severity of osteoporosis. The mechanism of Reduced PTH expression may vary; the most believed theory is that nano iso bioflavonoid acts upon bioavailability while Vitamin D acts directly upon calcium homeostasis[17] . PTH hormone plays a crucial role in bone remodeling. By decreasing the PTH hormone, bone density will increase, reducing the risk of bone fractures and avoiding osteoporosis[18] . The pathway of decreasing the expression of PTH hormone needs further study, but it's more likely through anti-inflammatory and anti-oxidant pathways, as those pathways regulate PTH hormone[19] .This study suggested that nano iso bioflavonoid has the potential ability to manage osteoporosis because it has enhanced bioavailability, anti-inflammatory abilities, and the ability to reduce oxidative stress.

Conclusion

This study highlights the therapeutic potential of Ginkgo biloba isoflavonoids, their nanoformulation, and vitamin D in mitigating glucocorticoid-induced osteoporosis. The findings indicate that nanoisoflavonoids were the most effective treatment, significantly restoring vitamin D levels, reducing parathyroid hormone concentrations, and downregulating parathyroid hormone gene expression, followed by isoflavonoid extract and vitamin D. These results suggest that nanoisoflavonoids enhance bioavailability and therapeutic efficacy, providing a promising alternative for osteoporosis management. The implications of this research extend to the development of novel, plant-derived nanotherapeutics that could be integrated into clinical osteoporosis treatment strategies. However, further studies are required to explore the long-term effects, optimal dosages, and underlying molecular mechanisms of nanoisoflavonoids in bone health. Additionally, clinical trials are necessary to validate these findings in human populations and assess potential interactions with conventional osteoporosis therapies.

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