A fraction of Pueraria tuberosa extract, rich in antioxidant compounds, alleviates ovariectomized-induced osteoporosis in rats and inhibits growth of breast and ovarian cancer cells

Abstract

Pueraria tuberosa (Roxb. ex Willd.) DC., known as Indian Kudzu belongs to family Fabaceae and it is solicited as "Rasayana" drugs in Ayurveda. In the present study, we analyzed the efficacy of an ethyl acetate fraction from the tuber extract of Pueraria tuberosa (fraction rich in antioxidant compounds, FRAC) against menopausal osteoporosis, and breast and ovarian cancer cells. The FRAC from Pueraria tuberosa was characterized for its phenolic composition (total phenolic and flavonoid amount). Antioxidant property (in vitro assays) of the FRAC was also carried out followed by the analysis of the FRAC for its antiosteoporotic and anticancer potentials. The antiosteoporotic activity of FRAC was investigated in ovariectomy-induced osteoporosis in rats. The cytotoxicity effect was determined in breast and ovarian cancer cells. Gas chromatography/mass spectrometry (GC/MS) analysis of the FRAC was performed to determine its various phytoconstituents. Docking analysis was performed to verify the interaction of bioactive molecules with estrogen receptors (ERs). The FRAC significantly improved various biomechanical and biochemical parameters in a dose-dependent manner in the ovariectomized rats. FRAC also controlled the increased body weight and decreased uterus weight following ovariectomy in rats. Histopathology of the femur demonstrated the restoration of typical bone structure and trabecular width in ovariectomized animals after treatment with FRAC and raloxifene. The FRAC also exhibited in vitro cytotoxicity in the breast (MCF-7 and MDA-MB-231) and ovarian (SKOV-3) cancer cells. Furthermore, genistein and daidzein exhibited a high affinity towards both estrogen receptors (α and β) in the docking study revealing the probable mechanism of the antiosteoporotic activity. GC/MS analysis confirmed the presence of other bioactive molecules such as stigmasterol, β-sitosterol, and stigmasta-3,5-dien-7-one. The FRAC from Pueraria tuberosa has potential for treatment of menopausal osteoporosis. Also, the FRAC possesses anticancer activity.

Fig 1. Effect of FRAC from Pueraria tuberosa on biochemical parameters of serum.

Data were average ± SEM (n = 6). *** p < 0.001 significantly different from sham control group. ## p < 0.01, ### p < 0.001 significantly different from OVX group. Ca, calcium; P, phosphorus; ALP, alkaline phosphatase; TRAP, tartrate resistant acid phosphatase; TG, triglycerides; TC, total cholesterol.

Fig 2. Effect of FRAC from Pueraria tuberosa on biochemical parameters of urine.

Data were average ± SEM (n = 6). *** p < 0.01 significantly different from sham control group. # p < 0.05, ## p < 0.01, ### p < 0.001 significantly different from OVX group. Ca, calcium; P, phosphorus; HP, hydroxyproline.

Fig 3. Effect of FRAC from Pueraria tuberosa on femur biomechanical parameters.

Data were average ± SEM (n = 6). ** p < 0.01, *** p < 0.001 significantly different from sham control group. # p < 0.05, ## p < 0.01, ### p < 0.001 significantly different from OVX group.

Fig 4. Effect of FRAC from Pueraria tuberosa on breaking strength of femur and 4^th lumbar vertebrae.

Data were average ± SEM (n = 6). *** p < 0.001 significantly different from sham control group. ## p < 0.01, ### p < 0.001 significantly different from OVX group.

Fig 5. Effect of FRAC from Pueraria tuberosa on body and uterus weight.

Data were average ± SEM (n = 6). *** p < 0.001 significantly different from Sham control group. ## p < 0.01, ### p < 0.001 significantly different from OVX group.

Fig 6. Effect of FRAC of Pueraria tuberosa on the histopathology of the femur.

A, Photomicrography of the femur of sham control group showing typical bone architecture; B, Photomicrography of the femur of OVX control group showing disruption of trabeculae; C, Photomicrography of the femur of raloxifene treated group showing improved trabecular thickness, and compactness of cells indicating mineralization of bone; D, Photomicrography of the femur of FRAC-100 mg/kg treated group showing the improved trabecular thickness and bone architecture; E, Photomicrography of the femur of FRAC-200 mg/kg treated group showing the restoration of typical bone architecture and an increase in width of trabeculae.

Fig 7. In vitro cytotoxicity of FRAC of Pueraria tuberosa against different cancer cell lines.

Values are mean ± SEMs (n = 3).

Fig 8. Docking study of genistein in estrogen receptors.

A, Co-crystalized ligand of 1x76 (genistein) showing hydrogen bond with Arg346, Leu339, and His475. Hydrophobic interactions were also seen near the benzene rings with different amino acid residues of estrogen receptor α (PDB: 1x76). The ligand showed a docking score of -26.1648. B, Co-crystalized ligand of 1x7R (genistein) showing hydrogen bond with Leu346, Arg394, Gly521, Glu353, and His524. Hydrophobic interactions were also seen near the benzene rings with different amino acid residues of estrogen receptor β (PDB: 1x7R). The ligand showed a docking score of -32.4084.

Fig 9. Docking study of daidzein in estrogen receptors.

A, Docking pose of daidzein in estrogen receptor α (PDB: 1x76) active site with a docking score of -28.3129. Daidzein formed a hydrogen bond with Arg346, Glu305, and His475. Hydrophobic interactions were also seen near the benzene rings with different amino acid residues of estrogen receptor α (PDB: 1x76). B, Docking pose of daidzein in estrogen receptor β (1 x 7R) active site with a docking score of -31.8923. Daidzein showed a hydrogen bond with Arg394, Glu353, Gly521 and His524. Hydrophobic interactions were also seen near the benzene rings with different amino acid residues of estrogen receptor β (PDB: 1x7R).

Fig 10. Docking study of internal ligands on Ix76 and 1x7R receptors.

A, Docking pose of daidzein in estrogen receptor α (PDB: 1x76) active site with a docking score of -28.3129. Daidzein formed hydrogen bond with Arg346, Glu305, and His475. Hydrophobic interactions were also seen near the benzene rings with different amino acid residues of estrogen receptor α (PDB: 1x76). B, Docking pose of 5-hydroxy-2-(4-hydroxyphenyl)-1-benzofuran-7-carbonitrile in estrogen receptor β (1 x 7R) active site with a docking score of -28.9356. 5-hydroxy-2-(4-hydroxyphenyl)-1-benzofuran-7-carbonitrile showed a hydrogen bond with Arg394, Glu353and His524. Hydrophobic interactions were also seen near the benzene rings with different amino acid residues of estrogen receptor β (PDB: 1x7R).