Identification and biological characterization of bioactive ingredients derived from erxian decoction and Chinese yam for alleviating menopausal syndrome in vitro and in vivo

Abstract

Menopause is characterized by a gradual decline of ovarian estrogen secretion associated with the menopausal syndrome. Around 75% of women in the USA in the late stage of menopause report symptoms such as hot flashes or night sweats. In southern China, 37.2% of menopausal women suffer from insomnia, 37.2% complain joint and muscle pain, and 17.5% suffer from hot flashes. The standard conventional method to treat menopausal syndrome is Hormone Replacement Therapy (HRT). However, various potential risks including breast cancer, ovarian cancer and even stroke were observed after HRT. To discover safer drug candidates for treating menopausal syndrome, Erxian decoction (EXD) and a functional food, Chinese yam, was used in the present study to screen for estradiol-stimulating (E2-stimulating) ingredients. EXD has been clinically used for over sixty years to manage menopausal syndrome without side effects reported. Various pharmacological activities of EXD have been studied. However, the bioactive ingredients and mechanism of EXD for treating menopausal syndrome have not been fully unraveled. Thus, in the present study, the biological activity-guided strategy (BAGS) was developed to screen fractions with good E2-stimulating effect from EXD. The data demonstrated that TW, TE and BUTE extracts of EXD exhibited E2-stimulating effects without stimulating proliferation of estrogen-positive breast cancer cells in vitro. To further investigate the mechanism of EXD in alleviating menopausal syndrome, network pharmacological study and large-scale screening strategy by proteomics were performed. Results from network pharmacological study disclosed that 20 phytochemicals with a good drug-likeness parameter contributed to the management of menopausal syndrome via 34 significant pathways. To mimic the therapeutic effects of EXD, jatrorrhizine (JA) from EXD was screened in the further study by using network pharmacology and primary ovarian granulosa cell culture model. JA stimulated E2 in vivo via upregulating ovarian aromatase protein expression. Moreover, JA improved cognitive function in naturally menopausal rats. The proteomics data demonstrated that Premarin (PRE: HRT) increased the risk of venous thrombosis via upregulating COL5A2 and COL5A3, and targeting APOA1 to increase the risk of breast cancer. EXD upregulated FTH1 to attenuate oxidative stress, and targeted TPM1 and SERPINB5 to suppress breast cancer progression. Therefore, EXD may be a safer alternative therapy for the menopausal syndrome. A novel patented protein DOI isolated from Chinese yam by our research team demonstrated estrogenic and osteogenic effects in vitro and in vivo. Data in another part of this thesis indicated that trypsin-digested DOI but not proteinase K-digested DOI stimulated E2 biosynthesis, suggesting that peptide(s) derived from tryptic DOI may also have E2-stimulating effects. Thus, a reverse phase HPLC method was developed to separate the peptides and a BAGS was used to screen for E2-stimulating peptides from DOI. The peptides obtained by using in-silico analysis of tryptic digest of DOI with known partial amino acids were synthesized. Peptide TSP7 showed a better E2-stimulating effect in vitro. Therefore, JA from EXD and TSP7 from DOI protein may be potential new candidates for treating the menopausal syndrome. However, new study will be designed for further evaluating their E2-stimulating effect and safety in vivo.