Ameliorative Effect And Molecular Mechanism Of Aqueous Extract Of Red Ginseng And γ-Aminobutyric Acid Derivatives On Premature Ovarian Failure Caused By D-gal

Red ginseng is a very common cooked ginseng product,which is warm and slightly bitter in nature,and has many effects such as tonifying the vital energy,benefiting the blood,brightening the eyes,and calming the mind.In recent years,many studies have reported that the saponins in ginseng are effective in relieving the symptoms of premature ovarian failure,maintaining normal oestrogen levels,and promoting healthy development of follicles at all levels of growth.Based on the rich research base of the pharmacological activity of red ginseng,we speculate that the active ingredients in red ginseng may have an ameliorative effect on D-gal-induced ovarian dysfunction.Therefore,in this study,we investigated the effect of red ginseng extract(RGE)on premature ovarian failure(POF)through a combination of in vivo and ex vivo models,and revealed the possible molecular mechanism of RGE in the treatment of POF to provide a theoretical basis for the clinical treatment of POF.Theoretical basis for the clinical treatment of POF.The results showed that γ-aminobutyric acid monosaccharide had a protective effect on D-gal-induced KGN cell damage.However,due to the low content of γ-aminobutyric acid in red ginseng and the low content of γ-aminobutyric acid monoglycoside produced during processing,this study also synthesized GABA monoglycoside from glucose and γ-aminobutyric acid by simulating the principle of melad reaction produced during the processing of red ginseng,and analyzed its pharmacological effects superficially,which laid a certain foundation for making full use of the active ingredients of red ginseng.The main findings of this paper are as follows:1.Protective effect of RGE on D-gal induced premature ovarian failure in mice and its mechanismAfter one week of acclimatization of ICR female mice,the model was constructed by intraperitoneal injection of 400 mg/kg D-gal for 42 days.Vaginal secretions were collected daily at regular intervals to observe the estrous cycle.The model was successfully constructed when the erogenous cycle was disrupted at approximately 15 days.RGE was administered by oral gavage for up to four weeks.The results showed that RGE(400 mg/kg)was able to restore the Dgal-induced disruption of the oestrous cycle,increase the level of estradiol(E2)and decrease the level of follicle stimulating hormone(FSH)in the serum;at the same time,RGE ensured normal ovarian function and reduced the number of atretic follicles induced by D-gal.In addition,RGE maintains the oxidative balance of the body and improves the antioxidant defence capacity of the body by reducing the MDA content of lipid peroxidation products and increasing the activity of superoxide dismutase SOD in ovarian tissues.According to the analysis of Western blot results,RGE proadministration significantly increased the expression levels of antioxidant factors Nrf2 and HO-1 protein and inhibited the expression of proteins related to senescence pathway and apoptosis pathway.In conclusion,these results suggest that RGE may inhibit oxidative stress by modulating the Nrf2/HO-1-mediated signaling pathway and ameliorate ovarian dysfunction in POF mice through PI3K/Akt-mediated granulosa cell apoptosis and senescence.2.Protective effect and mechanism of RGE on D-gal induced KGN cell damageUsing KGN cells as the in vitro injury model,cell proliferation and toxicity assays were used to firstly determine the protective effect of RGE on D-gal-induced KGN cells;subsequently,the effects of saponins and amino acid derivatives in red ginseng on cytotoxicity were determined,and MTT results showed that ginsenoside Rg3 and GABA monoglycoside had a protective effect on D-gal-induced KGN cell The MTT results showed that ginsenoside Rg3 and GABA monosaccharide had a protective effect on D-gal-induced damage in KGN cells.In order to further investigate the effect of RGE on D-gal-induced KGN cytotoxicity and the molecular mechanism,immunofluorescence staining and Western blot were used to verify the effect.The results showed that RGE at doses of 80,160 and 320 μg/m L could improve the viability of KGN cells,and it effectively inhibited the excessive accumulation of ROS in KGN cells,increased SOD activity and decreased MDA levels.RGE inhibited oxidative stress in KGN cells.At the same time,RGE pretreatment reduced the loss of mitochondrial membrane potential(MMP)and decreased the red fluorescence intensity of Caspase 3 in the cytoplasm of KGN cells by immunofluorescence staining.9 and Bax)expression levels,all results confirmed that RGE improved D-gal-induced apoptosis in ovarian granulosa cells.In addition,RGE proadministration reduced intracellular senescence-related protein expression and inhibited their onset of senescence.In summary,both in vivo and in vitro studies confirmed that RGE ameliorated ovarian damage by targeting and modulating the Nrf2/HO-1-mediated signaling pathway and PI3K/Aktmediated granulosa cell apoptosis and senescence signaling pathway,and that the multiple pathways of oxidative stress and apoptosis work together to protect against D-gal-induced ovarian dysfunction.This study provides a valuable reference for the prevention and treatment of POF.3.Study on gamma-aminobutyric acid derivative simulation synthesis and pharmacological activity in Red ginsengDuring the processing of red ginseng,amino acids react with reducing sugar components under high temperature conditions to produce maillard reaction products.It was previously reported that a small amount of γ-aminobutyric acid was present in red ginseng at about 0.0829%,so it could react with glucose to form GABA monoglycoside during the steaming process.According to the mechanism of the Merad reaction,GABA monosaccharide was synthesized artificially by using citric acid as the reaction medium at 75 ℃ for 20 min under high temperature conditions.The retention time of GABA monosaccharide was 21.199 min by HPLC-ELSD,and its structure was identified by 1H-NMR,13C-NMR,HMBC,HSQC NMR and high resolution mass spectrometry,and its chemical formula was finally obtained as C9H19O6 N.ROS staining showed that GABA monoglycoside prevented the excessive accumulation of reactive oxygen species in KGN cells and reduced the red fluorescence intensity of Caspase 3 in the cytoplasm.gal-induced apoptosis in KGN cells.This study provides a superficial analysis of the pharmacological activity of GABA monoglycoside and lays the foundation for further exploration.