| Object:Menopause is an oestrogen dysfunction endocrine disorder and is characterised by psychological and physiological symptoms. Over the past decades, several studies have been performed in an attempt to reveal the mechanisms of menopause. Hormone replacement therapy with oestrogen and progesterone is currently the most successful approach. However, in response to reports outlining the negative outcomes of menopause hormone therapy from the Women’s Health Initiative, the number of women receiving conventional oestrogen treatments has declined dramatically.Since there has been an increased interest in phytotherapy due to the risk of hormone therapy for menopausal symptoms, a number of botanical remedies specifically for women menopause, are beginning to come to researchers attention. Hypericum perforatum L. is a traditional herbal supplement that has been widely used in female metabolic disorder treatments, which be considered as a safe and effective alternative medicine. Hypericum perforatum L. was used widely for depression, Alzheimer’s disease, rheumatic arthritis, inflammatory disease, bacterial infections and skin injury treatments. Although many researches concerned on its anti-depressive effect, there still many ambiguous points about the mechanisms.This study was intended to test the assumption that menopausal insulin resistance is responsive to interruptions in the metabolite pathway. Ovariectomy was performed on adult female SD rats to mimic the lifespan of middle-aged women who suffer from ovarian hormone deficiency accompanied by various syndromes. A metabolite-profiling fingerprint was then prepared to clarify the connections between those syndromes. Moreover, some biomarkers can be selected to reveal which was the most important or the most responsible metabolic pathway. This study also used body weight, tail skin temperature and endometrial pathological examination to try to understand how pathological states are reflected in abnormal metabolite profiles. Additionally, this study aimed to identify metabolite changes to explain how NIL confers its beneficial effects to the endocrine system using a novel chemometric diagnostic model which may help determine normal, oestrogen-loss or treatment groups.Methods:Firstly, an ecidence-based medicine literature method was carried out for Hypericum perforatum effects evaluation which could support the dosage design processes. A systematic review and meta-analysis processed by searching from:Pubmed, Cochrane Library, Embase and Web of Science database. Hypericum perforatum L. extracts and its combination herbs were significantly superior to placebo (standard mean difference=-1.08;95%confidence interval-1.38to-0.77) and extracts of Hypericum perforatum L. proved to be more effective than placebo in the treatment of menopause. Adverse events occurred in53(17.4%) patients on Hypericum perforatum L preparations and45(15.4%) patients on placebo (relative risk=1.16;95%confidence interval0.81to1.66), indicating that the extracts of Hypericum perforatum L. have possibly fewer side effects than placebo.Secondly, on the basis of preliminary studies, the1H-NMR analysis parameters were optimized to fit targeted metabolomic researches. Meanwhile, an integral process of HPLC-MS/MS analysis were chose and established for global metabolomic researches metabolic studies which concerning on the urinary characters. Besides, the surgical procedure of ovariextormy on SD male rats was optimazied and developed for either global or targeted metabolomic studies.Third, based on the Design of Experiment (DOE) theory, an experimental design optimization method for further optimization of chromatographic conditions were established for Hypericum perforaturm fingerprint analysis. In order to clarify the antioxidant function of flavonols on the human body, their biological effects and action mechanisms were generally identified and evaluated using spectrometry or chromatographic tandems technologies. Compared to UV, GC or GC/MS detections, antioxidative abilities of flavonols or their products are frequently identified and quantified using HPLC-MS techniques, which are be able to identify unknown reaction products through MS fragment analysis. Also, their antioxidant ability could be quantified in this way due to their high selectivity and sensitivity without the need for time-consuming fractionation, purification or derivation processes. In addition, to increase selectivity and accuracy in reaction structural elucidation and free radical scavenging capacity comparison, the QqQ HPLC-ESI-MS technology was used. In this paper, the reactions between flavonols (rutin, hyperoside, quercitrin and quercetin) and ABTS+were determined by UV spectrophotometric analysis at fixed absorbance values in a time-functional mode. To improve the selectivity and sensitivity of free radical scavenging activity screening, a HPLC-ESI/MS QQQ quantitative method was established and validated. Together, the products of the reaction were identified and the reaction mechanisms were speculated.Forth, by using1H-NMR and HPLC-MS/MS technologies, the global metabolic analysis were performed by compared the variances of bio-fluid metabolites amomg the OVX surgery, aging, positive control treatment, tri-dose Hypericum perforatum extracts groups, then the targeted metabolites were identified and quantified via’H-NMR which could provided the clue for mechanism explanation of menopausal syndroms. Combining with those external indicators like weight, hot flashes indicators and uterine pathology and multivariate statistical analysis, the metabolic fingerprint were characterized, the potential biomarkers were chosen, the possible mechanisms were proposed and the metabolomic-based drug treatment evaluation model was established finally.Results53metabolites were identified as the most related biomarkers for menopause mechanisms exploration:glucose, creatine, carnitine, acetyl carnitine, citric acid, taurine, glutamine,3-indoxysulfate, palmitic acid, methionine, alanine. Those markers could be used to predict the multi-disease which induced by OVX surgery, such as hot flashes, dyslipidemia, depression, kidney disease and cancer. Wherein the3-indoxysulfate was consided as a most potential biomarket for menopausal chronic kidney disease diagnosis. In addition, arginine, urea, fumaric acid, nicotinamide, phenylalanine were identified as a highly age-related metabolites that these abnormal levels of oxidative metabolites could help to explain the result of aging shock, inflammation and degenerative disease pathogenesis.Under nilestriol treatment, OVX-induced hyperglycemia and insulin resistance generates a plurality of markers exception were reversed. Interestingly, the markers identified in the OVX+NIL treatment group included lactate; the lactate serum and urine levels quickly increased in this group. Lactate overload, however, may result in some side effects. Whereares, the establishment of a drug treatment efficacy OVX-induced metabolic evaluation model. On this basis, we found that OVX rats after different doses of Hypericum perforatum, arginine metabolic pathway and pyruvate metabolic pathways in abnormal metabolite levels has been significantly restored. Acetic acid, glutamine, carnitine, histidine, urea, glucose, and can be selected as ornithine quantify biomarkers. The acetyl-lysine, phenylacetyl glycine,5-HT, GABA, tryptophan is a qualitative metabolites may Hypericum treatment mechanism to provide additional basis for interpretation. Clinical evaluation model based on the establishment of the above can be proved, Hypericum perforatum capable of oxidizing respiratory chain and pyruvate metabolism pathway by arginine metabolic pathways related interventions to reduce the excessive production of reactive oxygen species in the body, inhibit fatty acid synthesis a lot to ease the process of menopause syndrome.ConclusionIn this study, metabonomics surgery in ovariectomized rat model of menopause syndrome caused by similar changes in metabolites, a comprehensive assessment of ovarian estrogen effects on the body caused by decay. Combined1H-NMR and HPLC-MS/MS, the global and targed metabolomics approaches were integral for briged the link between menopause and metabolites changing induced by OVX model that mimic the human menopausal syndromes. In the study of OVX induced metabolic disorders intervention by Hypericum extracts,29differential metabolites were screened from NMR and HPLC-MS/MS based metabolomic processe, which highly retaed to menopaul weight abonormal, hot flashes, depression, arthritis and cancer. It was found that these endogenous small molecule metabolites could provide an evidence to explain the Hypericum treatment mechanism from molecular level. Meanwhile, the metabolomic model for Hypericum efficacy evaluation could also be applied in further researches about traditional Chinese medicine. |
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