Study On Resource Chemistry Of Lycium Barbarum Leaves And Flowers

This thesis is divided into four chapters,mainly summarized as follows:Chapter one,Literature reviewThe chapter collects and collates the relevant literature,to carry out herbal textual research on Lycium leaves and Lycium flowers,and to systematically review the research progress of their chemical constituents and pharmacological activities,so as to provide references for the resource value mining and product development of Lycium leaves and Lycium flowers.Chapter two,Evaluation of resource chemical constituents and standard establishment of Lycium barbarum L.leaves and flowers.?1?To determine the types and contents of chemical constituents in Lycium barbarum leaves?LBL?and flowers?LBF?,modern analytical methods such as UV spectrophotometry,high performance liquid chromatography?HPLC?and ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry?UPLC-TQ-MS?were used to evaluate flavonoids,phenolic acids,saccharides,nucleosides,amino acids and alkaloids in LBL and LBF.The results showed that the contents of total phenolic acids in LBL and LBF were 10.64 mg/g and 22.72 mg/g,respectively,and the contents of total flavonoids were 20.18 mg/g and 62.36 mg/g,respectively.Ten and seven phenolic acids and flavonoids were detected in LBL and LBF,respectively.The content of neochlorogenic acid in flowers was the highest?1.634 mg/g?.The contents of chlorogenic acid?2.607 mg/g?and rutin?4.943 mg/g?in leaves were the highest,and the contents of chlorogenic acid in samples from Inner Mongolia were significantly higher than those from other three producing areas.Neutral polysaccharides?18.99 mg/g?and acid polysaccharides?18.69 mg/g?were detected in LBL,and neutral polysaccharides?22.22 mg/g?and acid polysaccharides?8.15 mg/g?were also detected in LBF.LBL and LBF both contain fructose,glucose,sucrose and maltose.The contents of fructose?41.87 mg/g?and glucose?47.42 mg/g?in flowers are higher than other saccharides,and sucrose?15.93 mg/g?in leaves is the highest.For LBL of different producing areas,the sucrose content in the samples from Gansu Province was significantly higher than that in the other three.There are 8 kinds of nucleosides?total 53.44 ?g/g?and 12 kinds of amino acids?total 384.7 ?g/g?in LBL,as well as 10 kinds of nucleosides?total 2.01 mg/g?and 19 kinds of amino acids?total 17.66 mg/g?in LBF.The contents of nucleosides and amino acids in LBL showed a tendency of Qinghai>Gansu>Ningxia>Inner Mongolia.The contents of betaine in LBL?47.83 mg/g?was higher than that in flowers?18.78 mg/g?,besides,there was no significant differences in the contents of betaine in LBL and LBF from different areas.?2?Based on UPLC-Q-TOF/MS and orthogonal partial least squares discriminant analysis?OPLS-DA?,the chemical constituents of LBL for fruits and LBL for leaves were studied.The results showed that rutin,chlorogenic acid,quercetin-3-O-sophoroside-7-O-rhamnoside,quercetin-3-O-glucoside-7-O-rhamnoside could be used as the index components to distinguish LBL for fruits and LBL for leaves.Using high performance liquid chromatography?HPLC?,the fingerprints of LBL for leaves and for fruits were established,and similarity evaluation was carried out.The results showed that the content of rutin in LBL for fruit was limited to 40%floating according to the average value of 1.0365%,that is,the content of rutin should not be less than 0.6%as the lower limit was identified as the LBL for fruits;and the content of chlorogenic acid in LBL for leaves was limited to 40%floating according to the average value,that is,the content of chlorogenic acid should not be less than 0.3%as the lower limit was identified as the LBL for leavesChapter three,Research on resource value discovery and resource utilization of LBL?1?DPPH,ABTS,FRAP were used to evaluate the antioxidant activities of the extracts of LBL.The results showed that LBL water extract?WL?had strong scavenging activity of DPPH free radical,its IC50 value was 0.017±0.002 mg/mL.LBL ethanol extract?EL?had strong scavenging activity of ABTS free radical,and Its IC50 value was 0.351 ±0.025 mg/mL.In FRAP method,EL showed the highest antioxidant activity,0.020±0.001 mmoL FeSO4.According to the above results,the ethanol extract of LBL leaves has strongest antioxidant capacity.Oxidative damage model of PC 12 cells induced by hydrogen peroxide?H2O2?and inflammatory damage model of PC12 cells induced by lipopolysaccharide?LPS?were used to evaluate the neuroprotective effect of LBL by MTT assay after drug administration.The results showed that EL and LBL polysaccharide extract?DT?had significant preprotective effects on the oxidative damage model of PC 12 cells induced by H2O2,and showed a certain concentration correlation.Under the same concentration gradient,the effect was EL>DT.Kaempferol-3-O-rutin,cryptochlorogenic acid,neochlorogenic acid,chlorogenic acid and rutin also had significant preprotective effects,among which rutin had the strongest activity.For the LPS-induced inflammatory injury model of PC12 cells,WL and EL had obvious protective effect at a certain concentration and showed concentration correlation,whereas the protective effect of DT was weak.Rutin had the strongest protective effect on LPS-induced PC 12 cell damage.The results can provide references for the development and application of LBL.?2?In this section,a type 2 diabetic mellitus?T2DM?rat model,induced by high-fat diet combined with low-dose streptozotocin?STZ?injections,was adopted.After 4 weeks of continuous administration of LBL extract,blood,urine,liver,kidney and pancreas tissues of the rats were collected for biochemical index determination and histopathological analysis.Biochemical analysis results showed there was abnormal blood glucose,blood lipids,ALT,AST and urinary protein levels in model group 5 and the pathological sections of liver,kidney and pancreas also showed significant pathological changes in the model group.After the administration of LBL extract,blood glucose,blood lipids,ALT,AST and urinary protein levels tended to be normal,significantly different from the model group,indicating that LBL could improve liver and kidney injury from T2DM,and the pathological sections of liver,kidney and pancreas also showed that the pathological changes of each group were improved.This section experiment used UPLC-QTOF/MS technique to perform metabolomics analysis of serum and urine in type 2 diabetic rats to find differential metabolites and potential biomarkers,and to construct metabolic pathways to validate them.A total of 22 metabolites were annotated?7 from serum and 15 from urine?,and the metabolic pathways involved mainly include:pantothenate and Co A biosynthesis,pentose and glucuronate interconversions,nicotinate and nicotinamide metabolism,and arachidonic acid metabolism.Nicotinate and nicotinamide metabolism was considered as the most important metabolic pathway modified by LBL extract,with an impact-value of 0.2381.Then,Western blot was used to verify the insulin receptor transduction pathway and arachidonic acid metabolism pathway.Results showed that the expression levels of relevant proteins in the liver of type 2 diabetic rats were abnormal,suggesting that insulin resistance and inflammatory reaction in T2DM rats.After administration,all groups showed different degrees of callback,which confirmed that LBL extract could improve T2DM rats through insulin receptor transduction pathway and arachidonic acid metabolism pathway.The regulation of LBL on the diversity of gut microbiota in T2DM rats was studied by 16S rRNA sequencing.Overall,45 genera were found to have significant differences,among them,the levels of six genera,including Marvinbryantia,Parasutterella,Prevotellaceae_NK3B31_group,Blautia,Ruminococcus₁,and Coprococcus₂,were reversed to the normal level after administration of LBL extract.The prediction of metabolic pathway of intestinal flora showed that carbohydrate,amino acid,energy,and abnormal lipid metabolisms,glucose biosynthesis,and metabolic pathways were disordered,and the intestinal environment of diabetic rats was out of balance,suggesting that LBL extract could alleviate the imbalance of gut microbiota in T2DM rats and ameliorate diabetes.In order to comprehensively analyze the relationships between 9 biochemical indexes,22 metabolites,and 47 genera of gut microbiota,a correlation matrix was established calculating Pearson correlation coefficient.These relationships suggested that biochemical indexes,metabolites,and gut microbiota could affect the development of T2DM together.Chapter 4,Preliminary study on antioxidant and neuroprotective activity of LBF?1?DPPH,ABTS,FRAP were used to evaluate the antioxidant activities of the extracts of LBF.The results showed that LBF ethanol extract?EF?had strong scavenging activity of DPPH and ABTS free radical,its IC50 value was 0.163±0.007 mg/mL and 0.380±0.018 mg/mL,respectively.In FRAP method,EF showed the highest antioxidant activity,0.021±0.001 mmoL FeS04.According to the above results,the ethanol extract of LBF have strongest antioxidant capacity.Oxidative damage model of PC 12 cells induced by hydrogen peroxide?H2O2?and inflammatory damage model of PC 12 cells induced by lipopolysaccharide?LPS?were used to evaluate the neuroprotective effect of LBF by MTT assay after drug administration.The results showed that LBF water extract?WF?and EF had significant preprotective effects on the oxidative damage model of PC 12 cells induced by H2O2,and showed a certain concentration correlation.Under the same concentration gradient,the effect was EF>WF.For the LPS-induced inflammatory injury model of PC 12 cells,WF,EF had obvious protective effect at a certain concentration and showed concentration correlation.The results can provide references for the development and application of LBF.