Extraction And Fraction Of Hypoglycemic Components From Rubus Chingii Hu And Identification Of Major Bioactive Compounds

Pupengzi?FPZ?,belong to genus rubus and family rosaceae,is the dry unripe fruit of rubus chingii Hu.As a traditional edible and medicinal fruit of China,it is documented to show a series of benefits,such as benefit kidney,solid essence,bright eyes et al.Modern pharmacological researches also found that FPZ extracts possess many biological activities such as anti-cancer,anti-inflammatory,et al.Diabetes mellitus is a chronic disease has damaged human beings health for a long time,?-glucosidase inhibitors is a popular approach used to treat diabetes by attenuating fasting and postprandial blood sugar levels,development of effective and low-toxic natural?-glucosidase inhibitors is meaningful for the treatment and prevention of diabetes.This research extracted the bioactive components of FPZ with different techniques and solvents.To obtain the optimal bioactive model,the potential of FPZ extracts against diabetes,pigmentation,gout,and alzheimer's disease were evaluated through free radical scavenging,?-glucosidase,tyrosinas,xanthine oxidase,and acetylcholinesterase inhibitory ability assays.The hypoglycemic active components were further fractionated and enriched with macroporous resin column,the hypoglycemic abilities were also measured in vivo and in vitro,the phytochemical profiling of major chemical compounds were constructed by UPLC-QTOF-MS/MS.Finally,the major chemical constituents were isolated and identified by chromatographic separation,mass spectrometry identification and NMR techniques.The main results of this paper were summarized as follows:?1?The ultrasonic extracts of 20% and 40% ethanol have the highest yield?P>0.05?,with the yield of 22.6%-23.00%.The 40%ethanol ultrasound and 80%methanol maceration extract showed the highest total phenol content with the value of146.57 and 144.88 mg GAE/g,respectively?P>0.05?.100%ethanol ultrasound?26.95 mg QuE/g extract?and maceration?25.72 mg QuE/g extract?extracts had the highest total flavonoid content?P>0.05?.The strongest DPPH free radical scavenging ability and?-glucosidase inhibition was found on 60%methanol and 40%ethanol ultrasonic extract,respectively,the corresponding IC₅₀0 value of was 11.14?g/mL and 0.08?g/mL.The later is much lower than its positive control acarbose(IC₅₀=70.20?g/mL).The extract prepared with 80%ethanol maceration,80%methanol ultrasound and 100%ethanol maceration exhibited the lowest xanthine oxidase,acetylcholinesterase and tyrosinase inhibition,with the IC₅₀0 value of 71.85?g/mL,44.22?g/mLand 17.23?g/mL,respectively,but they were much lower than corresponding positive control.Therefore,the extract yield,total phenolic and total flavonoid content,antioxidant and enzyme inhibition ability of samples depend on the extraction technique and solvent used.As compared with macetration,ultrasound could effectively improve the yield,total phenolic and total flavonoid content,antioxidant,?-glucosidase and acetylcholinesterase inhibition ability of extracts,the40%ethanol ultrasound extract of FPZ has promising potential in development of functional food or health food against diabetes.?2?The?-glucosidase inhibitors in FPZ was re-extracted by ultrasound assisted with 40%ethanol,and further enriched by macroporous adsorption resin to obtain 6eluting fractions,including water phase?156.19 g?,10%ethanol phase?17.87 g?,40%ethanol phase?67.97 g?,60%ethanol phase?39 g?,80%ethanol phase?4.05 g?and95%ethanol phase?1.3 g?.The 40%ethanol phase(FPZ₄₀)exhibited the strongest?-glucosidase inhibition,its acarbose equivalent value was 1188 g acarbose/g phase.High and low dose of FPZ₄₀0 were gavaged into C57BL/6 mice to investigate its ability against fasting and postprandial blood glucose level.The results indicated that the fasting blood glucose level of FPZ-H and FPZ-L group mice was significantly lower than that of acarbose group,31.5%and 30.9%lower than that of the positive group,respectively.In addition,the FPZ-H group showed the lowest postprandial blood glucose level,the value is?724±25.01 mM?is 84%and 71.2%of that of acarbose and control group,respectively.Which indicated that FPZ₄₀0 could significantly reduce the fasting and postprandial blood glucose level.Twenty-six compounds were identified from FPZ₄₀0 by HPLC-QTOF-MS/MS analysis combined with the mass spectrometery information such as parent ion,MS/MS fragmentation pattern and formula,including 15 ellagitannins?4 pedunculagin/casuariin and its isomers,4galloyl HHDP glucose,4 derivatives of ellagic acid,ellagic acid,casuarinin and casuaricin?,7 phenolic acids?salvianolic acid C,caffeoylethreonic acid,brevifolin carboxylic acid,rubusin A,courmaroylthreonic acid,darendoside B,ethyl gallate?and4 flavonoids?procyanidins dimer,epicatechin/catechin,vanillin-O-hexoside and apigenin-O-rhamnoside?.?3?Sephadex LH-20,silica gel column,ODS medium pressure column and semi preparative liquid chromatography were used to separate and purify the compounds in FPZ₄₀,and a total of 14 pure compounds were obtained.The structures of these compounds were accurately characterized by high-resolution mass spectrometry and NMR,and the accurate structures of five compounds were obtained,They were p-hydroxybenzoic acid?1?,gemin D?2?,brevifolincarboxylic acid?3?,ethyl gallate?13?,ellagic acid?14?,among which gemin D was first identified from FPZ.Due to the insufficient precision of the equipment and the low amount of compounds,the precise structure of 8 tannins can not be determined at present.The exact structure of8 ellagitannins is under identified,but their structure can be proposed as galloyl-HHDP-glucose,galloyl-Ellagic acid-HHDP-glucose,roshenin C or its isomers,rhoipteleanin E,and pedunculagin/casuariin,etc.based on MS/MS data.Through the evaluation of?-glucosidase activity inhibition ability of the monomers isolated from rubus chingii Hu,it was found that ellagitannins had a strong activity.The above research results can provide important technical references and theoretical basis for the application of FPZ in functional food and health care products against diabetes,and for its further study in anti-diabetes activity,which is of great significance.