Tracing The Origin Of Lycium Barbarum And Its Hypoglycemic Mechanism Of Anthocyanins

Lycium ruthenicum Murray(LRM)is a unique therapeutic plant that contains various functional components such as anthocyanins,polysaccharides and pectin.It possesses an extensive range of biological and pharmacological activities including antioxidant,antifatigue,and antitumor.LRM possesses strong drought-and salt-resistance properties,which makes it an ideal plant for preventing soil desertification and alleviating soil salinity-alkalinity.Therefore,LRM is hopeful in maintaining public health and protecting ecological environment.The cultivating areas of LRM are mainly distributed in northwestern China,including Qinghai Province,Gansu Province,Inner Mongolia,Xinjiang Uygur Autonomous Region,Ningxia Hui Autonomous Region,and etc.The nutritional quality of the LRM from different regions is different,as the geographical,environmental and climatic conditions influence the quality of the fruit.The trade of inferior quality LRM has been increased in the market due to economic considerations.As the LRM from Qinghai Province has the superior quality level so it is more likely to be adulterated.However,no effective geographical origins traceability method for LRM has been developed.Besides,many scholars have investigated the anti-oxidation,blood lipids regulation,and anti-aging effects of LRM anthocyanins which are the characteristic components of LRM and are responsible for its various biological activities.However,limited information is available on exploring its hypoglycemic effect and potential mechanism.The issues above seriously limited the further utilization of LRM on industrial level.In this paper,the anthocyanin composition of LRM from five different provinces was systematically analyzed by adopting high resolution mass spectrometry and LRM traceability methods were established by adopting the multivariate statistical analysis of the anthocyanins.Then,high purity LRM anthocyanins were obtained by adopting methanol extraction method combined with XAD-7HP macroporous resin purification,and the hypoglycemic effect and the potential mechanism of LRM anthocyanins were studied by in vitro and in vivo experiments.This study is of great practical significance to maintain the trading market stability and promote the development and utilization of LRM.The main outcomes are described as follows:1.The pattern of anthocyanin composition of LRM from different provinces was the same.The anthocyanins in LRM were found mostly in the glycosylation form and were connected with sugar moieties including galactoside,glucoside and rutinoside at 3rd position in the C ring and at 5th position in the A ring(aglycones)of petunidin,delphinidin and malvidin.Furthermore,most of the anthocyanins in LRM were detected in the acylation form.The acylated groups(linked with the sugar moieties at 3rd position in the C ring)involve coumaric acid,caffeic acid,malic acid and ferulic acid.Among the anthocyanins,petunidin-3-O-rutinoside(trans-p-coumaroyl)-5-O-glucoside(2.48-18.83 mg/g DW)was appeared as the most abundant anthocyanin and accounted for more than half of the total anthocyanins(53.44-66.19%).In addition,the concentrations of individual and total anthocyanins in LRM from different provinces were found significantly different from each other.LRM from Qinghai Province showed higher concentrations of total anthocyanins(29.39 mg/g DW)9 followed by Gansu(20.82 mg/g DW),Inner Mongolia(17.33 mg/g DW),Xinjiang(16.72 mg/g DW)and Ningxia(4.59 mg/g DW).The major anthocyanins in all LRM samples presented the same order.2.Anthocyanin composition could be a credible index for LRM classification based on geographical origins.Principal component analysis(PCA)combined with linear discriminant analysis(LDA)of the anthocyanin concentrations satisfactorily categorized the LRM samples by providing a 100.0%success rate based on geographical origins.The color of LRM infused fluids could also be a clear index for LRM classification based on geographical origins.PCA combined with LDA of the color values satisfactorily categorized the LRM samples by providing a 93.3%success rate based on their geographical origins.Multivariate statistical analysis of the color values could be a simplified and reliable method of tracing the geographical origins of LRM.It has great advantages over the method of multivariate statistical analysis of the anthocyanin concentrations as well as the previously reported methods(based on e-nose and e-tongue and anthocyanidin composition analysis)due to the availability of the instruments,analysis time,required reagents and suitability of the field testing.3.After acid-methanol solid-liquid extraction,ethyl acetate liquid-liquid extraction,and chromatographic separation on macroporous resins,LRM anthocyanins were obtained with a purity of about 66%.Then,the hypoglycemic effect of LRM anthocyanins was studied and the results showed that LRM anthocyanins can effectively reduce the postprandial blood glucose levels of normal SD mice.4.LRM anthocyanins can inhibit the activity of ?-glucosidase.LRM anthocyanins can effectively inhibit ?-glucosidase activity in vitro with IC50 value of 1.35 ?g/mL.The positions of C4,C7,C3',C4' and C5' of the anthocyanidin are the potential sites for binding of LRM anthocyanins to ?-glucosidase.LRM anthocyanins can also effectively inhibit the activity of ?-glucosidase in Caco-2 cells with a similar inhibition effect as acarbose(their IC50 values are 25.26 and 21.36 ?g/mL,respectively).5.LRM anthocyanins can alleviate insulin resistance of Hep-G2 cells.LRM anthocyanins can activate P13K signal pathway via phosphorylating IRS2 and Akt and inhibit the activity of downstream GSK3? and FOXO1 by phosphorylating,improving glycogen synthesis and reducing gluconeogenesis and thus improving glucose consumption;LRM anthocyanins can also improve mitochondrial function via reducing the accumulation of mitochondrial ROS,enhancing intracellular ATP production and mitochondrial membrane potential level.