Study On The Preparation And Hypoglycemic Activity In Vitro Of Quebrachitol From Leaves Of Seabuckthorn(Hippophaea Rhamnoides L.)

Hippophae rhamnoides L.,a genus of Hippophae from the Elaeagaceae family,is widely cultivated in Shanxi,Inner Mongolia,Liaoning,Xinjiang and Tibet in China.Hippophae rhamnoides is regarded as one kind of medicinal and edible plant,and it is rich in nutrients and physiological active ingredients.In Chinese pharmacopoeia,it is recorded that seabuckthorn has various physiology functions,including invigorating spleen and eliminating food,relieving cough and removing phlegm,promoting blood circulation and dispersing blood stasis,etc.Modern pharmacological studies have also shown that seabuckthon has significant pharmacological activities of regulating blood lipid,anti-inflammatory,antioxidant,hypoglycemic,antibacterial and anti-tumor.However,the current application and development of seabuckthorn are generally concentrated in the fruit of seabuckthorn,while the seabuckthom leaves has always been abandoned as waste,causing a heavy resource waste.As a kind of methyl ether derivative of inositol,quebrachitol possesses a variety of important physiological activities and of great value to lucubrate.It has been proved to be abundant in the leaves of seabuckthom.In this paper,modern analysis technology and different activity research methods were applied to systematically study the extraction process,separation and purification,structure identification and activity evaluation of the leaf of seabuckthom.The main results are as follows:1.Establishment and evaluation of determination of quebrachitol in seabuckthom leaves.Comparing the two methods of gas chromatography(GC)and liquid chromatography(LC),we found that LC is more conformable for the determination of quebrachitol,and the LC showed higher accuracy and reliability than GC.After a systematic investigation of the method,it was found that the method showed high precision(RSD value<1%),high accuracy(the standard recovery was 95.9%-98.7%,RSD value<2%)and good repeatability(RSD value<2%).2.Study on the space-time change of quebrachitol in the leaves of seabuckthomThe space-time change law of quebrachitol content in seabuckthorn leaves were explored by collecting samples from five different months from May to September in Gansu and Liaoning provinces.The results showed that the quebrachitol content in the samples from both provinces showed a trend of continuous accumulation and increase,and the content achieved the highest(64.70-73.27mg/g dry weight)in the samples picked in September.3.Preparation of quebrachitol from the seabuckthorn leavesUltrasonie assisted solvent method was used to prepare quebrachitol from seabuckthorn leaves,the degree of crushing,solvent concentration,solid-liquid ratio and extraction time were used as the influencing factors,and the extraction rate of plantainol from seabuckthom leaf was regarded as the evaluation index to carry out single factor and response surface test,so as to optimize the extraction process of quebrachitol.The results showed that,the extraction yield was higher(5.18%),when the extraction conditions were set as follows:crushing degree was 20 mesh,the extraction solvent was pure water,the solid-liquid ratio was 1:8 and the extraction time was 40 min.4.Purification and structure identification of purified extract from seabuckthorn leavesResin and sephdex gel column chromatography were used to conduct the purification,and GC-MS,GC×GC-TOF-MS and UHPLC-QTOF-MS/MS were applied to identify the components structure of the purified extract from seabuckthorn leaves.After the optimization of the punfication,the purified extract of seabuckthorn leaf with a purity of 72.7%was finally obtained.And 13 compounds were identified:quebrachitol,1,3-phthalate,1,4-phthalate,tagerfuranose,galactose,glucose,mannose,gulonic,lyxose,2-O-L-Leucyl-D-glucopyranose,(1R)-1,5-Anhydro-1-{[(4-hydroxybutanoyl)amino]methyl}-D-glucitol,2-O-[2-(Benzylamino)-2-oxoethyll-a-D-glucopyranose.5.Evaluation of hypoglycemic activity in vitroThe in vitro hypoglycemic activity was evaluated by measuring the inhibition of a-amylase.The results showed that both the standard quebrachitol and the purified extract of seabuckthom leaf had inhibitory effect on a-amylase,and the IC50 of the standard quebrachitol and purified extract on a-amylase was 0,604ng/mL and 2.407mg(purified substance)/mL,respectively.The standard quebrachitol and the purified extract of seabuckthorn leaf can improve the insulin sensitivity in the Hep-G2 insulin resistance cell model.The results showed that the glucose intake of the model cells increased after the addition of the tested samples,and the intake increased with the increase of drug concentration.When the standard quebrachitol concentration reached 20 ?g/mL,can be shown to significantly promote the increase of the effect,and the seabuckthorn leaf purified substance at the concentration of 40 g/mL,can achieve a significant increase in glucose intake effect.