Comparative Study On The Components Of Chinese Seabuckthorn And Tibetan Seabuckthorn Leaves And Preliminary Screening Of Their Activities

ObjectiveChinese sea buckthorn is a variant of Hippophae rhamnoides L.,which has been listed in the Chinese Pharmacopoeia since 1977.It has the functions of invigorating spleen and eliminating digestion,relieving cough and expelling phlegm,promoting blood circulation stasis and other effects.H.tibetana Schlecht.,which is similar to H.rhamnoides L.,was originally merged into H.rhamnoides L..Because of its morphological characteristics,geographical distribution and growth environment are peculiar,it was classified as an independent species eventually.Tibetan sea buckthorn has the effect of invigorating the lung and promoting blood circulation and can rule irregular menstruation,uterine disease,stomach,tuberculosis,gastric ulcer,etc.The local Tibetan people also use it for the treatment of hepatitis in folk medicine.As a medicinal and edible plant,sea buckthorn is rich in nutrients and bioactive components.The sea buckthorn leaves are usually discarded as wastes,but the researches have confirmed that the content of flavonoids was the highest in the leaves?Mean value is 792 mg,the mean value of fresh fruit is 533 mg?,while flavonoids was deemed as an important index to measure the quality and physicochemical identification of sea buckthorn products.It is estimated that the annual output of sea buckthorn leaves can reach 5 tons per hectare,resulting in a great waste of resources.In addition,compared with the fruits,the sea buckthorn leaves also have the advantages of high yield,easy collection,long acquisition cycle,simple production and process,and easy storage and so on.The development prospect is attractive through the deepening research and reasonable utilization of its leaf components.Therefore,based on the advantages of resources,the following studies were carried out in this paper:?1?To analyze the chemical components in leaves of Chinese and Tibetan sea buckthorn qualitatively,and compare the differences between them by using ultra performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry?UPLC/Q-TOF-MS?.?2?UPLC-LTQ-Orbitrap-MS was applied as the detection means combined with metabonomics technology,to construct the discriminant model of leaves of Chinese sea buckthorn and Tibetan sea buckthorn,and find out the characteristic components that cause the difference of the two kinds of sea buckthorn leaves.?3?To collect numerous samples of leaves of Chinese and Tibetan sea buckthorn of different origins,and establish a method for determination of the flavonoid glycosides of two kinds of sea buckthorn and explore the differences of the contents in the leaves of two sea buckthorn species.?4?H₂O₂ model was used to induce the cytotoxicity in PC 12 cells,the morphology of PC 12 cells was observed by microscope,the cell damage was measured by MTT assays,to observe the protective effects of total flavonoids of sea buckthorn?TFH?,different polar parts of leaves of Chinese and Tibetan sea buckthorn.Methods1.UPLC/Q-TOF-MS was applied to establish a rapid identification method for the analysis of chemical components in leaves of Chinese and Tibetan sea buckthorn with a good separation of all the ingredient and optimizing MS conditions for stable components cleavage fragments.2.UPLC-LTQ-Orbitrap-MS was adopted to fully analyze and achieve enough components of the two kinds of sea buckthorn leaves,then the metabonomics technology was applied to set the dimension data using PC A and PLS-DA,obtain the typical classification of two kinds of sea buckthorn leaves,and through the PLS-DA identification of the characteristics of the differences in two kinds of sea buckthorn leaves.3.Reflux was used to extract Sea buckthorn samples,and HPLC was applied to establish a method for quantitative determination of flavonoid glycosides in all.The difference of the contents was analyzed.4.H2O2-induced PC 12 cell oxidative damage model was established by analyzing the effects of H2O2 on the survival of PC 12 cells under different concentrations and time periods.The protective effects of TFH and different polarity of leaves of two kinds of sea buckthorn on oxidized PC 12 cells were analyzed.Results and conclusion1.Through the analysis of mass spectrometry data and with the help of literature data,a total of 35 compounds were detected and most of them were flavonoids.Among these compounds,29 were the common components of the two species,2 components were unique to Chinese sea buckthorn and 4 were characteristic components of Tibetan sea buckthorn.The results indicated that the compositions of the two kinds of sea buckthorn leaves were quite similar and the UPLC/Q-TOF-MS method could be applied to rapidly and effectively analyze and speculate the compounds in two kinds of sea buckthorn leaves.For the flavonoid nucleus,RDA cleavage and the loss of some small molecular fragments such as CO,CHO,C3O2 and CO2,mainly occurred.In negative ion mode,the glycosylation position of flavonol 3-0-glycosides and flavonol 7-O-glycosides were differentiated and determined through investigating the fragmentation behavior of[M-H]-ions resulting from the different glycosylation position.The radical aglycone ion[Y0-H]-· is more pronounce for flavonol 3-O-glycosides.For the flavonol 3,7-di-O-glycosides,the[Y30-H]-· ion is formed by the hemolysis cleavage of 3-O glycosidic bond and the[Y0-2H]-· ion formed with the elimination of 3-O and 7-O glycosidic bonds.The relative quantitative information of 35 chemical constituents in the leaves of two kinds of sea buckthorn were obtained by Masslynx 4.1.The results showed that the contents of main components of the Chinese sea buckthorn leaves were about 1.5 times as high as that of Tibetan sea buckthorn.In addition,relative to the main components,the contents of 6 specific components were rather low.2.Using UPLC-LTQ-Orbitrap-MS analysis technique,41 components were identified and elucidated based on molecular ion peak,retention time and cracked fragment information.The metabonomics technology was applied to set the dimension data using PCA and PLS-DA,and the PC A analysis model R2X=69.2%,Q2 = 53.6%,PLS-DA analysis model R2Y=99.9%,Q2 = 98.5%,which successfully got the typical classification and constructed the distinction model of two kinds of sea buckthorn leaves.In addition,the ingredients linked with VIP value>1 in the PLS-DA model and p<0.05 in the paired sample t-test as the differences of the two kinds of leaves and a total of 10 differential components were obtained by statistical analysis.3.Based on the HPLC method,the determination method of flavonoid glycoside in the leaves of Chinese and Tibetan sea buckthorn of different origins was established.The results showed that there were significant differences in the content of flavonoids between two kinds sea buckthorn leaves.The content of flavonoids in Chinese sea buckthorn leaves was 1.13%,while in Tibetan sea buckthorn leaves varied greatly from 0.6%to 1.4%,suggesting that the results may be related to their original habitats.Tibetan sea buckthorn is often distributed in the alpine region above 5000 meters.However,the temperature,light,altitude and ultraviolet radiation of the Tibetan sea buckthorn samples collected in this experiment are quite different from their original habitats,which have a great influence on the synthesis and accumulation of flavonoids.4.The effects of H2O2 on the survival rate of PC 12 cells under different concentrations and time were analyzed.The results showed that H2O2 could impair PC 12 cells and decrease the survival rate in a time and dose dependent manner.Compared with the control group,when PC 12 cells were treated with 300?M H2O2 for 4 h,the cell viability decreased to 54.67%,indicating that the model was successful.The protective effects of TFH and different polarity parts of two kinds of sea buckthorn leaves on oxidized PC 12 cells were analyzed.The results indicated that different polarity components could inhibit the decrease of cell viability induced by oxidative damage of H2O2 in different degree.The activity order was as follows:n-butanol fraction<ethanol fraction<ethyl acetate fraction,and the activity of ethyl acetate fraction of Chinese sea buckthorn was obviously higher than that of other polarities.The content of total flavonoids in polar parts showed that the oxidative stress activity of sea buckthorn was positively correlated with them.It could significantly improve the survival rate of oxidative damage cells when the content of TFH was increased to 29.63%.Compared with the model group,0.4,0.25 mg/ml TFH protection group could significantly increased A570 OD value and the survival rate of PC12 cells,and the protection rate was 35.87%?p<0.05?.