Metabolomics Research And Anti-inflammatory Study Of Different Parts Of Hippophae Rhamnoides L.

Sea buckthorn(Hippophae rhamnoides L.)is a kind of hardy deciduous shurb belonging to family Elaeagnaceae,mainly distributed in countries along the Silk Road.China’s sea buckthorn germplasm resources and reserves are the most abundant,and its reserves account for about 90% of the world’s sea buckthorn resources.Sea buckthorn is widely distributed in north and southwest China.It contains plentiful bioactive substances,such as flavonoids,tannins,triterpenoids,sterols,organic acids and volatile oils.In addition,it has a variety of biological activities,such as anti-oxidation,antibacterial and anti-tumor.Moreover,sea buckthorn plays an active role in controlling soil erosion,improving the ecological environment and increasing farmers’ income in China.The berries of sea buckthorn are recorded in Chinese Pharmacopoeia,which are widely used to treat diseases in Tibetan and Mongolian traditional medicines.The berries have the effects of strengthening spleen,promoting digestion,relieving cough,dispersing phlegm,promoting blood circulation and removing blood stasis.It has been reported that the leaves of sea buckthorn have many biological activities,such as anti-oxidation,anti-bacterial,anti-viral,anti-inflammatory,anti-cancer,and anti-obesity.The leaves are vey important raw materials for extracting sea buchthorn flavonoids,and they have been managed as new resource food.Sea buckthorn branches contain triterpenoids with anti-inflammatory and antioxidant activities.In order to further study the chemical constituents and biological activity of sea buckthorn,this project carried out the metabolomics and anti-inflammatory activity study of sea buckthorn berries,leaves and branches by multidisciplinary methodology including phytochemistry,metabolomics and pharmacology.Firstly,the chemical constituents of sea buckthorn berry were isolated and purified by open column chromatography and semi-prepare HPLC.As a result,six compounds were isolated and identified as tamarixetin-3-O-β-D-glucoside-7-O-α-L-rhamnoside(1),isorhamnetin-3-O-β-D-glucoside-7-O-α-L-rhamnoside(2),1-methyl-1,2,3,4-tetrahydro carboline-3-carboxylic acid(3),isorhamnetin-3-O-β-D-sophoroside-7-O-α-L-rhamnoside(4),rutin(5)and isorhamnetin-3-O-β-D-glucoside(6).Among them,compound 3 was obtained from the genus of Hippophae for the first time.Secondly,using the isolated compounds as standards,the metabolomics study of sea buckthorn berries,leaves and branches were carried out by ultra high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry(UHPLC-QTOF-MS)technology.Thirty four compounds were identified,including eleven tannins,and twenty three flavonoids.The discriminative models of sea buckthorn berries,leaves and branches were constructed based on metabolomics data processing technology,such as principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA)and hierarchical cluster analysis(HCA).It was found that the chemical components of sea buckthorn berries,leaves and branches were significantly different,and the similarity of chemical components of sea buckthorn leaves and branches was higher.Twenty four significant markers were found to distinguish these parts based on the PLS-DA model,combining the variable importance in projection(VIP)and the t-test P value.In addition,the inhibition of nitric oxide(NO)production of different parts in macrophage RAW 264.7 cells was carried out.At the concentration of 10 μg/mL,different parts of sea buckthorn extracts showed NO inhibition values from 73% to 98%,indicating that sea buckthorn berries,leaves and branches have good anti-inflammatory activities.The branch,leaf and berry in Dingxi of Gansu province,leaf and berry in Gilgit-Baltistan of Pakistan showed better anti-inflammatory activities.We found that isorhamnetin-3-O-rutinoside,isorhamnetin-3-O-glucoside-7-O-rhamnoside,isorhamnetin-hexoside-rhamnoside and isorhamnetin-3-O-glucoside of the berries,galloyl-HHDP-hexoside,ellagic acid and stachyurin of the leaves,dimeric procyanidin B and epicatechin of the branches were significantly correlated with anti-inflammatory activity via S-loadings plot and VIP in Orthogonal projections to latent structures(OPLS).Moreover,the anti-inflammatory mechanisms of sea buckthorn leaves were preliminary investigated based on network pharmacology.It was predicted that MAPK1,AKT1 and MAPK3 were the key targets of anti-inflammatory effects of sea buckthorn leaves.PI3K-Akt signaling pathway,HIF-1 signaling pathway and TNF signaling pathway were the key pathways of anti-inflammatory effects of sea buckthorn leaves.In conclusion,the metabolomics and anti-inflammatory sudy of different parts of sea buckthorn will enrich people’s understanding of the chemical constituents and pharmacological activities of sea buckthorn,and provide scientific basis for promote further effective comprehensive development and utilization of each parts of sea buckthorn.