Hepatoprotective Effects Of The Constituents Of Agriophyllum Squarrosum Seed And Xanthoceras Sorbifolia Xylem From Inner Mongolia

As a pathological mechanism,oxidative stress is involved in the occurrences and developments of liver diseases.Using antioxidants is a rational way in aid for preventing and curing oxidative stress-related liver diseases.Natural antioxidants contained in food and medicinal plants have strong antioxidant and free radical scavenging abilities.The main constituents of Agriophyllum squarrosum（A.squarrosum）seed and Xanthoceras sorbifolia（X.sorbifolia）xylem were used as research materials in this research,in order to provide scientific and theoretical bases for the development and utilization of these two plants which grow abundantly in Inner Mongolia.As a Mongolian medicine,A.squarrosum possesses the functions of clearing heat,detoxifying,diuresis etc.The plant seed（Shami in Chinese）which has high nutritional value and a long history as food material was considered by traditional Chinese medicine to be green and natural and has the effect of reducing weight.However,little research had been done to reveal the chemical constituents and bioactivities of A.squarrosum seed.Thus,the secondary metabolites of A.squarrosum seed and their biological activities were studied in this research in the following five aspects:（1）The extracts of A.squarrosum seed coats and kernels were analyzed by ultra high performance liquid chromatography-triple quadrupole mass spectrometry（UPLC-QQQMS）,and the results showed that the coats contained much more secondary metabolites than the kernel did.Therefore,the seed coats were extracted in a larger scale,twelve chemicalconstituents,3-O-[α-L-rhamnopyranosyl-（1→6）-β-D-glucopyranosyl]-7-O-（β-D-glucopyranosyl）-quercetin（1）,quercetin-3-O-β-D-apiosyl（1→2）-[α-L-rhamnosyl（l→6）]-β-D-glucoside（2）,allantoin（3）,rutin（4）,isorhamnetin-3-O-rutinoside（5）,isorhamnetin-3-O-β-D-glucoside（6）,naringenin（7）,protocatechuic acid（8）,p-coumaric acid（9）,chrysoeriol（10）,isoquercitrin（11）,and luteolin-6-C-glucoside（12）were obtained after repeated column chromatography.（2）As most of the components are flavonoids,the contents of total flavonoids and each compound in A.squarrosum seed coats and kernels were determined and compared with 8 conventional grain seeds（millet,wheat,rice,peanut,sesame,flaxseed,soybean,and corn）.It was found that the flavonoid content in A.squarrosum seed coats（335.7μg/g）was only next to soybean（351.2μg/g）and was greater than those in the other7 crop seeds.UPLC-MS quantification revealed that the major constituents in A.squarrosum seed coats were compound 1（440.0μg/g）,4（110.9μg/g）,2（99.6μg/g）,5（93.6μg/g）,and 3（51.5μg/g）,versus isoflavonoids-genistin（16,565.4μg/g）,daidzin（14,401.9μg/g）,and glycitin（18,97.3μg/g）in soybean.（3）DPPH radical scavenging activity showed that compounds 1,2,4,8,11,and 12 exhibited good activity with their EC₅₀ being 15.9,22.2,16.9,4.6,10.8,8.9μg/mL,respectively.These 6 compounds were further tested for their activity on protecting DNA from damaging by peroxy radical and all these compounds showed strong protective effects,and they also could protect HepG2 cells from tert-butyl hydroperoxide（t-BOOH）induced oxidative damage（P<0.05 or P<0.01）,attenuate intracellular ROS generation and increase the expression of Nrf2,pp38,pJNK and Bcl-2（P<0.05 or P<0.01）.（4）Flavonoid-rich antioxidant fraction（AF）had protective effect on CCl₄ induced liver injury in mice.The AF could effectively decrease the levels of alanine transaminase（ALT）and aspartate aminotransferase（AST）in serum（P<0.05 or P<0.01）;reduce the content of malonaldehyde（MDA）（P<0.01）,and increase the level of superoxide dismutase（SOD）（P<0.01）in liver;improve liver histopathological changes,increase the level of Nrf2 and HO-1（P<0.01）.（5）The inhibitory effects of the 12 compounds on human hepatic stellate cell（LX2）were screened.Compound 9 showed the greatest inhibition on LX2 cell proliferation and migration（P<0.01）and down-regulated the expressions of CollagenⅠ,α-SMA and p-Smad2/3（P<0.01）.Xanthoceras sorbifolia Bunge（Sapindaceae）is another dominant plant in Inner Mongolia,and its xylem is a kind characteristic Mongolian medicine called Wenguanmu in Chinese.Earlier studies in our laboratory found that procyanidin A2（A2）was one of the major antioxidant constituents of X.sorbifolia xylem.However,little is known about its antioxidant capability in cells.So,the mechanism of A2 against oxidative damage in L02 cells was determined in this study,and the results are as follows:（1）A2 significantly protected the cells from oxidative damage induced by t-BOOH（P<0.01）,and reduced the lactate dehydrogenase（LDH）release（P<0.01）,cell death and intracellular ROS production（P<0.01）.A2 significantly increased the expressions of HO-1 and NQO1,and promoted the nuclear translocation of Nrf2,increased the expressions of pp38 and pJNK.（2）A2 had the ability to penetrate into cells and part of it converted to its isomer A1,and the cellular concentration of the dimers peaked at 24h.A2 and A1 were detected in mouse serum and liver,30 min after gavaging to mice of A2 at a dosage of 83.3 mg/kg.However,no degradation of the dimers to monomers was observed,as epicatechin or catechain were not found in the cells and mice treated with A2.So,A2exerted the bioactivity in the dimeric form.Through these experiments,we found that both the characteristic grain crop A.squarrosum seed and the medicinal plant X.sorbifolia in Inner Mongolia contain natural antioxidants.They protected cells from oxidative damage through reducing intracellular ROS and activating the transcription factor Nrf2.The experiments revealed the antioxidant and liver protection effect and mechanism of the main compounds in A.squarrosum Seed coat and X.sorbifolia xylem.The results provided scientific bases for better use of these two plants to develop health food or pharmaceutical products,which could in turn promote the cultivation of these plants in desert and north areas of our country.