Studies On Extraction And Isolation Of Chemical Constituents From Forsythia Suspensa Leaves And Their Antioxidant And Anti-tumor Effects

Forsythia is the fruit of Forsythia suspensa(Thunb.)Vahl,a plant of Oleaceae.It has the effect of clearing away heat and detoxification,detumescence and dispersing knot.It mainly treats warm heat,erysipelas,macular rash,carbuncle swollen toxin,dyspnea,and urine gonorrhea.As the non-medicinal part of Forsythia suspensa,Forsythia suspensa leaves are rich in resources,and the contents of triterpenoid acids such as ursolic acid,phenylethanol glycosides and lignans such as phillyrin glycosides in Forsythia suspensa leaves are significantly higher than those in fruits.However,as non-medicinal parts,Forsythia suspensa leaves are mostly abandoned,resulting in a serious waste of resources.In order to further develop the medicinal and feeding value of Forsythia suspensa leaves,the chemical components of Forsythia suspensa leaves were extracted and separated,and their antioxidant and anti-tumor activities were screened and studied.in order to provide experimental and theoretical basis for the application and development of forsythia leaves in animal husbandry and veterinary clinic.Part one: Extraction and isolation of Chemical constituents from Forsythia suspensa leavesThe chemical constituents of Forsythia suspensa leaves were extracted,separated and purified by D101 macroporous resin,silica gel column chromatography and semi-preparative high performance liquid chromatography(HPLC).Ten compounds were obtained and their structures were identified by mass spectrometry(MS)and nuclear magnetic resonance(NMR).Ten compounds were identified as forsythoside I(FSI,1),forsythoside H(FSH,2),forsythoside A(FSA,3),forsythoside B(FSB,4),phillyrin(PHI,5),pinoresinol-β-D-glucoside(PIN,6),phillygenin(PHG,7),betulinic acid(BA,8),ursolic acid(UA,9)and oleanolic acid(OA,10).At the same time,the eluted parts of Forsythia suspensa leaves with different polar solvents were obtained by water extraction and macroporous resin,and the contents of the above components in different polar parts of Forsythia suspensa leaves were determined.the results showed that the main components in 30% ethanol elution parts were phenylethanosides such as phillyrin A,and the main components in 60% ethanol elution parts were lignans such as phillyrin.The main components in 90% ethanol are triterpenoids such as ursolic acid.Part two: Study on Antioxidant effect of Chemical constituents of Forsythia suspensa leavesThe antioxidant activities of 10 chemical components extracted from Forsythia suspensa leaves were screened by chemical method and RAW264.7 cell model induced by hydrogen peroxide(H2O2).The results showed that FSA,FSB,FSH and FSI had good scavenging ability to DPPH free radical,ABTS free radical and superoxide anion radical,which was significantly better(P < 0.05)than that of PHI,PHG,PIN,UA,OA and BA.At the same time,FSA,FSB,FSH and FSI could significantly increase(P < 0.05)the activities of superoxide dismutase(SOD)and catalase(CAT)in H2O2-induced macrophage RAW264.7,increase the content of reduced glutathione(GSH)(P < 0.05),decrease the content of malondialdehyde(MDA)(P < 0.05),and alleviate the oxidative damage induced by H2O2.Through heat map analysis and principal component analysis,it was found that phenylethanol glycosides of FSA,FSB,FSH and FSI 4 were positively correlated with their antioxidant capacity.The results showed that phenylethanol glycosides in forsythia leaves had good antioxidant activity.Part three: Effects of phenylethanol glycosides of Forsythia suspensa leaves on growth performance and antioxidant capacity of broilersLivestock and poultry are easily affected by a variety of stress factors in the process of breeding,so there is an urgent need to develop safe,effective and stable natural antioxidants.The effect of phenylethanol glycoside(F30)from Forsythia suspensa leaves on the antioxidant capacity of broilers was studied in this paper.One-day-old AA white-feathered broilers were randomly divided into 4 groups with 6replicates in each group.The control group was fed with basic diet,and the experimental group was fed with 125,250 and 500 mg/kg phenylethanol glycoside extract(F30)respectively.The experimental period was 42 days.The results showed that compared with the control group,500 mg/kg F30 could significantly increase the average daily gain(ADG)and decrease the feed-weight ratio(F / G)of 21-day-old and 42-day-old broilers,and the thymus index of 21-day-old broilers and the thymus,spleen and bursa index of 42-day-old broilers were significantly increased by adding250,250 and 500 mg/kg F30.Addition of 500 mg/kg F30 significantly increased the activities of antioxidant enzymes(SOD,CAT,GSH-Px)and decreased the level of MDA in serum and liver of 21-day-old and 42-day-old broilers.To sum up,the addition of forsythia leaf phenylethanol glycoside extract F30 to the basic diet of broilers could improve the growth performance,promote the development of immune organs and improve the antioxidant capacity of broilers,and the effect of adding 500mg/kg was the best.Part four: study on the anti-tumor effect of chemical constituents from Forsythia suspensa leavesThe antitumor activity of chemical constituents from Forsythia suspensa leaves was screened by MTT method.The results showed that UA,OA,BA and triterpene extract of Forsythia suspensa leaves(F90)had good antitumor activity.In order to further explore the anti-tumor effect of chemical constituents from Forsythia suspensa leaves.In this paper,the effect of triterpene extract of Fructus Forsythiae(F90)on breast tumor cells was studied.Apoptosis was detected by Annexin V-FITC/PI double staining,mitochondrial membrane potential(MMP)was detected by rhodamine 123(Rh123)staining,intracellular reactive oxygen species(ROS)was detected by DCFH-DA staining,and F90 uptake and localization in cells were detected by fluorescence confocal microscope.The effect of F90 on the expression of apoptosis-related proteins was detected by Western Bolt.The results showed that the triterpene extract of Forsythia suspensa leaves(F90)could significantly inhibit the proliferation of MCF-7 and MDA-MB-231 breast tumor cells,and the results of Annexin V-FITC/PI double staining,Rh123 staining and DCFH-DA staining showed that F90 could induce(P < 0.01)apoptosis of MCF-7 and MDA-MB-231 cells,decrease MMP and increase intracellular ROS content in a concentration-dependent manner(P < 0.01).The results of fluorescence confocal microscope showed that F90 mainly acted on mitochondria and lysosomes in a dose-and time-dependent manner.The results of Western Blot assay showed that F90 could activate Caspase-3 protein and up-regulate the expression of Cleavedcaspase-3,pro-apoptotic protein(BAK),mitochondrial power-associated protein 1(DRP1)and mitochondrial mitoprotein 1(FIS1).To sum up,F90 can inhibit the proliferation of breast tumor cells MCF-7 and MDA-MB-231 and promote tumor cell apoptosis.Its mechanism may be related to reducing MMP,increasing intracellular ROS level,targeting mitochondria and lysosomes,activating caspase-3,and up-regulating the expression of Cleavedcaspase-3,BAK,DRP1 and FIS1.In recent years,the incidence of tumor disease in veterinary clinic,especially in pet clinic,is increasing year by year,and tumor disease has become one of the main reasons that threaten the life of pets.therefore,the results of this study provide a certain experimental basis for the research and development of forsythia leaf as an antineoplastic drug.To sum up,in this study,D101 macroporous resin,silica gel column chromatography and HPLC preparation were used to separate and purify the chemical constituents of Forsythia suspensa leaves,and 10 compounds were obtained.at the same time,different polarity extracts of Forsythia suspensa leaves were obtained by water extraction macroporous resin,and their antioxidant and anti-tumor effects were studied.The results showed that phenylethanol glycosides of FSA,FSB,FSH and FSI4 showed good antioxidant activity,and the addition of phenylethanol glycoside of forsythia leaf in broiler diet could improve the growth performance,immune organ development and antioxidant capacity of broilers,and the effect of adding phenylethanol glycoside of 500mg/kg forsythia leaf was the best.The triterpenoids of UA,OA and BA 3 showed good anti-tumor activity,and the triterpenes of Forsythia suspensa leaves could significantly inhibit the proliferation of breast tumor MCF-7cells and MDA-MB-231 cells,induce and promote apoptosis.Its mechanism may be related to reducing MMP,increasing intracellular ROS level,targeting mitochondria and lysosomes,activating caspase-3,and up-regulating the expression of Cleavedcaspase-3,BAK,DRP1 and FIS1.