Study On The Chemical Constituents And Biological Activities Of Two Species Of Euphorbia Plants And Two Species Of Sophora Plants

Isolation and screening of active components in medicinal plants is an effective way to search for new drugs.In this paper,the chemical constituents and biological activities of four medicinal plants,Sophora alopecuroides,Sophora flavescens,Euphorbia neriifolia and Euphorbia kansui,were studied systematically.Through a variety of chromatographic separation techniques and methods（such as MCI,ODS,RP-18,silica gel,Sephadex LH-20 gel and semi-preparative HPLC,etc.）,comprehensive utilization of MS,HR-ESI-MS,1D-and 2D-NMR,and X-ray single crystal diffraction and other spectroscopic methods,combined with electronic circular dichroism and computational ¹³C-NMR methods,195 compounds were isolated and identified from the above four medicinal plants.The structure types involved diterpenes,triterpenes,alkaloids and steroids and so on.Among them,47 compounds were new ones and the NMR data of 3compounds were assigned for the first time.In addition,we screened the biological activity of the isolated compounds,including anti-viral,anti-inflammatory and anti-tumor activities,which provided a certain scientific basis for further scientific research.Sophora alopecuroides is a traditional Chinese medicine commonly used with a long history.The whole plant of S.alopecuroides has a bitter taste and a cold nature.Its application parts include roots,stems,whole plants and their seeds,and it has functions such as clearing away heat and detoxification,removing wind dryness and dampness,relieving pain and killing insects,etc..Alkaloids are its important active ingredients,which have various pharmacologicalm effects such as anti-inflammatory,anti-cancer,anti-viral,anti-arrhythmia,and immune regulation.In this study,a total of 49 quinolisidine alkaloids were isolated and identified from the chloroform alkaloid phase of 95%ethanol extract of S.alopecuroides.The types include 37 matrine type,9 spartine type,and 2 cytisine type and one other alkaloid.Including 16 new compounds,the new compounds are named:Sophalode A（1）,Sophalode B（2）,Sophalode C（3）,Sophalode D（4）,Sophalode H（5）,Sophalode I（7）,Sophalode E（12）,Sophalode F（13）,Sophalode G（14）,Sophalode N（15）,Sophalode P（16）,Sophalode M（17）,Sophalode O（28）,Sophalode J（33）,Sophalodes K（34）和Sophalode L（35）.Among them,the absolute configuration of Neosophoramine（6）is determined by comparing its CD spectrum with the ECD of compound Sophalodes H（5）.The structures and absolute configurations of new compounds were elucidated by extensive spectroscopic techniques,X-ray diffraction analysis,and quantum chemical calculation.Compounds 1-4 were the first examples of C-11 oxidized matrine-type alkaloids from Sophora plants.In addition,the NMR data and absolute configuration of compound 18 was reported for the first time.Bioactivity assays showed that 7,34,44 and 48 exhibited the most significant NO inhibitory effects with IC₅₀ values of 18.26³3.30μM,respectively,better than that of（+）-matrine（8）(IC₅₀=38.90μM),the representative alkaloid with anti-inflammation from S.alopecuroides.The anti-inflammatory activity and mechanism of action of the new compound 34 were further evaluated.In Western blot analysis,compound 34 not only significantly inhibited the expression of nitric oxide synthase（i NOS）induced by lipopolysaccharide（LPS）treated macrophages（RAW 264.7）.They also significantly reduced the expression of cyclocxygenase-2（COX-2）,which plays a key role in mediating inflammation and assisting in the production of inflammatory mediators.The expression of cycoxidase-2（COX-2）,which plays a key role in assisting the production of inflammatory mediators.The results showed that compound 34 produced anti-inflammatory activity by inhibiting the expression of i NOS and COX-2.Moreover,compound 26 exhibited marginal cytotoxicity against He La cell lines.As a traditional Chinese medicine,Sophora flavescens has been used for more than 2000years.It mainly uses the root of S.flavescens as medicine,which has the functions of clearing away heat and dampness,removing wind and killing insects,and diuresis.It is used for heat dysentery,blood in the stool,jaundice,urinary obstruction,leucorrhea,vaginal itching,eczema,eczema,skin itching,scabies and leprosy,and external use can also treat trichomonas vaginitis.A total of 19 alkaloids were separated and identified from the chloroform alkaloid phase of the 95%ethanol extract of S.flavescens.The structure types include 13 matrine type,3 cytisine type,and 3 spartine type.Among them,2 new compounds:Sophcence A（1）and Sophcence B（13）.The NMR data of（-）-（35）⁷-Dehydrosophoramine（11）and Oxy-N-methylcytisine（14）were reported for the first time.Activity screening results showed that compound 4 has obvious NO inhibitory activity against LPS-induced RAW 264.7,with IC₅₀value of 22.14μM,Nitric oxide synthase inhibitor（L-NMMA）was used as positive control with IC₅₀ value of 21.80μM.When compound 19 is 20mg/ml,it has a significant inhibitory effect on glioma stem cell GSC-3#.As a kind of Dai medicine,Euphorbia neriifolia has a lot of medicinal value.It is cold in nature,sweet in taste,and toxic.It has the effects of clearing heat and detoxification,reducing swelling and pain,astringing sores and promoting muscles,moistening the intestines and laxatives,relieving cough and relieving asthma.However,there are few researches and reports on the E.neriifolia at home and abroad.In order to further clarify the active ingredients and the mechanism of action of E.neriifolia,and at the same time to find active compounds with novel structures,sixty compounds,were isolated from the stem barks of E.neriifolia.The structure types include 10 rosinane-type diterpenes,16 ent-isopimarane-type diterpenes,1 ent-primrane-type diterpenes,and 2 isopimarane-type diterpenes.14enantiomers-atisanane diterpenes,1 kaureane diterpene,7 ingenane diterpenes,6 sequonane diterpenes and 3 small molecule compounds.Including 21 new compounds:Eupneria A（1）,Eupneria B（2）,Eupneria C（3）,Eupneria D（4）,Eupneria E（5）,Eupneria F（7）,Eupnejca A（10）,Eupneria J（11）,Eupneria K（12）,Eupneria M（13）,Eupneria N（14）,Eupneria O（19）,Ent-isopimara-8（14）,15-dien-3β,7α,12β-triol（20）,Eupneria L（22）,Eupnejca C（23）,Eupnejca D（24）,Eupneria P（25）,Eupnejca E（26）,Eupneria G（30）,Eupneria H（31）,Eupneria I（32）.Compounds 11-13,22 and 26 were the first examples of C18（19）-norditerpenoid with ent-isopimarane skeleton from the E.neriifolia,and their absolute configuration have been determined by X-ray single crystal diffraction or calculated ECD.In addition,compound 30 is the first diterpenoid of 18（19）-nor ent-artisantane type isolated from the E.neriifolia.Activity tracking found that 5 compounds have significant anti-HIV activity,of which compounds 46 have the most significant activities,with EC₅₀ value of0.0028μg/ml,Zidovudine（AZT）was used as positive control with EC₅₀ value of 0.0086μg/ml.Compound 35 had significant inhibitory effect on Hep G2 and Hep G2/ADR cells with IC₅₀ values of 13.70μM and 15.57μM,respectively.And compound 44 have the strongest inhibitory effect on Hep G2 cells with IC₅₀ value of 0.01μM.In the study of the inhibitory activity of NO production,the compounds 47 and 48 have significant IC₅₀ values of 6.37 and5.78μM,respectively,L-NMMA was used as the positive control and the IC₅₀value was21.17μM.Further mechanism studies showed that 47 and 48 showed stronger anti-inflammatory potential by inhibiting LPS-induced pro-inflammatory mediators NO,IL-1β,IL-6 and i NOS in RAW 264.7 cells.In addition,when compound 18 is 10mg/m L,it has obvious protective effect on influenza A virus infection infected by MDCK cells,with IC₅₀value of 3.86μM,and as the positive control,the IC₅₀value of Nucleozin was 0.87+/-0.14μM.The medicinal history of Euphorbia kansui can be traced back to the"Shen Nong’s Materia Medica"2000 years ago.It belongs to the genus Euphorbia and is a unique plant in my country.Traditional Chinese medicine believes that E.kansui is poisonous,cold in nature,bitter in taste,and is attributed to the lung,kidney,and large intestine meridian.In clinic,it is mostly used for malignant tumors such as liver cirrhosis,ascites,pleural effusion,edema,cough,esophagus cancer,breast cancer,polar pancreatic cancer,lung cancer,melanoma,asthma,chronic bronchitis,etc.A total of 67 compounds were separated and identified from the roots of E.kansui,including 20 triterpenoids,10 japiramane diterpenes,and 15 ingenane diterpenes,15 steroids,1 flavonoid and 6 other ingredients.Among them including 8 new compounds and named:Eupokanu A（3）,Eupokanu B（4）,Eupokanu C（9）,Eupokanu D（10）,Eupokanu E（11）,Eupokanu F（21）,Eupokanu G（46）and Eupokanu H（56）.Activity studies have found that both ingenane-type diterpenoids and steroids have better NO inhibitory activity.Among them,ingenane-type compounds 30,33,35-36 and 38 have the greatest effect on the production of NO in RAW 264.7 cells induced by LPS,with IC₅₀ values of 0.55,0.57,0.56,0.30 and 2.98μM,respectively.In addition,steroid compounds 45 and 58 also have strong inhibitory activity,with IC₅₀ values of 9.10 and 1.71μM,respectively,L-NMMA was used as positive control(IC₅₀=21.80μM);16 compounds have anti-Hep G2 activity(IC₅₀=12.55⁷1.23μM).Among them,compounds 10,28 and 33 have the most significant activity,with IC₅₀ values of 18.24,12.55 and 12.61μM,respectively,Adriamycin as positive control(IC₅₀=4.37μM);Compound 33 has significant cytotoxic activity against human breast cancer cell MCF-7 and lung cancer cell A549,with IC₅₀ values of 17.12 and 21.97μM,respectively.And we studied the anti-liver cancer mechanism of compound 33 in vitro,and the experimental results showed that compound 33 could inhibit the survival and proliferation of Hep G2 cells by down-regulating Akt/m TOR,up-regulating PKC-δ,promoting cell apoptosis,and up-regulating the ERK/MAPK pathway,showing a dose-response relationship.In addition,88 g of triterpenes（1）were isolated,which has a significant selective inhibitory effect on glioma tumor stem cells.compound 1 also showed strong cytotoxic activity against lung cancer cell A549,with IC₅₀ value of 25.96μM.