Study On The Constituents Of Illicium Majus Hook.f.et Thoms.

Objective: By studying the chemical constituents from the twigs and leaves of Illicium majus,a series of compounds were isolated with novel structures and better activities,and it provided a scientific basis for the development and application of the Illicium majus.Methods:(1)Separation and identification of chemical constituents: Firstly,The twigs and leaves of Illicium majus were extracted with 85 % ethanol,and then the ethanol extract was separated and purified by using column chromatography over macroporous resin D101,silica gel,polyamide column,MCI,Sephadex LH-20,and flash chromatography coupled with preparative HPLC.Finally,the chemical structures were elucidated by spectroscopic analysis including 1D-NMR(NMR,DEPT),2D-NMR(HSQC,HMBC,COSY,NOESY,ROESY),HRESIMS,X-ray,and physicochemical constants analysis.(2)DPPH radical scavenging assay for antioxidant activity evaluation: The antioxidant activity of the compounds can be judged by evaluating the scavenging ability of the isolated compounds on DPPH free radicals.(3)Evaluation of tyrosinase activity in vitro: The compound solution,tyrosinase solution,and L-dopa solution were mixed and reacted for a period of time using the dopa oxidation method,and then the compounds’ effect on tyrosine was evaluated according to the change in the amount of dopa pigment.(4)Evaluation of antibacterial activity: The inhibitory effects of the compounds on the standard strains S.aureus ATCC 29213 and E.coli ATCC 25922 were evaluated by the micro broth dilution method.(5)Evaluation of cytotoxic activity: The CCK-8 method was used to evaluate the effects of some diterpenoids on three tumor cells(HCT-116,MCF-7,MGC-803).Results and Conclusions:(1)The structures of 64 compounds were identified as 24diterpenoids(1-24),12 lignans(25-36),8 phenethyl alcohols(37-44),2phenylpropanoids(45-46),1 flavonoid glycoside(50),1 monoterpenoid(53),2sesquiterpenoids(54-55),2 steroids(63-64)and 12 other compounds.Among them,these were 15 new compounds,named as Majusane acid A(1),Majusane acid B(2),Majusane acid C(3),Majusane acid D(4),Majusane acid E(5),Majusane acid F(6),Majusane acid G(14),Majusane acid H(15),Majusane acid I(16),Majusanicol A(17),(7R,8S)-4,3′,9-trihydroxyl-3-methoxyl-7,8-dihydro-benzofuran-1′-propylneolignan-9′-O-β-D-gluco-pyranoside(25),(7R,8S)-4,3′,9′-trihydroxyl-3-methoxyl-7,8-dihydrobenzofuran-1′-propylneolignan-9-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside(26),(7R,8S)-4,3′,9′-trihydroxyl-3-methoxyl-7,8-dihydrobenzofuran-1′-propylneolignan-9-O-α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside(27),(7R,8S)-4,3′-dihydroxyl-3-methoxyl-9′-acetyl-7,8-dihydrobenzofuran-1′-propylneolignan-9-O-α-(4′-acetyl)-L-rhamnopyranosyl-(1→2)-β-D-xylopyranoside(28),4-hydroxyphenylethyl 4-O-β-D-glucopyranosyl-(1→3)-O-β-D-glucopyranoside(38).(2)New compounds 25-27 and known compounds 36(Isodunnianol),45(1-Ocaffeoyl-β-D-glucopyranose),46(Soulieana acid 1),60(Protocatechuic acid)showed significant free radical scavenging activity with the clearance rates(%)of 66.01±0.38,53.90±1.61,55.17±1.47,72.25±0.56,84.84±0.29,80.28±2.15,and 78.15±0.43,respectively.Further structure-activity relationship analysis showed that the antioxidant of lignans was related to the number of glycosides and hydroxyl groups,their connection positions.(3)Some diterpenoids had a certain inhibitory effect on tyrosinase,among them,new compound 3 had the best inhibitory effect with the inhibition rate of 20.1±1.29 %;because its structure contained similar structure fragment to the positive control drug kojic acid.Phenyl alcohol compounds had a strong activation effect on tyrosinase,among them,compounds 43(P-hydroxyphenethyl alcohol),45(1-O-caffeoyl-β-Dglucopyranose)and 46(Soulieana acid 1)had the stronger activation effect with the activation rates(%)of 231.4±5.53,229.0±4.34,and 213.6±5.33,respectively;Further structure-activity relationship analysis found that the 4-hydroxyl group was an essential group for the activity of phenethyl alcohol compounds.(4)Compounds 35(Dunnianol)and 36(Macranthol)had the stronger inhibitory effect on S.aureus,with MIC values of 1 μg/mL and 16 μg/mL,respectively;compounds 11(Macrophynin E),17(Majusanicol A),and 18(Isopimara-7,15-dien-19-ol)had moderate strength inhibitory effects on S.aureus,with MIC values of64 μg/mL,64 μg/mL,and 32 μg/mL,respectively.(5)Abietane-type diterpenoids 9(Jiadifenoic acid K)and 11(Macrophynin E)had the better inhibitory effects on the three types of tumor cells,and the survival rates were11 % ～ 21 % under the concentration of 50 μg/mL.