Chemical Investigation Of Triterpenoids From The Fruits Of Akebia Trifoliata And Their Anti-Neuroinflammatory,Neuroprotective Activities

Akebia trifoliata,listed in "Chinese Pharmacopoeia" 2015 edition,is the dried fruits of Akebia quinata(Thunb.)Decne.,A.trifoliata(Thunb.)Koidz.var.australis(Diels)Rehd.and A.trifoliata(Thunb.)Koidz.Fructus Akebiae has the effects of relieving liver,helping pain,anti-tumor and diuresis.Previous studies on the chemical constituents of Fructus Akebiae showed that the main constituents were triterpenoids and triterpenoid saponins.In our previous study,we found that the crude extract of Fructus Akebiae showed antidepressant activity in vivo.However,the active antidepressant components and their mechanisms are unclear.Our research mainly focused on the following aspects.The crude extract of the Akebia trifoliata was subjected to column chromatography over ODS,Sephadex LH-20,and silica gel,and semi-preparative high performe liquid chromatography.24 compounds were obtained.Their structures,including 19 triterpenes and 5 phenolic acids,were identified by 1H NMR,13C NMR,and MS.They were identified to be akebia saponin PA(1),hederagenin 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside(2),akebia saponin B(3),akebia saponin E(4),decaisoside B(5),akebia saponin PK(6),akebia saponins C(7),akebia saponins D(8),akebia saponin PD(9),oleanolic acid(10),yuzhizioside Ⅳ(11),akebia saponin PE(12),oleanolic acid 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside(13),arjunolic acid(14),hederagenin 3-O-α-L-arabinopyranosyl-28-O-β-Dglucopyranoside(15),3β-[(α-L-arabinopyranosyl)oxy]-23-oxo-olean-12-en-28-oic acid(16),hederagenin(17),maslinic acid(18),hederagonic acid(19),3-caffeoylquinic acid methyl ester(20),4-caffeoylquinic acid methyl ester(21),3,4-O-dicaffeoyl-1-O-methylquinic acid methyl ester(22),protocatechuic acid(23),and caffeic acid methyl ester(24).Among them,15,16 and 19 were isolated from the Akebia trifoliata for the first time.Their antidepressant activity was evaluated using two classic cell models:(1)Corticosterone-induced PC-12 cell model:The protective effect of compounds on corticosterone-induced PC-12 cell was evaluated by MTT method.(2)LPS-induced BV2 cell neuroinflammation model:The effects of compounds on the expressions of iNOS and COX-2 on LPS stimulated BV2 cells were detected by Western Blot.It was revealed that on the PC-12 cell,14 could increase the survival rate of PC-12 cells.In comparison with the corticosterone group,compound 14 increased the cell survival rate by 16%at a concentration of 25 μM.On the BV2 cell,in comparison with the LPS group,compound 14 inhibited the expression of iNOS(P<0.05)and COX-2(P<0.05).17 derivatives were obtained by structural modification of four different positions of 14(C-11,C-12,C-23,and C-28).The antidepressant activity of the derivatives was evaluated on the two cell models.A-9 exhibited the most significant anti-neuroinflammatory and neuroprotective activities.On the PC-12 cell model,A-9 increased the cell survival rate by 27%at a concentration of 6.25 μM,with high efficiency and low toxicity comparing with precusor 14.On the BV2 cell model,comparing with the LPS group,the derivative A-9 inhibited the expression of iNOS(P<0.001)at a concentration of 25 μM.Based on the above results,the structure-activity relationship was summarized as following:(1)The oxidation at the C-11 to a carbonyl group reduced the activity;(2)The hydroxyl groups at the C-2,C-3,and C-23 simultaneously substituted with acetyl group could maintain the activity;(3)the oxidation at the C-12 to a carbonyl groupincreased the activity;(4)The hydroxyl on C-2,C-3,C-23 isprotected,and the activity was maintained;(5)The C-23 was oxidized to an aldehyde group,and the activity is maintained.In present study,chemical investigation on the fruits of Akebia trifoliata was carried out.A total of 19 triterpenoids and 5 phenolic compounds were obtained.These triterpenoids were evaluated for antidepressant activity,and compound 14 showed significant anti-neuroinflammatory and neuroprotective activities in both cell models.Compound 14 was selected as the lead for further structure modification and activity evaluation.The activity of derivative A-9 is enhanced.