STUDIES ON THE BIOACTIVE CONSTITUENTS OF Curculigo Orchioides Gaertn.&Callicarpa Macrophylla Vahl

(Ⅰ)Curculigo orchioides Gaertn.,a traditional Chinese medicine known as "Xian-Mao" in China,belongs to the Curculigo genus of Amaryllidaceae family,widely distributed in Sichuan,Guizhou,Yunnan and Guangxi province of China and grows at an altitude of 1600 meters of the grassland under the bushes and leafy glades.The efficacy of Rhizoma Curculiginis are dissipate carbuncle swelling,invigorate yang of kidney,strong physique,dissipate cold-dampness,benefit essence and blood and so on.The chemical constituents of the Curculigo genus have been studied,and cycloartane triterpenes,norneolignans and their glycosides,phenolic glycosides,phenolic compounds,chlorophenolic glucosides and alkaloids are regarded as their main ingredients.In recent years,immune regulation,anti-oxidation,anti-fatigue,anti-aging and antiosteoporotic activity of Curculigo genus had been reported.From the 70%ethanol extract of the dried rhizomes of Curculigo orchioides Gaertn.,35 compounds were isolated using solvent extraction,silica gel c.c.,ODS c.c.,Sephadex LH-20,pTLC,pHPLC and recrystallization ect..On the basis of their physico-chemical properties and spectroscopic analysis,thirty compounds have been identified as curculigine E(CO-1),curculigine F(CO-2),curculigine G(CO-3),orcinoside H(CO-4),curculigine K(CO-5),curculigine L(CO-6),curculigine H(CO-7),curculigine I(CO-8),orcinol glucoside(CO-9),orcinol glucoside B(CO-10),curculigine J(CO-11),orcinol-1-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside(CO-12),anacardoside(CO-13),curculigoside I(CO-14),curculigoside(CO-15),curculigoside B(CO-16),curculigoside C(CO-17),curculigoside G(CO-18),glucosyringic acid(CO-19),benzyl-O-β-D-glucopyranoside(CO-20),breviscapin C(CO-21),p-Hydroxy-cinnamic acid(CO-22),2,6-dimethyoxybenzoic acid(CO-23),3-methoxy-4-hydroxy benzoic acid(CO-24),3,5-dimethoxy-4-hydroxy benzoic acid(CO-25),3,4-dihydroxy-benzoic acid(CO-26),3,5-dihydroxy-4-methoxy benzoic acid(CO-27),5-hydroxymethylfurfural(CO-28),levoglucosan(CO-29),daucosterol(CO-30).Among them,compound CO-1～8,CO-11 and CO-14 were new compounds.Compound CO-20,CO-22,CO-27,CO-29 were obtained from the Amaryllidaceae family for the first time.The proliferation activity of promoting osteoblastic bone formation of the 20 phenolic glycosides from Curculigo orchioides Gaertn.were tested in vitro by means of MTT and their structure-activity relationships were discussed.It was found that chlorophenolic glucosides had better proliferation activity of promoting osteoblastic bone formation than phenolic glycosides obviously,and chlorophenolic glucosides(CO-1～3、5～7)showed certain activity against MC3T3-E1 cell line.Among the curculigosides series of compounds,there are no compounds showed the proliferation activity of promoting osteoblastic bone formation against MC3T3-E1 cell line,except CO-17 with certain activity.Thus,the influence factors of proliferation activity of promoting osteoblastic bone formation are the number of chlorine atoms and phenolic hydroxyl and their replace positions.Through consulting great deal of Curculigo orchioides Gaertn.literature,we make a review on the chemical constituents,pharmacological activity,clinical application,content determination method,pharmacognostic identification and processing progress of Curculigo orchioides Gaertn..(Ⅱ)Callicarpa macrophylla Vahl.belongs to the Callicarpa L.genus of Verbenaceae family,a kind of folk medicine in Yunnan,the root,the stem,and the leave of which were all used in medicine,widely distributed in Guangdong、Guangxi、Guizhou、Yunnan provinces of China and grow at an altitude of 110-2000 meters of the hill road,sparse forests or thickets.Callicarpa macrophylla Vahl.is a bitter taste,slightly acrid and flat,with the actions to eliminate stasis to activate blood circulation and to stop bleeding,also had the actions of detumescence and analgesia.From the 95%ethanol extract of all the grass of Callicarpa macrophylla Vahl.,compounds were isolated using solvent extraction,silica gel c.c.,ODS c.c.,Sephadex LH-20,pHPLC,and recrystallization etc..On the bases of IR,UV,MS,NMR,CD spectra and physico-chemical evidence,the structures of the 34 compounds were identified as diterpenoids,flavonoids and other compounds.The structures of diterpenoids were identified as callicapene M1(CM-1),callicapene M2(CM-2),14α,18-dihydroxy-7,15-isopimaradiene(CM-3),isopimaradien-3β,18-diol(CM-4),14α-hydroxyisopimaric acid(CM-5),7α-hydroxysandaracopimaric acid(CM-6),8(14),15-sandaracopimaradiene-7α,18-diol(CM-7),callicarpol M1(CM-8),16α,17-dihydro xy-phyllocladane(CM-9),calliterpenone 17-acetate(CM-10),hoffmanniaketone(CM-11),calliterpenone M2(CM-12),callicapene M3(CM-13),callicapene M4(CM-14),callicapene M5(CM-15),13-dien-16,15-olide(CM-16),callicapoic acid M1(CM-17),callicapoic acid M2(CM-18),callicarpol M3(CM-19),labda-8(17),13(Z)-diene-15,16-diol(CM-20),17-hydroxy-13-methyl-labda-7,13Z-diene-15-oic acid(CM-21),6-hydroxynidorellol(CM-22),callicapoic acid M3(CM-23),callicapoic acid M4(CM-24).The isolated flavonoids are 5-hydroxy-3 ’,4’,3,7-tetramethoxyflavone(CM-26),3,5-dihydroxy-3’,4’,7-trimethoxyflavone(CM-27),5,7-dihydroxy-3’,4’,3-trimethoxyflavone(CM-28),3’,4’,5-trihydroxy-3,7-dimethoxyflavone(CM-29),4’,5-dihydroxy-7-methoxy flavone(CM-30),3,5,7-trihydroxy-4’-methoxyflavone(CM-31),Naringenin(CM-32),5,4’dihydroxy-7,3 ’-dimethoxyflavone(CM-33)。Other compounds:asperglaucide(CM-25),β-sitosterol(CM-34),daucosterol(CM-35).Compounds CM-1,2,8,10,12-15,17-19,23,24 are new compounds.Compounds CM-3,4,6,7,11,16,20-22,25,27-29,30 were isolated from the family Verbenaceae for the first time.Compounds CM-5,30,32 were isolated from the genus Callicarpa L for the first time.The inhibitory activities on nitric oxide production of 24 diterpenoids from Callicarpa macrophylla Vahl.were tested in vitro by means of MTT and their structure-activity relationships were discussed.The results showed that labdane-diterpenes CM-16、17、19 and isopimarane-diterpenes CM-6、7 had strong inhibitory activity on nitric oxide production against RAW264.7 cell line,the rest were weak or no activity.Thus,the influence factors of labdane-diterpenes inhibitory activity may be the connection of unsaturated lactone ring and the number of double bonds and their replace positions in the side chain.However,the influence factors of isopimarane-diterpenes inhibitory activity may be double bonds,hydroxyl and their replace positions.The AChE,BuChE inhibition activities of 24 diterpenoids from Callicarpa macrophylla Vahl.were tested in vitro by means of Ellman.The results showed that labdane-diterpenes CM-16、21 had strong AChE and BuChE inhibition activity compared with other compounds,phyllocradane-diterpene CM-10 had moderate activities.Through consulting great d’eal of literature,we make a review on the chemical constituents,pharmacological activity,pharmacognostic identification and clinical application.