Study On The Pharmacophylogenetic Significance Of The Chloranthaceae In China.

Chloranthaceae is a small family of four genera,i.e.,Sarcandra,Chloranthus, Hedyosmum and Ascarina,and ca.70 species occurring in tropical and subtropical areas. Three Genera and 16 species are distributed in the southern part of China.The whole plants or roots of plants in this family are widely used in folk medicine as antispasmodic, antiseptic,and anticancer drugs,or for relieving ache,boils,and dermatopathia in eastern Asia.Reviewed the previous studies of the family Chloranthaceae including distribution, traditional usage,chemical constituents and bioactivities,we choose the family of Chloranthaceae as an objective of our pharmacophylogenetic investigation.S. hainanensis,a shrub distributing only in the south of China,is widely used to invigorate blood circulation and eliminate blood-stasis as traditional Chinese medicine.However,to the best of our knowledge,there was no phytochemical and pharmacological report about S.hainanensis.So we initiated an investigation on the chemical constituents and bioactivities of S.hainanensis with the purpose to study the chemical constituents and differentiate S.hainanensis from S.glabra and better understand the chemotaxonomic relationship of the family of Chloranthaceae.Kinds of chromatographic methods were employed to isolate and purify compounds from Sarcandra hainanensis.43 Compounds were isolated from the 95%ethanol extract of S.hainanensis.The structures of 36 compounds were identified by combination of spectroscopic strategies,including 18 flavanoid and derivates,12 organic acids and derivates,2 anthraquinones,1 coumarin,1 triterpenoids and 2 sterols.All the compounds were isolated from the title plant for the first time.6 compounds of them were new compounds,20 were isolated from genus Sarcandra for the first time and 18 were reported from the family of Chloranthaceae for the first time. The six new compounds were named as sarcandrones A - F(15-18,5,6).Structures of the known compounds were identified as 2',3'-hydroxy-4',6'-dimethoxychalcone(1), 2'-hydroxy-4',5'-dimethoxychalcone(2),cardamonin(3),boesenbergin B(4),7-hydroxy -5,6-dimethoxyflavanone(7),7-hydroxy-5-methoxyflavanone(8),7-hydroxy-5,8 -dimethoxyflavanone(9),naringenin-4',7-dimethylether(10),pinostrobin(11), 3,4',5,7-tetra-hydroxyflavanone-3-O-glucoside(12),kaempferol(13),kaempferol 3-O-glucoside(14),palmitic acid(22),icosanoic acid(23),ctadecanoic acid(24), (E)-3-hydroxy-4-methoxycinnamal-dehydeacrylaldehyde(25),2,6-dihydroxy -3,5-dimethoxy-benzaldehyde(26),caffeic acid(27),rosmarinate acid(28),methyl rosmarinate(29),ethyl rosmarinate(30),3,4-dihydroxyl-phenyl-ethanol(31), (3,4-dihydroxyl-phenyl)-ethanol-O-galactoside(32)(3,4,6-trihydroxyl-phenyl)-ethanol -O-galactoside(33),acidchrysophanol(19),emodin(20),isofraxidin(21), 9,19-Cycloarta-24-en-3-ol(34),13-sitosterol(35) and daucosterin(36).A proper chromatographic pattern for S.hainanensis based on high performance liquid chromatography coupled with diode-array detector(HPLC-DAD) was developed for the first time.Four main characteristic constituents of S.hainanensis were assayed as 2'-hydroxy-4',5'-dimethoxychalcone(2) 1.2555 mg/g,7-hydroxy-5,6-dimethoxy -flavanone(7) 0.4687 mg/g,isofraxidin(21) 0.2248 mg/g,rosmarinate acid(28) 0.5290 mg/g,respectively.The chromatographic pattern was also employed for screening the four constitunents in the 13 species of Chloranthaceae.The amounts of the constituents exited in different plants were assayed,respectively.A MRM analytical method with LC-MS was established for screening the new biflavanoids(15-17) with trace amounts in the plants of Chloranthaceae.The result provided scientific evidences to the differnences between the constituents of S. hainanensis and that of S.glabra.Characteristic compounds(2,3,7,15,21) isolated from S.hainanensis were detected for their bioactivities against non-small lungcancer cell lines A549,H322M,H157,H358, Hop62,H1792 and breast carcinoma cell lines T47D,MCF7.The chalcones(2,3) and biflavanoids(15) tested could significantly inhibit the survival of the above cell lines. Compounds 15 and 16 were assayed for their HIV-1 integrase inhibition activities with a microplate screening method using magnetic beads.They showed weak activities for the integrase strand transfer reaction.The different portions of S.hainanensis as well as the water and MeOH extracts of 8 different plants of Chloranthaceae were assayed for their antibacterial activity.A Gram-positive bacteria(Staphylococcus aureus) and Gram-negative bacteria(Bacillus dysenteriae,Bacillus aeruginosus and Bacillus coli) were used.In the four different portions of S.hainanensis,EtOAc and H₂O portions were active to Staphylococcus aureus. Compared the two kinds of extrats,the MeOH extracts were more active than the H₂O ones.Among the MeOH of the 8 plants tested,Chloranthus fortunei was active to the four bacterias,S.hainanensis was active to S.aureus and Bacillus dysenteriae,S.glabra was active Staphylococcus aureus and C.serratus was active to Bacillus aeruginosus.The results of this thesis may support the view of S.hainanensis as an independent species since its chemical profile was different from that of S.glabra.The standards with different contents in the plants of Chloranthus were determined while almost no standard was detected in the only spices of Hedyosmum.The chemotaxonomic relationship between the three genera of Chloranthaceae in China may be deduced as the species of Chloranthus having a close affinity with Sarcandra and Hedyosmum being the sister group of Sarcandra and Chloranthus according to the characteristics of chemical constituents.In the view of the medical usage,S.hainanensis was not suitable to be used as a succedaneum of S.glabra.However,the special biological activities of S.hainanensis suggested it may be potential as a natural source of antitumor compounds.Since the species of Chloranthus were different from each other by their chemical constituents, they should be used carefully when used as folk medicine.The plants of genus Hedyosmum in China may not have the same action as S.glabra and S.hainanensis.