Studies On The Constituents From Water Extract Of Alismatis Rhizoma And Their Differences Of Origins

Alismatis Rhizoma(AR),known as "Zexie",has been used as a traditional medicine to treat dysuria,edema,obesity,diabetes,hyperlipidemia and hypertension in China for thousands of years.Water decoction was mostly used in traditional Chinese clinical treatment.However,most of the known bioactive constituents in AR were lipophilic and seldom detected in water decoction.The main bioactive constituents in it were still unknown.AR was called "Jian Zexie","Chuan Zexie.and"Guang Zexie" according to different origins.The controversy over species between Jian Zexie and Chuan Zexie has a long story,but there was no complete comparison up to now.According to the questions mentioned above,the main research contents of this thesis were listed as follows:First,the chemical compositions as well as their proportions in water extract were analyzed.The water extract was precipitated by 75%ethanol,of which the precipitate was 28%in water extract and the main constituents of it were polysaccharides.The remainder was concentrated and diluted by water,and adsorbed by macroporous resin overnight,then eluted by water and different concentrations of ethanol.These eluent were analyzed by UPLC-MS and HPLC.The water-eluted contained fructose,glucose and saccharose and the weight ratio of it was 60%.Nucleosides,nucleobase,amino acids as well as some unknown compounds were detected in 20%ethanol-eluted and the weight ratio of this part was 4.8%.The main compositions of 50%ethanol-eluted were sesquiterpenes and the weight ratio of it was 3.7%.The main compositions of 70%and 95%ethanol-eluted were triterpenoids and sesquiterpenes as well as some phenylpropanoids and nitrogen compounds,and the weight ratio of it was 2.7%.These portions showed significant hypolipidemic effects and could significantly decrease the content of TG in liver of hyperlipidemic rats.Different portions acted different characteristics in fat index,liver index,the content of LDL-C in serum and liver,as well as the content of HDL-C in serum.Second,the chemical compounds in 50%ethanol-eluted were studied.The total of fourty-one compounds were isolated from it by VLC,Silica gel,Sephadex LH-20,ODS,as well as preparative HPLC and recrystallization.Their structures of were elucidated by a combination of 1D and 2D NMR,HRESIMS spectroscopic data and electronic circular dichroism(ECD),calculated ECD or MO2(OAc)4-induced ECD methods.These compounds included twenty-four sesquiterpenes,two protostane-type triterpenoids,two diphenylpropanoid ethers,one 2,4,6-cycloheptatrien ketone skeleton compound,six phenolic acid,one cyclopeptide as well as five other ones,of which there were eighteen new compounds,two new natural products,and twelve compounds were isolated from Alismatis Rhizoma for the first time.Besides,eleven pairs of enantiomers were found and seperated by chiral HPLC chromatographic column.In addition,anti-inflammatory effects of some isolated compounds were evaluated by LPS-induced NO production in RAW 264.7 cells.Two protostane-type triterpenoids,compounds ZX-14 and ZX-15,showed significant anti-inflammatory effects with the IC50 values of 39.26 ± 2.75 and 63.87 ± 1,95 μM,respectively.Compounds ZX-14 and ZX-15 could also significantly inhibit CuSO4-induced migration of macrophages toward to neuromast at the concentrations of 10 and 20 μM in zebrafish model,and their potent anti-inflammatory effects were further confirmed.Third,the differences of AR from different origins in plant phytomorph,morphological characters,DNA barcoding,chemical compositions and hypolipidemic effects were compared.According to the descriptions in Flora of China,the plant phytomorph of Chuan Zexie was more similar with Alisma plantago-aquatica and Jian Zexie was more similar with Alisma orientale.One nucleobase was different between the leaves of Chuan Zexie and Jian Zexie in ITS2 sequence.Accordingly,Chuan Zexie was identified as A.plantago-aquatica and Jian Zexie was identified as A.orientale.As to the chemical compositions,the contents of polysaccharides of AR from different origins were in descent order of Guang Zexie(23.93～46.99%),Chuan Zexie(8.51～14.51%)and Jian Zexie(5.58～13.25%),and there were significant differences among them.Chuan Zexie and Jian Zexie were gathered in a group in HCA in light of the content of nucleosides,whereas Guang Zexie were gathered in another group,which meant Chuan Zexie and Jian Zexie were very similar in nucleosides and they were both different from Guang Zexie.The content of five nucleosides(vernine,cytidine,uridine,adenine and adenosine)in Chuan Zexie and Jian Zexie were 0.81～1.30 mg·g-1,in Guang Zexie were 0.35～0.50 mg·g-1,and the content of five nucleosides in Chuan Zexie were higher than Jian Zexie,except adenine.About triterpenoids,Chuan Zexie,Jian Zexie and Guang Zexie were gathered into three groups in HCA,indicating significant differences among them.The peak areas of Alisol B and Alisol B 23-acetate in Guang Zexie were significantly lower and the peak areas of Alisol A were significantly higher than that in Chuan Zexie and Jian Zexie.Besides,the peak areas of Alisol A,B and C in Chuan Zexie were significantly higher than those in Jian Zexie and the differences in the peak areas of Alisol B 23-acetate between them were not remarkable.In terms of hypolipidemic effects,the water and ethanol extract of Chuan Zexie and Jian Zexie both showed therapeutical effects in reduction the content of TG in liver,fat index,the content of LDL-C in serum and liver as well as raise the content of HDL-C in serum.As a whole,Jian Zexie acted better than Chuan Zexie,but with no significant differences.In sum,the chemical compositions of different proportions of water decoction and their weight ratios were revealed,which provided important references for the further study of effective components and quality evaluation of AR.Some new compounds including multiple chemical types were isolated from the water extract,which enriched the pharmacodynamic substances of AR.The differences of AR from different origins were revealed in species,chemical compositions and hypolipidemic effects and the results provided important scientific foundation for revision the Chinese pharmacopoeia and the instruction clinical medication.