Study On Chemical Constituents And Pharmacological Activities Of Merrillanthus Hainanensis

Merillanthus hainanensis Chun et Tsiang belongs Merrillanthus of the family Asclepiaceae,which has a narrow distribution in China.According to the specimen records,it is only distributed in Guangdong,Hainan and Yunnan provinces of China.It grows in low to mid-altitude in the forest valleys and belongs to the second-level protected species.This species was first discovered in Baisha County,Hainan Province in 1941 by the famous taxonomists,i.e.Professor Chen Huanyong and Professor Jiang Ying.M.hainanensis not only has important scientific value in the plant classification of Asclepiaceae family,but also shows broad ornamental and medicinal values.Although the discovery of M.hainanensis is more than 80 years,there is no systematic study on it except for Hou Jun et al.study on the tissue culture and plant regeneration.The chemical components and medicinal value of M.hainanensis is still unknown.From the perspective of biodiversity,the existence of each species plays an important role in the ecological environment.As a rare vine,it is of great significance to protect its population and develop its value.In this thesis,we compared the DNA sequences and HPLC fingerprints of the cultivated and wild-type of M.hainanensis.We successfully obtained the ITS sequences of cultivated and wild-types M.hainanensis.The sequencing results showed that the cultivated and wild-types of M.hainanensis have the same ITS sequences,the highest similarity to this sequence is the ITS sequence of Tylophora,up to 96.7%.Based on the ITS sequences,a phylogenetic tree was constructed using the Neighbor-Joining method.The results showed that M.hainanensis and Tylophora tenuis had close phylogenetic relationship and was far from Myriopteron extensum and Gymnanthera oblonga.Through a comparision of the HPLC fingerprints of the cultivated and wildtype of M.hainanensis,we found that they were consistent to each other,and both contained 12 common chromatographic peaks.To study the chemical constituents of M.hainanensis,the 95 % Et OH extract of the plant was systematically investigated,and 20 compounds were obtained through various chromatography methods.Their structures were elucidated on the basis of NMR,HR-ESI-MS,UV and IR analysis and chemical techniques.These compounds were identified as 3-O-[α-L-cymaropyranosoyl-(1→4)-β-D-digitoxopyranosoyl-(1→4)-β-D-oleandropyranosoyl] amplexicogenin A(TFT-1),3-O-[α-L-oleandropyranosoyl-(1→4)-β-D-digitoxopyranosoyl-(1→4)-β-D-oleandropyranosoyl] amplexicogenin A(TFT-2),Amplexicoside G(TFT-3),Atratcynoside F(TFT-4),Avellanedae A(TFT-5),Urs-12-en-3β-O-9E,12E-octadecadienoate(TFT-6),3,4-seco-lup-20(29)-en-3-oic acid(TFT-7),β-sitosterol(TFT-8),Ferulic acid(TFT-9),Dibutyl phthalate(TFT-10),phydroxybenzaldehyde(TFT-11),Dihydroactinidiolide(TFT-12),Tocopherol quinone(TFT-13),α-linolenic acid(TFT-14),9(S)-hydroxy-10 E,12Z-octadecadienoic acid(TFT-15),1-[(12E,16E)-12,16-eicosadienoyl]-2-[(E,E)-7,11-octadecadienoyl]-3-stearoylglycerol(TFT-16),Chlorophyllide a(TFT-17),Ethoxyphaeophorbide a(TFT-18),3-indole formaldehyde(TFT-19),Cinnamic acid(TFT-20).Compounds TFT-1and TFT-2 were new compounds,and all others compounds were isolated from M.hainanensis for the first time.At the same time,the chemical constituents of petroleum ether extracts from the stems,flowers and leaves of M.hainanensis were investigated by gas chromatography-mass spectrometry(GC-MS).The results showed that the three extracts had the same main components,including β-amyrenyl acetate and 3α-oleanen-12-acetate.On the basis of chemical composition study,some compounds were submitted to cytotoxic,antimicrobial and anti-inflammation activities.The results showed that all compounds had weak inhibitory activity against Hep G2,A549,He La and MCF-7 tumor cells,and they were more sensitive to MCF-7 than other cells.The antibacterial activity of each compound was studied and it was found that these compounds were more sensitive to Escherichia coli than Staphylococcus aureus.At the same time,we found that the stem,flower and leaves of M.hainanensis showed certain antibacterial activity against both Escherichia coli and Staphylococcus aureus,and all three parts were more sensitive to Escherichia coli.All the fractions and compounds were tested for the antiinflammation activity.It was found that the ethyl acetate extract,compounds TFT-5and TFT-9 had certain anti-inflammatory activity,while other extracts and compounds had no obvious anti-inflammatory activity.In addition,we developed a HPLC method for the content determination of Amplexicoside G(TFT-3)in M.hainanensis for the first time.The content of TFT-3in 12 batches of M.hainanensis samples in different seasons in Guangdong area was determined.The content ranged from 4.31～14.79 μg/m L,which indicated that there were some differences due to the different collection time of M.hainanensis.The content of compound TFT-3 collected in March was significantly higher than those collected in autumn.In addition,we also compared the stems and leaves of this plant,and found that compound TFT-3 showed much higher concentration in leaves.Our study on the content determination of compound TFT-3 would provide a scientific reference for the quality control of M.hainanensis.