Textual Research on Origin of Alismatis Rhizoma in China Pharmacopoeia / 中国实验方剂学杂志

This paper origin studies the origin of Alismatis Rhizoma in Chinese pharmacopoeia， and puts forward some suggestions for modification. Through the changes in the records of the source of Alismatis Rhizoma in the various versions of the Chinese Pharmacopoeia and the records of Flora of China and Materia Medica of China，it is found that the source of Alismatis Rhizoma in the Chinese Pharmacopoeia is confused. Specifically， the Chinese name of Alismatis Rhizoma does not correspond to the Latin name. As a common Chinese herbal medicine，Alismatis Rhizoma has a large market circulation. Many classic Chinese medicine prescriptions released by China Food and Drug Administration contain Alismatis Rhizoma. The development of the classic Chinese medicine prescriptions will further increase the market circulation of Alismatis Rhizoma. As a major national move to promote the development of traditional Chinese medicine， the study for classic Chinese medicine prescriptions requires defining the origin of the medicinal materials used，and the confused origin of Alismatis Rhizoma recorded in the Chinese Pharmacopoeia seriously hinder the development of the classics. Therefore，in order to regulate the origin of Alismatis Rhizoma， ensure the clinical efficacy and promote the development of classic Chinese medicine prescriptions，the confused origin of Alismatis Rhizoma in the Chinese Pharmacopoeia has to be resolved as soon as possible. Based on the analysis of the changes of Alismatis Rhizoma's producing areas in the past dynasties， it is found that the producing areas of Alismatis Rhizoma have continuous changed from Wei and Jin dynasties to present， and finally formed the current situation of Sichuan as the main producing area. In comparison of chemical composition，origin and market circulation of Alismatis Rhizoma in Sichuan Province that is the most productive， and Fujian Province that is the best quality， it is found that the two species are different in every aspects. Nowadays，Alisma plantago-aquatica occupies the majority of the market， which doesn't conform to Alisma orientale as specified in the 2015 edition of the Chinese Pharmacopoeia. Therefore， through textual research and analysis， it is suggested that both A. plantago-aquatica and A. orientale. Shall be used as the origin of Alismatis Rhizoma. In the 2015 edition of Chinese Pharmacopoeia，Cassiae Semen，Schizonepetae Herba，Aisaematis Rhizoma，Fibraureae Caulis and Ajugae Herba have the same problem. This paper provides ideas for the revision of sources of traditional Chinese medicine.

Correlation study between accumulation of triterpenoids and expression of relative genes in Alisma orientale / 中国中药杂志

To research the correlation between accumulation of triterpenoids and expression of key enzymes genes in triterpenoid biosynthesis of Alisma orientale,the study utilized UPLC-MS/MS method to detect eight triterpenoids content in the tuber of A. orientale from different growth stages,including alisol A,alisol A 24 acetate,alisol B,alisol B 23 acetate,alisol C 23 acetate,alisol F,alisol F 24 acetate and alisol G,and then the Real time quantitative PCR was used to analyze the expression of key enzymes genes HMGR and FPPS in triterpenoid biosynthesis. Correlation analysis showed that there was a significant positive relation between the total growth of these eight triterpenoids and the average relative expression of HMGR and FPPS(HMGR: r = 0. 998,P<0. 01; FPPS: r = 0. 957,P<0. 05),respectively. Therefore,the study preliminarily determined that HMGR and FPPS genes could regulate the biosynthesis of triterpenoids in A. orientale,which laid a foundation for further research on the biosynthesis and regulation mechanism of triterpenoids in A. orientale.

Prokaryotic expression, function verification and immunoassay activity of farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep / 药学学报

Farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep. (AoFPPS) is considered as one of the important rate-limiting enzymes in the biosynthetic pathway of protostane triterpenes. In order to investigate the expression and function of AoFPPS, the gene (accession No. HQ724508) was cloned into a bacterial expression vector pCzn1, then the combined plasmid pCzn1-AoFPPS was transformed into Escherichia coli BL21, and a fusion protein was obtained after induction. The fusion protein was purified by Ni resin, and the function was verified through in vitro enzymatic reaction. High performance liquid chromatography (HPLC) analysis revealed that the products were able to catalyze the synthesis of farnesyl pyrophosphate (FPP). High purity recombinant protein was used to immunize New Zealand rabbits to generate a polyclonal antibody. The titer of the antibody was determined by enzyme linked immunosorbent assay (ELISA), and Western blot results demonstrated that the antibody could specifically recognize the AoFPPS protein in A. orientale (Sam.) Juzep. So,the method of rapid immunoassay to detect AoFPPS was established. This study lays the foundation for further study of the AoFPPS gene expression, regulation and mechanism of action in A. orientale (Sam.) Juzep., and it also provides a scientific basis on improving the quality of Alismatis Rhizoma using the plant genetic engineering.

Prokaryotic expression, functional identification of squalene synthase in Alisma orientale and its immunoassay study / 中国中药杂志

Squalene synthase of Alisma orientale catalyzes farnesyl diphosphate (FPP) to form squalene, which is the key regulatory enzyme of the carbon source flow to protostane triterpenes biosynthesis. For further research on the function and expression of AoSS gene, the open reading frame (ORF) of squalene synthase gene (accession no. JX866770) from A. orientale was subcloned into a prokaryotic expression vector pCzn1 and induced the expression of AoSS gene in Escherichia coli BL21(Roseta). The fusion protein was mainly in the form of inclusion bodies and purified to obtain high purity protein. By verifying its functionality through vitro enzymatic reaction, the results showed that the catalytic protein had the catalytic activity of FPP into squalene. In order to research the expression of AoSS in A. orientale, the purified protein was used to immunized rabbits to prepare polyclonal antibody which was then purified, the titer of the antibody was greater than 1∶51 200 by ELISA detection, and displayed good specificity by Western blotting. The prepared antibody was used for immunoassay of AoSS in different organs of A. orientale, and the results showed that the AoSS expression level was the highest in tubers, followed by leaves, and lowest in root. Successful construction of prokaryotic expression vector, validation of gene functions and establishment of rapid immunoassay lay the foundation for further researches on the function and regulation of AoSS gene, and also provide scientific basis on the application of the protostane triterpenes of A. orientale in the field of synthetic biology.

Research on effective components compatibility of Alismatis Rhizoma resisting calcium oxalate calculus in vitro based on uniform design / 中草药

Objective: To investigate the inhibitory effect of six active triterenoids and their compatibility from Alisma orientale on the formation of urinary calcium oxalate calculus in vitro. Methods: A uniform design method was used to design the different compatibilities from six triterenoids. The inhibitory effects of the six triterenoids along with their different compatibility groups were evaluated in vitro using a standard seeded crystallization technique. Results: The six active triterenoids and their compatible preparations could significantly inhibit calcium oxalate crystal formation in vitro (P < 0.05), and the group of alisol A-alisol A 24-acetate-alisol B-alisol B 23-acetate-alisol F-alisol F 24-acetate (2.2∶3.8∶1∶3.5∶2.2∶1) was the most efficient with an inhibitory index of 188.29%. Conclusion: The triterenoids in A. orientale play a key role in the inhibition of calcium oxalate calculus formation and compatibility of alisol A, alisol A 24-acetate, alisol B, alisol B 23-acetate, alisol F, and alisol F 24-acetate can inhibit the calcium oxalate calculus well in vitro especially cooperated with each other, and the best compatibility proportion is 2.2∶3.8∶1∶3.5∶2.2∶1, respectively.

Simultaneous determination of four active components in Alisma orientale (Sam.) Juz. by HPLC-DAD using a single reference standard / 药物分析学报

A rapid, simple and practical high-performance liquid chromatography method coupled with diode array detector (HPLC–DAD) was developed to evaluate the quality of Alisma orientale (Sam.) Juz. through a simultaneous determination of four major active triterpenes using a single standard to determine the multi-components (SSDMCs). Alisol B 23-acetate was selected as the reference compound for calculating the relative response factors. All calibration curves showed good linearity (R240.9998) within test ranges. RSDs for intra- and inter-day of four analytes were less than 3.6% and 2.3%; the overall recovery was 92.1–110.2%(SSDMC). The proposed method was successfully applied to quantify the four components in 20 samples from different localities in China. Moreover, significant variations were demonstrated in the content of these compounds. In addition, hierarchical clustering analysis (HCA) and principal components analysis (PCA) were performed to differentiate and classify the samples based on the contents of Alisol C 23-acetate, Alisol A, Alisol A 24-acetate and Alisol B 23-acetate. This simple, rapid, low-cost and reliable HPLC–DAD method using SSDMC is suitable for routine quantitative analysis and quality control of A. orientale (Sam.) Juz.

Effect of Rhizoma Alismatis extracts on oxidative stress induced by cerebral ischemia-reperfusion injury in rats / 中华老年医学杂志

Objective To investigate the effects of Rhizoma Alismatis extracts on oxidative stress induced by cerebral ischemia-reperfusion injury in rats,and to explore its protective mechanism in cerebral ischemia-reperfusion injury.Methods A total of 60 male SD rats were randomly divided into sham operation group,model group,Alisma orientalis group and Nimodipine positive control group (n=15,each).Cerebral ischemia-reperfusion injury model was prepared by suture method after 14 days of intragastric administration.After 24 hours,scores of neurological dysfunction,the infarct size,the water content of the brain,the malondialdehyde (MDA),superoxide dismutase (SOD),nitric oxide (NO) levels in serum and brain tissues,and the activity of inducible NO synthase (iNOS)were detected.Results As compared with the model group,Alisma orientalis group showed that the scores of neurological dysfunction,cerebral water content,cerebral infarction size,contents of MDA and NO,and the activity of iNOS were significantly reduced,and the activity of SOD was significantly increased in respectively [(2.21 ± 0.38) vs.(2.78 ± 0.43),(81.18 ± 2.09)％ vs.(88.33±4.15)％,(0.26±0.07) ％ vs.(0.35±0.04)％,(5.92±1.64) μmol/L vs.(8.21±1.47)μmol/L,(115.48±18.65) mU/L vs.(75.52±20.78) mU/L,(28.23±4.32) μmol/L vs.(41.73±3.85) μmol/L,(15.31±1.68) mU/L vs.(23.49±3.53) mU/L,(5.41±0.68) μmol/L vs.(7.58±1.49) μmol/L,(168.57±10.65) mU/L vs.(150.11±13.62) mU/L,(14.37±0.77) μmol/L vs.(22.08±1.57) μmol/L,(9.83±0.75) mU/L vs.(13.28±1.84) mU/L,respectively,all P＜0.05]Conclusions Alisma orientalis extract has the protective effect on focal cerebral ischemia reperfusion injury,and the mechanism may be related to antioxidant and scavenging free radicals.