Quality Control of Anemone Raddeana Rhizome and Its Toxicity
|School||Hangzhou Normal University|
|Keywords||Anemone raddeana Rhizome Quality control Fingerprint Toxicity|
The Rhizome of Anemone raddeana Regel is a kind of traditional Chinese medicine (TCM). Traditionally, it is used to treat neuralgia and rheumatism. Recently, many triterpenoid saponins have been separated and determined from A. raddeana Rhizome, which could be responsible for many bioactivities of A. raddeana Rhizome such as antiinflammation, antitumor, antieclampsia, and antipyretic and analgesia. But the quality control of A. raddeana Rhizome has not been examined and the toxic compounds and toxicology of A. raddeana Rhizome have not been determined either. This thesis reported the first study to the quality evaluation and acute toxicity of A. raddeana Rhizome in order to promote the development of it.In this thesis, thin layer chromatography (TLC) was the first method of choice for herbal analysis, and was used for evaluation of the quality of11batch samples of A. raddeana Rhizome collected from northeast China. High performance liquid chromatography (HPLC) was employed to determine the contents of Raddeanin A in11samples. HPLC, FT-1R (Fourier-transform infrared spectroscopy) and UV (Ultra violet) methods were applied to establish the fingerprints of A. raddeana Rhizome for quality evaluation. Before constructing the fingerprint, the original data was standardized in order to eliminate the influence of the dimension. Then the standardized data was used to calculate the cosine of unit vector cos θ. In order to comprehensively describe the difference between the sample chromatogram and the referential fingerprint, the sample percentage of the reference was used to correct cosθ. The resultant value was defined as H. cosθ and H could provide qualitative and quantitative evaluation of the fingerprint. The values of cosθ and H in3fingerprints were used for cluster analysis performed with SAS9.1software. The acute toxicity of A. raddeana Rhizome was examined by determining the median lethal dose (LD50) or maximum tolerated doses (MTD) in mice. The LD50of the water extract of A. raddeana Rhizome was firstly determined by ICR mice. At the same time, MTD of petroleum ether, chloroform and n-butanol extracts and the ethyl acetate extract were determined, respectively. The petroleum ether, chloroform, ethyl acetate and n-butanol extracts were obtained by extracting the water extraction in due order. The LD50was estimated in mice. The LD50value and the95%confidence interval were statistically calculated with the Bliss’s method. The calculation was performed with SAS9.1software.The visualized TLC plates showed that the11sample solutions gave spots at the same distance.the Raddeanin A reference traveled. The percentage of RA (g/g, dried weigh) in Ⅱ samples was all higher than0.2%determined by HPLC. According to China Pharmacopoeia (Part I,2010Edition). It could be concluded that they were all up to standard. When HPLC fingerprint was performed, the chromatographic conditions were:the mobile phase, acetonitrile-0.1%phosphate buffer (v/v); flow.1.0mL/min; injection volume,10μL; detective wavelength,206nm; the column temperature,40℃; the gradient condition,0-7min, acetonitrile47%;7-40min, acetonitrile80%(v/v). The number of theoretical plates was calculated by RA. which were no less than4000. The cosine values of vectorial angle of11samples calculated were all above0.97. The cosθ values varied largely from0.115to0.951and the H values varied largely too from0.045to1.129. The values of cosθ and H of S9were the lowest and those of S3were the highest. The FT-1R fingerprint with scanning scope1500-800cm-1scanned the tablet sample. The cosθ values calculated were above0.997. The calculated values of cosθ and H of S8were the lowest whereas those of S5were the highest. An UV-vis spectrophotomcter was employed for spectra recording and was operated at the wavelength from210nm to400nm. The cosθ values calculated were above0.998. The cosθ and H values of S11were the lowest whereas those of S6were the highest. All the cosθ and H values of the three fingerprints were used to comprehensively analysze the H samples by cluster analysis performed with SAS9.1software. The resultant dendrogram showed thai S4 and S11were firstly clustered into one group, and then S1and S6were clustered into one group. S8had the longest distance from the others.The LD50of the water extract of A. raddeana Rhizome was estimated in mice. The LD50value and the95%confidence interval were statistically calculated with the Bliss’s method. The calculation was performed with SAS9.1software. The LD50and the95%confidence interval of the water extract were104.5and95.45-115.29g/kg (plant powder per body weight) respectively and those of the ethyl acetate extract were604.81and537.99-673.57(g/kg, plant powder per body weight) respectively. According to Blach well method, the dosage of A. raddeana Rhizome for people was10.45g plant powder per person, which demonstrated that the usual dosage1-3g is safe in clinic each time for adult. The toxic TCM is classified into "High acute toxicity","Acute toxicity" and "Slight acute toxicity". As for A raddeana Rhizome, the toxic dose one time for adult was10.45g/kg; the latent time was less than30min: and the LD50was104.5g/kg (plant powder per body weight, i.g. by mice). So the toxicity of A. raddeana Rhizome was between "Acute toxicity" and "Slight acute toxicity". When the MTD was determined, the petroleum ether, chloroform and n-butanol extracts apparently had no acute toxicity at the concentration of1000g/kg (plant powder per body weight).