ABSTRACT
OBJECTIVE To establish the fingerprint of Tibetan medicine Adhatoda vasica ，and determine the contents of vasicine and vasicinone ，so as to comprehensively evaluate its quality combined with chemical pattern recognition. METHODS Using vasicine as control ，HPLC fingerprints of 11 batches of A. vasica were established with Similarity Evaluation System for Chromatographic Fingerprints of TCM （2012 edition）. The common peaks were identified and their similarities were evaluated. Cluster analysis （CA），principal component analysis （PCA）and orthogonal partial least squares-discriminant analysis （OPLS-DA） were performed by using SPSS 25 software and SIMCA 14.1 software. The variable importance in the projection （VIP）value＞1.0 was used as the standard to screen the differential components affecting the quality of A. vasica ；the contents of vasicine and vasicinone were determined by HPLC simultaneously. RESULTS A total of 23 common peaks were found ，and peak 2 was identified as vasicine ，and peak 4 was identified as vasicinone. Their similarities ranged 0.920-0.994. The results of CA showed that 11 batches of samples were clustered into 3 categories（distance was 14）：S1-S8 as one category （origin：Yunnan，Tibet），S9 as one category （origin：Yunnan），S10-S11 as one category （origin：Sichuan）；the results of P CA and OPLS-DA showed that S 9 and S10-S11 were divided into one category respectively ，and S1-S8 were further divided into 2 categories：S1，S4 as one category，S2-S3，S5-S8 as one category ；the common peaks with VIP value ＞1.0 included peak 2，peak 16，peak 21，peak 17，peak 1 and peak 13. Among 11 batches of samples ， contents of vasicine and vasicinone were 4.12-10.22 and 0.60-3.26 mg/g， respectively. CONCLUSIONS Established edu.cn HPLC fi ngerprint and content determination method are simple and accurate ，and can be used for the quality evaluation of Tibetan medicine A. vasica ，by combining with chemical pattern recognition. Vasicine and other components may be the differential components that affect the quality of the drug.
ABSTRACT
Objective:To establish the quality standard for peganum harmala alkaloids cream ( CAPH) . Methods: The general quality of CAPH was inspected according to the general notices described in Chinese Pharmacopoeia volumeⅠ2010 edition. The qual-itative identification was carried out by TLC with harmine and harmaline as the index ingredients. The content determination was carried out by HPLC methods with harmine, harmaline and vasicine as the index ingredients. Results:The inspection items were all met the requirements. The experimental samples and the reference substances in TLC showed the identical spots with the same color and shape at the same position. The calibration curve of harmaline, harmine and vasicine was linear within the concentration range of 3. 440-110. 000 μg·ml-1 , 3. 340-107. 000 μg·ml-1 and 1. 380-22. 000 μg·ml-1 , respectively. The recovery was 98. 1%, 99. 8% and 99. 3% with RSD of 1. 75%, 1. 78% and 1. 95%, respectively (n=6). Conclusion: The established quality control methods meet the requirements of methodology, and the results lay foundation for the quality standard for CAPH.
ABSTRACT
OBJECTIVE: To develop the quality specification of seeds of Peganum harmala. METHODS: According to the Chinese Pharmacopoeia (2010 Version, Volume 1) and its appendix method, the water, total ash, acid insoluble ash, 50% ethanol extractives, and heavy metal were analyzed for seeds of P. harmala. TLC method was used to separate harmaline (HAL), harmine (HAR) and vasicine (VAS) in seed samples using mixture of ethyl acetate-methanol-ammonia water (10:1.5:0.5) as a developing solvent on high performance silica G pre-coated plate with 254 nm fluorescent (GF254) and to identify them inspected under UV 366 nm, 254 nm, visualized by spraying with both Dragendorff reagent and by bioautographic assay. In the HPLC method, HAL and HAR were separated on a C18 column with acetonitrile-ammonium acetate water (19:81) as the mobile phase and detected at 330 nm. The HPLC fingerprints were performed on the same C18 column and eluted by using a linear gradient of acetonitrile (A) and 0.1 mmol · L-1 ammonium acetate buffer under the flow rate at 0.7 mL · min-1 and detected at 280 nm. RESULTS: In the TLC procedures, 254 and 366 nm fluorescent, Dragendorff reagent, and bioautographic assay for the detection of acetylcholinesterase inhibitor can be used for qualitative identification of the active ingredients. For the HPLC quantitative method, the calibration curve of HAR displayed ideal linearity over the range of 1.97-198.68 μg · mL-1 with average recovery of 99.69% (RSD of 1.89%). HAL displayed ideal linearity over the range of 1.70-345.30 μg · mL-1 with average recovery of 100.66% (RSD of 1.78%). The contents of HAL and HAR in 11 batches of seeds of P. harmala were 3.234% and 3.755%. In the characteristic fingerprints of seeds of P. harmala, four common peaks were identified. CONCLUSION: The results indicated that the water, total ash, acid insoluble ash, and 50% ethanol extractives were not more than 9.0%, 8.0%, 1.0%, and 22.0%, respectively. The heavy metal of plumbum, cadmium, arsenic, mercury, and copper were not more than 5 × 10-6, 3 × 10-6, 2 × 10-6, 2 × 10-6 and 20 × 10-6, respectively. The content limit of the sum of HAL and HAR was not lower than 5.5%. With the peak of HAL as reference peak, the variance of relative retention time of the four common peaks, in the characteristic fingerprints of seeds off. harmala, should be fluctuated in the range of 5% of the specified value. The qualitative and quantitative method established was suitable for the quality evaluation and assessment of seeds of P. harmala.