Fingerprint establishment ，chemical pattern recognition and content determination of Tibetan medicine Adhatoda vasica / 中国药房

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.

Anti-Proliferative Activities of Vasicinone on Lung Carcinoma Cells Mediated via Activation of Both Mitochondria-Dependent and Independent Pathways

Vasicinone, a quinazoline alkaloid from Adhatoda vasica Nees. is well known for its bronchodilator activity. However its antiproliferative activities is yet to be elucidated. Here-in we investigated the anti-proliferative effect of vasicinone and its underlying mechanism against A549 lung carcinoma cells. The A549 cells upon treatment with various doses of vasicinone (10, 30, 50, 70 µM) for 72 h showed significant decrease in cell viability. Vasicinone treatment also showed DNA fragmentation, LDH leakage, and disruption of mitochondrial potential, and lower wound healing ability in A549 cells. The Annexin V/PI staining showed disrupted plasma membrane integrity and permeability of PI in treated cells. Moreover vasicinone treatment also lead to down regulation of Bcl-2, Fas death receptor and up regulation of PARP, BAD and cytochrome c, suggesting the anti-proliferative nature of vasicinone which mediated apoptosis through both Fas death receptors as well as Bcl-2 regulated signaling. Furthermore, our preliminary studies with vasicinone treatment also showed to lower the ROS levels in A549 cells and have potential free radical scavenging (DPPH, Hydroxyl) activity and ferric reducing power in cell free systems. Thus combining all, vasicinone may be used to develop a new therapeutic agent against oxidative stress induced lung cancer.

Evaluation of hepatoprotective activity of vasicinone in mice.

Justicia adhatoda (vasaka) leaves have long been used in Indian Ayurvedic system of medicine as antitussive. Its crude extract has been previously reported to have hepatoprotective activity. Vasicinone was isolated from leaves of J. adhatoda, column purified and characterized using, TLC UV, FT-IR and 1H NMR. The isolated vasicinone was evaluated for hepatoprotective activity using (CCl4)-induced acute hepatotoxicity model in mice. CCl4 treatments lead to significant increase in SGOT, SGPT, ALP levels. Pre-treatment with vasicinone and silymarin (25 mg/kg/day for 7 days) significantly decreased these enzyme levels. Histopathology of the livers from vasicinone and silymarin pre-treated animals showed normal hepatic cords and absence of necrotic changes suggesting pronounced recovery from CCl4 induced liver damage. Both vasicinone and silymarin significantly decrease the CCl4 mediated increase in pentobarbital indiced sleeping time in experimental animals, thus indicating recovery of liver function. Based on the above results it can be concluded that vasicinone may act as hepatoprotective in mice and warrants further investigation on human volunteers.