利用丹參葉片癒合組織生產隱丹參酮之研究

Abstract

丹參（Salvia miltiorrhiza Bunge）為我國民間常用的傳統藥材，常用於心血管疾病之治療，本研究利用丹參葉片所誘導出之癒合組織，探討大量繁殖癒合組織之培養基中auxins及cytokinins適合濃度，建立以癒合組織生產隱丹參酮之培養系統。 添加1 mg/l 2,4-D及2 mg/l BA之MS基本鹽類培養基，可成功從丹參葉片誘得癒合組織，將此癒合組織培養於含1 mg/l 2,4-D、2 mg/l BA、20﹪椰子汁、3﹪蔗糖及1﹪Difco agar之MS基本鹽類培養基，一個月後可得到大量癒合組織，但這種癒合組織不含隱丹參酮，若再將丹參之癒合組織繼代培養於含0.2 mg/l BA、3﹪蔗糖及1﹪Difco agar之MS基本鹽類培養基中，二個月後癒合組織轉變為磚紅色，並獲得4.04 mg/g dry wt.之隱丹參酮。

Salvia miltiorrhiza Bunge is an important ingredient of certain recipes, which widely used in traditional Chinese medicine for the treatment of some cardiovascular related diseases. The main objectives of the present research work were (1) To standardize procedures for induction of callus from the leaf explants of S. miltiorrhiza. (2) To study the influence of different auxins and cytokinins on callus multiplication. (3) Establishing the methods for the production of cryptotanshinone, an active diterpenoid from callus cultures of S. miltiorrhiza. The results obtained were summarized as follows (1) Primary calli were induced when leaf explants were cultured on Murashige and Skoog''s basal medium containing 1 mg/l 2,4-D, 2 mg/l BA, 3% sucrose and 1% Difco Agar. (2) Of all the hormonal combinations tested [NAA + BA, IAA + BA, 2,4-D + BA, 2,4-D + TDZ, 2,4-D + Kinetin and 2,4-D + Zeatin ] optimum callus proliferation was observed on MS medium supplemented with 1 mg/l 2,4-D and 2 mg/l BA. (3) The HPLC analysis of the calli multiplied on the above mentioned mediums revealed that cyrptotanshinone was not formed. (4) Production of cyrptotanshinone from callus cultures of S. miltiorrhiza has been achieved. Callus grown on MS medium supplemented with 0.2 mg/l BA for a period of two months yielded maximum amount of cryptotanshinone and were analyzed by HPLC. To summarize, an efficient method for induction and multiplication of callus and production of cryptotanshinone in callus cultures of S. miltiorrhiza has been developed. These results are useful for pharmacological studies.