Journal of Taiwan Agricultural Research

Author:Chi-Han Liu, Jui-Sheng Lai, Horng-Mo Lee, and Min-Tze Wu*

Abstract:

    Barks of Huang-Po (Phellodendron spp.) contain berberine and it is used as an important herbal medicine for the treatment of fever, infammation, stomach ache and intestinal illness.  Taiwan Huang-Po [Phellodendron amurense Rupr. var. wilsonii (Hayata& Kanehira)] is a native species in Taiwan and the bark of this plant is used as an important medicine because of its high berberine content.  However, it would take more than 6–8 years to grow Taiwan Huang-Po trees for harvesting barks.  In addition, the supply of barks of Taiwan Huang-Po from natural habitat is dwindling due to excessive harvesting of this plant.  The objective of this study was to establish a cell suspension culture method for production of berberine from Taiwan Huang-Po, as an alternative method for production of berberine from barks of plants grown in natural habitat.  Two months old seedlings were used as explants to induce callus in this study.  Leaf petiole was proven to be the best tissue for callus induction.  Leaf petiles were placed on Murashige and Skoog (MS) basal medium containing 0.5 mg/L 2,4-dichloro-phenoxyacetic acid (2,4-D) and 1.0 mg/L 6-benzylaminopurine (BA) and incubated in dark for 28 days to form callus tissues which were used as inoculum for cell suspension cultures.  Callus tissues were inoculated on a medium containing WPM basal salt amended with 0.5mg/L 2,4-D and 1.0 mg/L BA and the cell suspension was cultured on a shaker at 100 rpm for 20 days and then used for testing amount of cells and berberine in the cultures.  Results showed that the total amount of cells in the cell suspension cultures increased by 9 folds after incubation for 20 days but no berberine was detected when the cells were extracted by 75% ethanol and analyzed by high performance liquid chromatography (HPLC).  In contrast, suspension cells grown in the medium containing 2 mg/L BA and 6 mg/L α-naphthaleneacetic acid (NAA) for 18 days, resulted in production of berberine.  Berberine accumulation reached 188.68 μg/g dry weight in the 28-day-old, cell-suspension culture.  Thus, this method of employing growth regulators in cell suspension culture may be important in eastablishing protocol for industrial production of berberine from cell suspension cultures of the endemic species P. amurens.

Key words:Phellodendron amurense var. wilsonii, Callus, Cell suspension, Berberine, High performance liquid chromatograph (HPLC)

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