Paper Details

PJB-2021-723

OPTIMIZATION OF PHELLODENDRON AMURENSE CALLUS CULTURE BY RESPONSE SURFACE METHODOLOGY AND IN VITRO ANTIOXIDANT, ANTIMICROBIAL ACTIVITY

Yuhong Zhang
Abstract

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Phellodendron amurense is one of the important associated species in the broadleaved-Korean pine forests of northeast China, which has been used for many years as a medical plant in Traditional Chinese Medicine, its phloem is called “GuanHuangBai” in context of Chinese medicinal material, P. amurenseis the only source plant. Berberine is an essential active compound contained in P. amurense, that has shown great prospect in recent clinical trials for future applications. It is overexploited for its high valuable medicinal properties, which has led to the depletion of wild P. amurense resources. Therefore, tissue culture technology is used to produce the effective components of P. amurense and relieve the pressure on the utilization of wild resources. This study was based on the successful tissue culture of P. amurense in the early stage, the seedling of P. amurense was taken as the material and MS as the basic medium, different explants (stems segments, petioles, and leaf), sucrose concentration, nitrogen content and exogenous hormone combinations (2, 4-D 2.0mg/L + 6-BA 0.5-2.0 mg/L and 6-BA 1.0 mg/L+ 2, 4-D 0.5-2.0 mg/L), the basic culture conditions: the pH of MS medium is 5.8, and the light intensity is 108 μmolm-2s-1, temperature 25±0.5°C, the light and dark cycle is 16 h / 8 h, the index was callus induction rate, single factor experiments were conducted. The optimal conditions were obtained under single factor conditions: the explant was stem segment, sucrose concentration was 20g/L, the content in the medium was N, and the combination of exogenous hormones was 2, 4-D 2.0 mg/L+ 6-BA 1.0 mg/L. On the basis of single factor results, three factors including sucrose concentration, nitrogen content and exogenous hormone combination were selected and experiments designed by using Design Expert.v8.0.6.1 Software, and the callus induction rate was taken as the index to optimize callus culture conditions by response surface methodology. The optimal conditions of callus culture were determined by regression model equation: stem segment as explants, sucrose concentration 18g/L, nitrogen content 1.0 N, combination of exogenous plant hormone 2, 4-D 2.0mg /L+6-BA 1.4mg/L, under these conditions, the callus induction rate of P. amurense was about 97.06%±8.69. Berberine was extracted from the callus that cultured under the optimal conditions and its content was6.14±1.12 mg/g (DW), meet the berberine content not less than 0.6% standard of the Chinese Pharmacopoeia. In vitro antioxidant and antibacterial activities were tested for the extracts of the callus and TCM “GuanHuangbai”. The results shown that antioxidant activities of the two extracts were mostly related to concentrations of berberine. Compared with those of the Guanhuangbai, the antioxidant activity of callus extracts (IC50: 0.2166mg/ml±0.0387) was slightly higher than that of TCM extracts (IC50: 0.2169mg/ml±0.0392), but the difference was not significant (p<0.05). The TCM extracts of P. amurense exhibited much better antimicrobial activity than callus extracts for gram-positive bacteria, but the similar potency for the gram-negative bacteria and fungi in the present study. The extracts from callus of P. amurense in the experiment had better antimicrobial activity.

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