Abstract
Purpose: To evaluate the potential of Lonicera macranthoides Hand. -Mazz. Yulei1 suspension culture system for enhanced production of the main secondary metabolite, chlorogenic acid.Methods: The callus of L. macranthoides Hand.-Mazz. “Yulei1” was suspension cultured in B5 liquid medium supplemented with different plant growth regulators. Biomass accumulation was calculated by weight method and chlorogenic acid production was measured using high performance liquid chromatography (HPLC). HPLC was carried out on C18 analytical column at 35 °C and the detection wavelength was set at 324 nm.Results: The results showed that maximum accumulation of biomass and chlorogenic acid were achieved 15 days after culture growth. The optimized conditions for biomass accumulation and chlorogenic acid production were 50 g/L of inoculum on fresh weight basis, B5 medium supplemented with plant growth regulators, 30 - 40 g/L sucrose and initial medium pH of 5.5. Maximum accumulation of chlorogenic acid and biomass were observed when the culture medium was supplemented with 2.0 mg/L6-BA. Optimal accumulation of chlorogenic acid was observed using combination of hormones 2.0 mg/L 6-Benzyladenine (BA) + 0.5 mg/L2, 4-Dichlorophenoxyacetic acid (2,4-D), while optimal accumulation of biomass was observed with 2.0 mg/L 6-BA + 2.0 mg/L2, 4-D. In addition, phenylalanine also contributed to the synthesis of chlorogenic acid at a concentration > 50 mg/L.Conclusion: Cell suspension cultures of L. macranthoides Hand.-Mazz. “Yulei1” have successfully been established. The findings provide a potential basis for large scale production of chlorogenic acid using cell suspension cultures of L. macranthoides.Keywords: Lonicera macranthoides, Cell suspension culture, Chlorogenic acid, Phenylalanine, Optimization
Highlights
Lonicera is a pharmacologically important medicinal plant and have long been used in Chinese traditional medicines
The maximum accumulation of chlorogenic acid was 21.86 mg/g dry weight (DW) when medium was supplemented with 2.0 mg/L of 6-BA
It appears that higher concentration of 2,4-D and naphthaleneacetic acid (NAA) inhibited chlorogenic acid production when combined with 2.0 mg/L6-BA
Summary
Lonicera is a pharmacologically important medicinal plant and have long been used in Chinese traditional medicines. This study was designed to establish cell suspension culture system and investigate the production of chlorogenic acid in the suspension cultures of L. macranthoides Hand.-Mazz. To determine optimal inoculum density, different amount of inoculant (25, 50, 75, 100 and 125 g/L) were tested for the production of biomass and chlorogenic acid. The 60 % methanol extracts were filtered through injector and membrane filter, and used for chlorogenic acid determination by HPLC analysis by the method described previously with minor modification [1]. “Yulei1” was determined from the corresponding calibration curve and the content was expressed as milligram of chlorogenic acid per gram of L. macranthoides Hand.-Mazz.
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