In vitro strategy to enhance the production of bioactive polyphenols and caffeoylputrescine in the hairy roots of Physalis peruviana L

Abstract

The Rhizobium rhizogene-transformed root culture from Physalis peruviana L. (P. peruviana) may be a promising and novel source of valuable phenolics, including caffeoylputrescine (CP), which is known for antioxidant, antidiabetic, insect-resistant, disease-resistant, and neuroprotective properties. In this study, to improve the production efficiency of phytochemical components in P. peruviana hairy root cultures, we optimized various culture conditions, including the inoculum size, liquid volume, culture media type, carbon source, sucrose concentration, initial pH, and application of elicitors, to enhance the total phenolic content and CP yield in these hairy root cultures. The findings indicate that the use of sucrose as carbon source resulted in the highest biomass (13.28 g DW/L), total phenolic content (6.26 mg/g), and CP yield (2.40 mg/L). The White medium excelled in enhancing the total phenolic content (9.35 mg/g), whereas the B5 medium was most effective for the biomass (13.38 g DW/L) and CP yield (6.30 mg/L). A sucrose concentration of 5% was best for the biomass (18.40 g DW/L), whereas a sucrose concentration of 4% was ideal for the CP yield. Optimal culture conditions were as follows: an inoculum size of 0.5 g/100 mL, a liquid volume of 100 mL in a 250-mL flask, B5 medium, 4% sucrose, and a pH of 5.5. Among the tested elicitors, methyl jasmonate (MeJA) at 100 µM significantly increased the biomass (21.3 g/L), total phenolic content (23.34 mg/g), and CP yield (141.10 mg/L), which represent 0.96-, 2.12-, and 13.04-fold increases, respectively, over the control after 8 days. The optimized HR culture of P. peruviana provides a promising system to enhance the production of CP for pharmaceutical applications.

Keywords: Physalis peruviana L.; Caffeoylputrescine; Culture conditions; Hairy root; Polyphenols.

Fig. 1

(A) Fresh weight and dry weight of hairy root of Physalis peruviana L. (P. peruviana) on different days after a sucrose treatment. (B) Identified hairy roots. Growth of hairy roots in a liquid 1/2MS medium: (C) 4 days - adaptive phase; (D) 24 days - stationary phase. Mass spectra of the identified caffeoylputrescine (CP) in the positive ion mode. (E) HPLC chromatograms of standard CP and methanolic extracts of P. peruviana HRs. (F) Secondary mass spectrometry of CP.

Fig. 2

Optimization of culture conditions to enhance the biomass, total phenolic content (TPC), and caffeoylputrescine production from hairy roots. The effects of the inoculum size (1(a) and 1(b)), liquid volume (2(a) and 2(b)), and carbon source sucrose (3(a) and 3(b)) were investigated. The presented values are the mean ± standard error of three replicates; the data recorded were after 24 days of culture.

Fig. 3

Optimization of culture conditions to enhance the biomass, total phenolic content (TPC), and caffeoylputrescine production of HRs. The effects of the media (1(a) and 1(b)), sucrose concentration (2(a) and 2(b)), and initial pH (3(a) and 3(b)) were investigated. The presented values are the mean ± standard error of three replicates; the data were recorded after 24 days of culture.

Fig. 4

Elicitor effects on the HR parameters of P. peruviana HR. (A–D) Impact of MeJA, SA, and SNP at concentrations of 50, 100, 200, and 400 µM on the (A) biomass, (B) total phenolic content (TPC), (C) caffeoylputrescine (CP) content, and (D) CP yield in 20-day-old P. peruviana HRs after an 8-day exposure. (E–H) Impact of the same elicitors at a fixed concentration of 100 µM on the biomass (E), TPC (F), CP (G), and CP yield (H) under different exposure durations (2, 4, 6, and 8 days). (I–L) Morphological effects of various elicitors on P. peruviana HRs: (I) non-treated control roots, (J) MeJA (100 µM) treated, (K) SA (100 µM) treated, and (L) SNP (100 µM) treated. CK denotes the untreated control group. The presented values are the mean ± standard error of three replicates. Bars = 1 cm.