Evaluation of Polyphenolic Compounds and Pharmacological Activities in Hairy Root Cultures of Ligularia fischeri Turcz. f. spiciformis (Nakai)

Abstract

A considerable amount of bioactive compounds have been used for the biopharmaceutical engineering to help human health and nutrition. Hairy root culture (HRC) or transgenic root is a favourable alternative technique for phytochemical production. Ligularia fischeri is a significant source of pharmaceutically important active compounds with an enormous range of health care applications. HRC of L. fischeri was developed using Agrobacterium rhizogenes for the production of polyphenolic compounds with antioxidant, antimicrobial, antidiabetic, anticancer and anti-inflammatory pharmaceutical activities. Hairy roots (HRs) were selected by morphological assessment, genetic and molecular analyses. The maximum accumulation of fresh mass (94.15 g/L) and dry mass (9.45 g/L) was recorded in MS liquid medium supplemented with 30 g/L sucrose at 28 days. Furthermore, HRs successfully produced numerous polyphenolic compounds, including six hydroxycinnamic acids, seven flavonols, seven hydroxybenzoic acids, vanillin, resveratrol, pyrogallol, homogentisic, and veratric acids, which were identified by UHPLC analysis. HRs produced higher total phenolic (185.65 mg/g), and flavonoid (5.25 mg/g) contents than non-transformed roots (125.55 mg/g and 3.75 mg/g). As a result of these metabolic changes, pharmaceutical activities were found higher in HRs than non-transformed roots (NTRs). The present study indicates that HRC has the potential to increase the content of beneficial polyphenolic compounds with higher potential pharmaceutical activities. To the best of our knowledge, the present study is the first report on enhancing the production of polyphenolic compounds with pharmaceutical activities from the HRCs of L. fischeri.

Keywords: Agrobacterium rhizogenes; Ligularia fischeri; hairy root cultures; pharmaceutical activities; polyphenolic compounds.

Figure 1

Agrobacterium rhizogenes-mediated hairy root cultures in Ligularia fischeri. (a) Hairy roots induction, (b) Hairy root cultures in hormone-free liquid MS medium, c. PCR analysis of the rolC gene in the transgenic root lines. DNA ladder marker lane M, pRiKCTC2703 DNA C(+), transgenic root lines (HRs) induced by A. rhizogenes L1–L6, roots from a non-transgenic plant (NTRs) C(−).

Figure 2

Factors influencing the biomass accumulation in hairy root cultures (HRCs) of Ligularia fischeri. (a) HRCs as affected by different media, (b) HRCs as affected by different concentrations of sucrose in the MS medium. (c) Time profile of HRCs in MS medium supplemented with sucrose (30 g/L). Data represent as means ± SD of three replicates. If followed by different letters, results are significantly different at p ≤ 0.05.

Figure 3

Total phenolic and flavonoid contents (TPC and TFC) in hairy root cultures of Ligularia fischeri. (a) TPC, (b) TFC. Data represent as means ± SD of three replicates followed by different letters, are significantly different at p ≤ 0.05.

Figure 4

Antioxidant activities in hairy root cultures of Ligularia fischeri. (a) Percentage inhibition of DPPH radicals, (b) Nitric oxide (NO) scavenging activity, (c) Total Fe³⁺–Fe²⁺ reductive potential reference antioxidants (butylated hydroxytoluene), (d) Total antioxidant activities by phosphomolybdenum method [expressed as equivalents of α-tocopherol (μg/g of extract)], (e) Metal chelating activity. Data represent means ± SD of three replicates. If followed by different letters, values are significantly different at p ≤ 0.05.

Figure 5

Antidiabetic and anti-inflammatory activities in hairy root cultures of Ligularia fischeri. (a) In vitro α-amylase activity, (b) Non-enzymatic glycosylation of hemoglobin activity, (c) Lipoxygenase inhibition activity, (d) Albumin denaturation inhibition assay. Data represent as means ± SD of three replicates. If followed by different letters, values are significantly different at p ≤ 0.05.

Figure 6

Antimicrobial activity in hairy root cultures of Ligularia fischeri using disc diffusion method. Statistical analysis was performed for each microbial species separately. Data represent means ± SD of three replicates. If followed by different letters, values are significantly different at p ≤ 0.05.

Figure 7

Cell viability of MCF-7 and HT-29 cell lines in hairy root cultures of Ligularia fischeri. (a) MCF-7, (b) HT-29. Data represent as means ± SD of three replicates followed by different letters, are significantly different at p ≤ 0.05.