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<p><i>Ajuga bracteosa</i> is a medicinal herb, known for its rich reservoir of pharmacologically active secondary metabolites including flavonoids, diterpenoids and withanolides. Overharvesting and environmental stress have placed this species under threat, initiating alternative production methods. This study established in vitro callus and suspension cultures of <i>A. bracteosa</i>, employing methyl jasmonate (Me-J) elicitation to stimulate biomass and metabolites accumulation. Callus cultures derived from hypocotyl explants were initiated on MS solid medium supplemented with specific plant growth regulators (PGRs), while suspension cultures were maintained in liquid medium with 2.0&#xa0;mg/L benzyladenine (BA). Me-J was applied at concentrations of 1.0, 3.0 and 5.0&#xa0;mg/L to evaluate its dose-dependent effects on growth kinetics, cell cycle progression and phytochemical enrichment. Moderate Me-J doses (1.0–3.0&#xa0;mg/L) promoted cell proliferation as evident from a marked increase in the S-phase cell population via flow cytometric analysis, whereas higher concentrations (5.0&#xa0;mg/L) inhibited growth and biomass accumulation. Biochemical profiling revealed a substantial enhancement in total phenolic and flavonoid contents (up to ~ 2–3-fold vs. control), along with elevated antioxidant activity in cell cultures treated with 3.0&#xa0;mg/L Me-J. Extracts derived from cell cultures were tested against <i>Leishmania tropica</i> promastigotes. Extracts from 3.0&#xa0;mg/L Me-J treated cell cultures exhibited the strongest antiparasitic activity (IC₅₀=22&#xa0;µg/mL), significantly more potent than extracts from BA treated cell cultures (IC₅₀=54&#xa0;µg/mL). Microscopic analysis using acridine orange/ethidium bromide staining along with agarose gel electrophoresis indicated classical apoptotic features in treated parasites. Flow cytometric quantification of propidium iodide–stained promastigotes confirmed apoptosis through accumulation of Sub-G₁ DNA content. Hemolysis assays indicated negligible toxicity of extracts on human erythrocytes and thus affirmed the biocompatibility of extracts. These findings underscore the mechanistic role of Me-J in modulating hormone balance and stress signalling in <i>A. bracteosa</i> cell cultures, leading to enhanced metabolite biosynthesis and cytotoxicity against protozoan pathogens via apoptosis. This integration of elicitor based modulation with flow cytometric monitoring presents a robust platform for optimizing plant based bioproduction and discovering bioactive antiparasitic compounds.</p>

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Methyl jasmonate-elicited Ajuga bracteosa cell cultures: dual flow cytometric analysis of biomass distribution and antileishmanial potency

  • 期刊论文

  • 发表日期：

    2025年10月23日

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  • Huma Ali,
  • Samreen Baig,
  • Javiria Sanum,
  • Eman Fayad,
  • Dalal Nasser Binjawhar,
  • Hanadi A. Katouah,
  • Uthman Balgith Algopishi,
  • Mubarak Ali Khan

摘要

Ajuga bracteosa is a medicinal herb, known for its rich reservoir of pharmacologically active secondary metabolites including flavonoids, diterpenoids and withanolides. Overharvesting and environmental stress have placed this species under threat, initiating alternative production methods. This study established in vitro callus and suspension cultures of A. bracteosa, employing methyl jasmonate (Me-J) elicitation to stimulate biomass and metabolites accumulation. Callus cultures derived from hypocotyl explants were initiated on MS solid medium supplemented with specific plant growth regulators (PGRs), while suspension cultures were maintained in liquid medium with 2.0 mg/L benzyladenine (BA). Me-J was applied at concentrations of 1.0, 3.0 and 5.0 mg/L to evaluate its dose-dependent effects on growth kinetics, cell cycle progression and phytochemical enrichment. Moderate Me-J doses (1.0–3.0 mg/L) promoted cell proliferation as evident from a marked increase in the S-phase cell population via flow cytometric analysis, whereas higher concentrations (5.0 mg/L) inhibited growth and biomass accumulation. Biochemical profiling revealed a substantial enhancement in total phenolic and flavonoid contents (up to ~ 2–3-fold vs. control), along with elevated antioxidant activity in cell cultures treated with 3.0 mg/L Me-J. Extracts derived from cell cultures were tested against Leishmania tropica promastigotes. Extracts from 3.0 mg/L Me-J treated cell cultures exhibited the strongest antiparasitic activity (IC₅₀=22 µg/mL), significantly more potent than extracts from BA treated cell cultures (IC₅₀=54 µg/mL). Microscopic analysis using acridine orange/ethidium bromide staining along with agarose gel electrophoresis indicated classical apoptotic features in treated parasites. Flow cytometric quantification of propidium iodide–stained promastigotes confirmed apoptosis through accumulation of Sub-G₁ DNA content. Hemolysis assays indicated negligible toxicity of extracts on human erythrocytes and thus affirmed the biocompatibility of extracts. These findings underscore the mechanistic role of Me-J in modulating hormone balance and stress signalling in A. bracteosa cell cultures, leading to enhanced metabolite biosynthesis and cytotoxicity against protozoan pathogens via apoptosis. This integration of elicitor based modulation with flow cytometric monitoring presents a robust platform for optimizing plant based bioproduction and discovering bioactive antiparasitic compounds.