Elicitation using Jasmonates, Salicylic acid, and Sodium chloride in cell suspension cultures of Justicia gendarussa Burm f.: A route for enhanced and sustainable production of apigenin and apigetrin

Elicitation using Jasmonates, Salicylic acid, and Sodium chloride in cell suspension cultures of Justicia gendarussa Burm f.: A route for enhanced and sustainable production of apigenin and apigetrin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Elicitation using Jasmonates, Salicylic acid, and Sodium chloride in cell suspension cultures of Justicia gendarussa Burm f.: A route for enhanced and sustainable production of apigenin and apigetrin Vinaya Chandran, Maya Rajan, Shahena S, Linu Mathew This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6782131/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Oct, 2025 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract This study realized a high and sustained yield of apigenin and apigetrin in Justicia gendarussa cell suspension cultures by using elicitors: methyl jasmonate (MeJA), jasmonic acid (JA), salicylic acid (SA), and sodium chloride (NaCl). We cultured the leaf segments on MS basal medium with 2,4-Dichlorophenoxy acetic acid (2,4-D) at 2 mg L − 1 and Benzyl Adenine (BA) at 0.2 mg L − 1 for friable callus induction. We transferred the best-responding calli to liquid MS medium with 2,4-D at 1 mg L − 1 and elicited the 40th -day-old culture with varying doses of elicitors. Cultures treated with 200 µM MeJA recorded the highest accumulation of apigenin (123.98 µg g⁻¹ DW) and apigetrin (342.87 µg g⁻¹ DW) by the 2nd day of elicitation. Also, both compounds showed a significant increase upon JA elicitation, with the maximum accumulation (apigetrin 114.68 µg g⁻¹ DW; apigenin 73.93 µg g¹ DW) occurring at 100 µM JA by the 6th day. Likewise, SA at 100 µM boosted compound production (apigenin 106.57 µg g⁻¹ DW and apigetrin 231.43µg g⁻¹ DW) on day 4. Salt stress also promoted the accumulation of apigenin (107.13 µg g⁻¹ DW) and apigetrin (245.31 µg g⁻¹ DW), with the highest accumulation at 200 mM of NaCl. The elicitation capability of these four elicitors was in the order MeJA > NaCl > SA > JA. All elicitors produced higher concentrations of compounds than found in leaf extracts of field-grown plants (29.69 µg g⁻¹ DW of apigenin and 122.42 µg g⁻¹ DW of apigetrin). apigenin apigetrin in vitro cultures elicitation J. gendarussa Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key Message MeJA, JA, SA, and NaCl elicitation boosts bioactive flavonoids, namely, apigenin and apigetrin, in J. gendarussa cell suspension cultures, with MeJA at 200 µM being the most effective. INTRODUCTION Justicia gendarussa Burm f. is a perennial shrub of 1-1.5 meters growing in marshy areas across Southeast Asia. This plant has medicinal properties, with all parts utilized as medicines (Kavitha et al., 2014 ; Mnatsakanyan et al., 2018 ). The plant contains bioactive substances like phenolic compounds, steroids, terpenoids, alkaloids, and flavonoids. The leaves treat diarrhea, fever, persistent cough, hypertension, and bone pain as they contain friedelin, lupeol, ß-sitosterol, and aromatic amines (Pal & Rahaman, 2015 ) and possess anti-inflammatory, anti-bacterial, anti-fungal, antioxidant, and anti-angiogenic properties. Likewise, aerial parts of this plant showed HIV type-1 reverse transcriptase inhibition in vitro (Woradulayapinij et al., 2005 ). Apigenin (4′,5,7-trihydroxy flavone) is one of the key flavonoids present in fruits and vegetables, which is the aglycone of several natural glycosides with a chemical formula of C 15 H 10 O 5 and a molecular weight of 270.24 (Salehi et al., 2019 ). It has three hydroxyl groups, the first and second in the C5 and C7 positions and the third at C4′ of the B ring (Allemailem et al., 2024 ). Apigenin treats diseases of the liver, lung, heart, and kidney, neurological diseases, and diabetes, and maintains good oral and skin health (Bi et al.,2023; Hu et al., 2025 ; Thomas et al.,2023; Siddique et al.,2025). This compound suppresses cancers by inducing apoptosis and cell-cycle arrest, inhibiting cell migration and angiogenesis, and reducing inflammation (Zhu et al.,2013; Naponelli et al., 2024 ). Apigenin synergizes with other drugs, reduces the risk of side effects, and improves the chemotherapy response (Xu et al., 2011 ). However, due to its lipophilic nature, the acidic environment of the gastrointestinal tract may inactivate it, resulting in reduced bioavailability and limiting its potential use in medicines and nutraceuticals. The apigenin glycosides show greater aqueous solubility, biological activity, and binding affinity to specific targets (Kamel et al., 2025 ). The common apigenin glycosides are apigetrin (apigenin-7-O-glucoside), vitexin (apigenin-8-C glucoside), isovitexin (apigenin-6-C-glucoside), and rhoifolin (apigenin-7-O-neohesperidoside) (Ali et al., 2017 ). Among them, apigetrin and vitexin were reported in J. gendarussa (Raghu and Agarwal, 2016; Zhang et al., 2021 ). Apigetrin or apigenin 7-O-glucoside is the most commonly found 'glucoside' in plants, has significant anti-inflammatory properties, and inhibits the pro-inflammatory cytokines such as TNF-α and IL-6 (Guo et al., 2019 ). Also, it prevents oxidative stress-related diseases, such as cardiovascular diseases and neurodegeneration, and protects the liver (Wojdyło et al., 2007 ; Zhao et al., 2020 ). It induces apoptosis and prevents cancer cell proliferation (Chahar et al., 2011 ). In vitro plant cell, organ, and tissue cultures are one of the most preferred systems for producing bioactive compounds. Elicitation in plant cells or tissue culture produces various secondary metabolites in large quantities in a limited time and space, continuously turning out commercially valuable and pharmaceutically active bio-compounds. The technique is sustainable and independent of geographical or seasonal variation, and modifying culture parameters can upscale product accumulation. Elicitors induce a specific biological response in plants by triggering various cellular processes, including defense responses, signal transduction pathways, and the production of secondary metabolites (Akula & Ravishankar, 2011 ). In in vitro medicinal plant cultures, biotic and abiotic elicitors alter or stimulate the metabolic pathways to produce novel/existing bioactive compounds (Namdeo, 2007 ). Elicitors signal membrane-bound receptors, activate the signal transduction pathways, and change the expression of regulatory players, resulting in enhanced synthesis and accumulation of phyto-compounds (Petrova et al., 2024 ). Jasmonates, including jasmonic acid (JA) and its methyl ester methyl jasmonate (MeJA), are a family of cyclopentanone compounds from linolenic acid, modulating a wide range of plant responses (Creelman & Mullet, 1997 ) and act as effective elicitors to enhance secondary metabolites in vitro (Sembdner & Parthier, 1993 ). MeJA, a volatile methyl ester, is an effective signaling molecule in abiotic and biotic stresses (Wang et al., 2015 ) because it can permeate cell membranes easily and demethylates quickly to release free JA (Fattorini et al., 2018 ). It is a vital player in intra- and inter-communications in plant cells, increasing the antioxidant enzyme activity and the expression of defense genes (Ho et al., 2020 ). Salicylic acid is a phenolic compound that has roles in various developmental and physiological processes, including ethylene production, stomatal movements, photosynthesis, membrane functions, pigment accumulation, enzyme activities, plant growth, and development (Ali, 2021 ). Hence, SA and its related precursors are classified as phytohormones (Koo et al., 2020 ). SA upregulates the genes for Phenylalanine Ammonia Lyase (PAL), Chalcone Synthase (CHS), and Flavonol Synthase (FLS), enhancing flavonoid accumulation (Ali et al., 2007 ). It acts early in stress, sometimes with synergistic effects with other elicitors (Woch et al.,2023). Rajan et al. ( 2020 ) reported that MeJA and SA enhance rhamnetin production in Vernonia anthelmintica cultures. Also, MeJA and SA stimulated the biosynthesis of alkaloids and flavonoids in Piper cumanense , with MeJA producing a noticeable effect (Rodríguez et al., 2020). NaCl is a readily available and inexpensive abiotic elicitor with proven efficacy (Kitayama et al., 2019 ). Salinity induces ionic and osmotic stress in plants. The intensity of the salt stress elicits a salinity response in plants with reversible or irreversible changes in plant function (Hawrylak-Nowak et al., 2021 ). Although excess salt can be toxic to plants, moderate salinity has a stimulatory effect on plant growth and the accumulation of health-promoting antioxidants and nutrients (Gupta & Huang, 2014 ). Different concentrations of NaCl (50–200 mM) enhanced the synthesis of soluble sugars, proteins, and bioactive phenolics in Inula crithmoides suspension cultures (Rodríguez et al., 2024). The present study explores the enhanced and sustainable production of apigenin and apigetrin by using abiotic elicitors in cell suspension cultures of J. gendarussa. MATERIALS AND METHODS Plant Collection We collected asymptomatic plants of J. gendarussa from the garden premises of Mahatma Gandhi University, Kottayam, Kerala. For the taxonomical identification, the herbarium was submitted with an accession number 7640 at the Regional Herbarium of Kerala, St Berchmans College, Changanassery, Kerala, India. Media preparation and Callus induction Leaf explants were disinfected using 70% (v/v) ethanol for 1 min, followed by 0.005% aqueous mercuric chloride solution for 1.5 min, repeatedly rinsed in sterile distilled water to remove excess mercuric chloride, and kept in sterile filter paper to eliminate excess water. We used MS media with 30 g L − 1 sucrose and 8 g L − 1 agar with different concentrations and combinations of plant hormones for the callus induction. The P H of the media was 5.6–5.8, adjusted using 1 N NaOH before adding agar. After adding syringe-filtered hormones, 10 ml of autoclaved media was dispersed into boiling tubes and stored. Surface-sterilized explants were cut into small squares approximately 1 cm x 1 cm, inoculated into stored MS medium, and incubated under a 16/8 h photoperiod at a Photosynthetic Photon Flux Density ( PPFD) of 45–50 µmol m − 2 s − 1 provided by 40 W cool white fluorescent lamps, at 25 ± 2°C and 70% relative humidity until callus induction. Friable calli generated from the leaf culture were maintained aseptically by continuous subculture with hormones in an MS medium. Identification of apigenin and apigetrin from in vitro -grown callus We suspended one gram of dried powdered callus in 10 ml of ethyl acetate for 48 hours. The supernatant was collected, evaporated to dryness, dissolved in HPLC-grade methanol (Merck, India), and filtered through a 0.22 µM nylon syringe filter to eliminate cellular debris. These cell extracts were subjected to High-resolution liquid chromatography-mass spectrometry (HR-LCMS) to identify the compounds. We performed HR- LCMS using the 1290 Infinity UHPLC System, 1260 Infinity Nano HPLC with Chipcube, 6550 iFunnel Q-TOFs (Agilent Technologies, USA), and a Hypersil GOLD C18 column (100 x 2.1 mm-3 MICRON). The method used is reverse-phase gradient UHPLC-MS, starting from aqueous to organic and returning to initial conditions for separating flavonoids. The mobile phase A comprised 0.1% formic acid in water, and B comprised 90% acetonitrile and 10% 0.1% formic acid in water. Ten microliters of the sample were injected with a flow rate of 0.3 mL min − 1 and a pressure of 1200 bar throughout the process. We recorded the mass spectra in positive ion mode. For HPLC, we used a Shimadzu Prominence HPLC system equipped with an SPD M20 A PDA detector, a Prominence 20 AD pump, and a Phenomenex Gemini C18 column for the separation. The mobile phase was water: acetonitrile (92:8) with 2% orthophosphoric acid at a flow rate of 0.7 mL/ min and an injection volume of 20 µL, and apigenin and apigetrin were identified using their Reference standards (Sigma-Aldrich, Saint Louis, USA), by monitoring the peaks at 337 nm. Establishment and determination of the growth curve of cell suspension cultures The callus formed from the leaf explants was inoculated (2.5g) into a 250 ml flask containing 50 ml fresh medium supplemented with 2,4-D (1mg L − 1 ). They were kept on a rotary shaker at 80 rpm in complete darkness. The biomass was filtered using filter paper (Whatman No. 1), and fresh weight ( FW g L − 1 ) and dry weight (DW g L − 1 ) were measured every 8-day interval. Growth in cell suspension was monitored by weighing fresh and dry biomass, and growth curves were plotted. The filtered cells were washed with deionized water, and the FW was determined. Cells were dried below 60°C for 7–8 hours till the water was removed entirely to calculate the dry weight and powdered using a mortar and pestle. At each 8-day interval, the dried biomass was extracted with ethyl acetate, and the compound production was estimated through HPLC analyses using the genuine apigenin and apigetrin reference standards. Preparation of elicitors We used JA, MeJA, SA, and NaCl as elicitors for the present study. JA and MeJA were purchased from Sigma Aldrich, dissolved in 96% ethanol to prepare a stock solution, and sterilized by microfiltration. At the late-log phase, various elicitor concentrations were added individually to 40-day-old suspension cultures. For MeJA and JA elicitation studies, we selected concentrations of 20 µM, 50 µM, 100 µM, and 200 µM. We chose 50 µM, 100 µM, 200 µM, and 300 µM for SA elicitation, and 50 mM, 100 mM, 200 mM, and 300 mM concentrations for NaCl elicitation. We set different exposure times (2, 4, 6, and 8 days) for each set of treatments. After the specified exposure time, biomass was collected and extracted, and HPLC was performed. All the treatments were repeated thrice. Statistical analysis We did a one-way analysis of variance (one-way ANOVA) at a 0.05 significance level to check the influence of the above elicitors on apigenin and apigetrin accumulation in suspension cultures. A post-hoc test—Duncan's Multiple Range Test- was performed to compare the means. We performed all statistical analyses using SPSS Ver.20 (SPSS Inc., Chicago, IL, USA). All experimental data were expressed as mean ± SE of three independent replications. RESULTS We selected leaf explants for callus induction in J. gendarussa . Different growth regulators at various concentrations were applied to induce callus. We obtained the best calli on MS media enriched with 2 mg L − 1 2,4-D and 0.2 mg L − 1 BA, which were homogeneous, friable, and light green coloured (Fig. 1 ). We initiated cell suspension cultures from these calli. The growth-synchronized cells proliferated, and the growth parameters, such as viability and growth curve based on the fresh and dry weight of the filter-harvested cells, were measured for 64 days (Fig. 2 ). These data gave essential information for further culture management. We monitored biomass accumulation at an 8-day interval for 64 days (Fig. 2 ). Fresh weight increased significantly from day 24 to day 48, reaching a peak of 13.78 g, indicating an active log-growth phase of J. gendarussa in vitro cultures. Dry weight increased modestly throughout, with the highest accumulation (0.8 g) observed on day 56. Identification and quantification of apigenin and apigetrin HR- LC-MS analysis of the callus extract identified apigenin and apigetrin based on retention time, molecular ion peak, and fragmentation pattern. The ESI-MS spectrum of callus in positive ionization mode (Fig. S1b) showed a prominent molecular ion peak at m/z 271.0605 at the retention time of 3.606 min( Fig. S1a), which matched the protonated molecular ion of apigenin [M + H] + , with a molecular mass of 270.05 Da. In addition to the main ion peak, another notable peak was observed in the MS zoomed spectra at m/z 293.0622 [ M + Na] + , representing the sodium adduct. Water adducts were observed at m/z 275.0315 → [M + H + H2O] + . The absence of fragment ions corresponding to the glycosidic moiety confirmed apigenin. The second compound (Fig. S1c) was detected at a retention time of 13.551 min. The compound exhibited a key molecular ion peak at m/z 433.1064 [M + H] + , corresponding to the protonated form of apigetrin (C₂₁H₂₀O₁₀). An additional isotope peak at m/z 434.1181[M + H] + was detected, consistent with the natural ^13C abundance. In the fragmentation patterns (Fig. S1d), sodium adducts were observed at m/z 455.0852 [M + Na] +, and a dehydrated sodium adduct at m/z 437.0948 [M + Na − H2O] + , indicating the presence of an O-glycosidic moiety, confirming the compound as apigetrin. The HPLC chromatograms showed a single dominant peak at a retention time of 15.957 (Fig. S2a) and RT at 13.788 (Fig. S2 b), representing apigenin and apigetrin standards, respectively. Different plant parts, such as stem bark, leaves, and roots, were extracted with ethyl acetate to analyze and quantify apigenin and apigetrin. Apigenin and apigetrin accumulation was relatively high in leaves compared to the root and the stem bark (Fig. S2c, S2d, S2e). HPLC analysis of callus extracts divulged the presence of apigenin and apigetrin (Fig. S2g and S2h). Negligible amounts of these compounds were detected in the culture filtrate (Fig. S2f); hence, it was omitted from further studies. These compounds were quantified by plotting a calibration curve based on peak area (y-axis) versus standard concentration (x-axis) (Fig. S3a and S3 b). Kinetics of Apigenin and Apigetrin accumulation in cell suspension cultures Apigenin recorded a concentration of approximately 9 µg g⁻¹ DW, slightly higher than apigetrin (7 µg g⁻¹ DW) on day 8 (Fig. 3 ). From days 8 to 32, both apigenin and apigetrin showed a consistent and slow increase in their concentration. During this initial phase, the production of apigenin was higher than apigetrin in suspension cultures. By day 32, both compounds had increased to 16–17 µg g⁻¹ DW. Thereafter, apigetrin accumulated more efficiently and in higher concentrations than apigenin, with the highest quantities observed on day 56. Apigetrin content peaked at 45 µg·g⁻¹ DW, significantly higher than apigenin (23 µg·g⁻¹ DW) at the same time point, showing a 2-fold increase in the production of apigenin glucoside compared to its aglycone. The overtaking of apigenin production by apigetrin happened on the 40th day. After the 56th day, there was no significant increase in apigenin and apigetrin production. Enhancement of apigenin and apigetrin production by eliciting the suspension cultures with various abiotic elicitors Elicitation is one of the most efficient strategies for augmenting secondary metabolite content in medicinal plants. Here, we used methyl jasmonate, salicylic acid, jasmonic acid, and sodium chloride to test their relative abilities to enhance the production of apigenin and apigetrin in cell suspension cultures of J. gendarussa. We added 20–200 µM of MeJA to 40-day-old cultures. We monitored the output of apigenin and apigetrin every 2-day interval for 8 days (Fig. 4 ). Across all days, apigenin and apigetrin accumulated in greater quantities in methyl jasmonate-elicited cultures than in control cultures. We noticed a significant dose and time-dependent increase up to 200 µM; beyond that, the effect decreased or led to browning and cell death. Maximum apigenin and apigetrin production occurred on day 2 at 200 µM MeJA (apigenin 119.25 µg·g⁻¹ DW and apigetrin 342.87 µg·g⁻¹ DW). Compared to the control cultures, 200 µM MeJA showed approximately a 6-fold increase in the case of apigenin and an 11-fold rise in apigetrin. Likewise, 100 µM MeJA effectively accumulated eapigenin ( 117.79µg·g⁻¹ DW ) and apigetrin (332.72 µg·g⁻¹ DW ) on the 4th day of elicitation. JA elicitation influenced the accumulation of apigenin and apigetrin in J. gendarussa suspension cultures (Fig. 5 ). Among the tested concentrations, 100 µM JA enhanced the production of both compounds, with maximal accumulation observed on day 6 (apigetrin 114.68 µg g- 1 DW; apigenin 73.93 µg g¹ DW). Apigetrin levels were notably higher than apigenin across all treatments and time points. JA 100 µM, applied for 6 days, was optimal for secondary metabolite induction in the present study. In SA elicitation (Fig. 6 ), maximum concentrations of both compounds were recorded on day 4 at 100 µM SA, with apigetrin reaching 231.43µg g¹ DW (7-fold) and apigenin 106.57 µg g¹ DW (5-fold) compared to the control. Apigetrin levels were consistently higher than apigenin at all SA doses and time points. We noted a dose-dependent increase up to 100 µM, after which compound accumulation declined. 200µM SA induced the highest compound accumulation (apigenin – 146.31 µg g¹ DW and apigetrin 220.66 µg g¹ DW) on day 4, slightly lower than 100 µM SA. Day 4 is the optimal harvesting time for maximum flavonoid production. NaCl significantly boosted the production of apigenin and apigetrin in a time- and dose-dependent manner (Fig. 7 ). The peak in concentration for both metabolites was observed on day 6, particularly under 200 mM treatment, where apigetrin reached 245.31 µg g⁻¹ DW and apigenin 107.13 µg g⁻¹ DW. We consider 200 mM NaCl for 6 days the most effective NaCl treatment. A decline in metabolite levels was observed by day 8, indicating that day 6 represents the optimal harvest point under NaCl elicitation. 300 mM NaCl did not enhance the accumulation of compounds, possibly due to the toxicity. DISCUSSION The present study overproduces apigenin and apigetrin in J. gendarussa in vitro cultures. Apigenin is a safe biomolecule with multiple pharmacological effects and does not cause severe toxicity, even at high doses. In an acute toxicity assessment of apigenin in rats or mice, no mortality or signs of toxicity were observed at oral dosages up to 5000 mg/kg (Shoubaky et al., 2016 ). Furthermore, in vitro evaluation has shown that apigenin has no carcinogenic or mutagenic effects (Czeczot et al.,1990; Birt et al., 1986 ). Nevertheless, at high doses, it can cause sedation and muscle relaxation (Ross & Kasum, 2002 ). Apigetrin is an O-glucoside of apigenin at the 7-hydroxyl position, glycosylated by UDP-glucosyl transferases. The presence of this sugar group alters its chemical and metabolic properties (Jones & Vogt, 2001 ). Usually, glycosylation improves solubility, stability, and transport within the plant tissues (Manach et al., 2004 ). So, this sugar moiety makes apigetrin more water soluble than apigenin, ensuring that apigetrin is better suited for aqueous formulations and oral delivery. Active transport can absorb glycosides more efficiently in the small intestine (Walle, 2004 ). Moreover, the gut microflora or the enzymatic activity of β-glucosidases hydrolyze the weaker O-glycosidic bond of apigetrin to apigenin, making it biologically active (Del et al., 2010). Therefore, we can use apigetrin as a prodrug for the more biologically active apigenin. The selected plant, J. gendarussa , is recognized as a rich source of flavonoids, particularly apigenin. 6,8-di-C-α-Larabinosyl-apigenin, 6-C-α-L-arabinosyl-8-C-β-D-xylosyl-apigenin, and justidrusamides Apigenin and vitexin were isolated from methanolic extracts of J. gendarussa leaves ( Raghu and Agarwal, 2016). We find variations in metabolic content across different plant parts and their in vitro cultures in several plant species. For example, an indole alkaloid, ajmalicine, was found at higher levels in shoot cultures of Catharanthus roseus , whereas it was significantly lower or undetectable in root and callus cultures (Loyola & Ochoa, 2018). Similarly, diosgenin was present in plantlet and shoot cultures but absent in callus and root cultures of Costus speciosus , indicating tissue-specific metabolite accumulation (Indrayanto et al., 1994 ). Hitherto, no reports identified apigenin and its glycoside apigetrin in J. gendarussa in vitro cultures. Therefore, we initiated friable calli from J. gendarussa leaf explants. We optimized callus induction using MS medium with 2,4-D and BA based on previous reports of callus induction in J. gendarussa leaf explants ( Wahyuni et al., 2017 ; Wahyuni et al., 2019 ). The fresh and dry weight discrepancy in the growth of the callus suspension indicated water accumulation in the calli during culture. Fresh biomass reduced slightly after day 48, suggesting the onset of cell senescence and nutrient depletion. Subsequently, we confirmed the presence of apigenin and its glycoside in the callus through HR LC-MS. J. gendarussa suspension cultures (both stem and leaf) showed equal or slightly increased phenolic yields compared to plant tissues (leaf and stem) when extracted with methanol (Bhagya and Chandrasekhar (2013). Here, HPLC analyses of the leaf sample gave higher concentrations of apigenin and apigetrin than unelicited cell cultures. The decline of compound production on day 64 in suspension cultures suggests a possible metabolite conversion and degradation of the cells. Usually, seasonal variation, explant maturity, and soil composition significantly influence the biosynthesis of plant secondary metabolites. In J. gendarussa , collected from various locations in Indonesia, the metabolite profile was influenced by soil nutrient composition (Ningsih et al., 2015 ). We observed that the glycoside form was higher than its aglycone apigenin in the leaf tissues and cell suspension cultures, in alignment with previous reports where glycosides were found to be more abundant than aglycones, with ratios ranging from 5:1 to 10:1 depending on the plant species (Fabjan et al., 2003). Many secondary metabolites like flavonoids, terpenoids, and phenolics are more biologically active in the aglycone form, so plants glycosylate them to regulate their activity (Xiao, 2017 ). Also, plants frequently convert aglycones (reactive or toxic) into glucosides to detoxify and store them safely in vacuoles (Le Roy et al., 2016 ). Glucosides are more water-soluble than aglycones, which makes them easier to store in aqueous compartments like vacuoles or cytosol (Song, 2018). Thus, the accumulation of apigetrin in cell suspension cultures may be an intrinsic mechanism to maintain metabolic stability and mitigate adverse effects like nutrient depletion. Elicitation upregulates flavonoid biosynthesis through transcriptional activation of key enzymes like Flavonol Synthase (FLS) and Chalcone Synthase (CHS). An early accumulation of aglycones by elicitation paves the way for improved glycoside formation as and when glycosyl transferase enzymes (UGTs) are activated. MeJA between 100–200 µM concentrations effectively enhanced flavonoid production in various plant cell cultures. For instance, in Glycyrrhiza inflata , 100 µM MeJA elicitation increased glycyrrhizin production in hairy root cultures (Li et al., 2013). In Hypericum perforatum , 100 µM MeJA treatment on day 15 resulted in the highest flavonoid production, i.e., a 2.7-fold increase compared to control cultures (Wang et al., 2015 ). Also, maximum quercetin content was observed at 100 µM MeJA concentration in Allium cepa (Iqbal et al., 2019 ). Likewise, 200 µM MeJA enhanced ginsenoside accumulation in Panax ginseng suspension cultures (Yu et al., 2005 ). In the present study, 200 µM MeJA for 2 days resulted in the highest accumulation of apigenin and apigetrin. Similarly, in Cardiospermum halicacabum , 200 µM MeJA effectively enhanced apigenin production (7.3-fold increase) in embryogenic suspension cultures (Harisaranra et al., 2008). Moreover, a considerable difference between apigenin and apigetrin content was observed after MeJA treatment, primarily due to the upregulation of glucosyl transferases triggered by MeJA. Similar effects were observed in Glycyrrhiza uralensis , where MeJA significantly increased flavonoid aglycones initially; over time, its glucoside was dominated by the activation of glycosylation enzymes ( Chen et al., 2019 ). Likewise, MeJA initially increased aglycone baicalein and glycoside baicalin; later, baicalin accumulated over time in Scutellaria baicalensis (Zhao et al., 2005 ). In our study, Apigetrin levels vastly overtook apigenin across all treatments. However, the apigenin and apigetrin production varied depending on the type of elicitor, its concentration, and the exposure time. We gave the same concentrations and exposure time for MeJa and jasmonic acid elicitation. However, MeJA gave better results due to its volatile nature and efficient membrane permeability. Hence, we can surmise that it is an effective elicitor for the rapid induction of apigenin and apigetrin in the cell suspension culture of J. gendarussa compared to JA. Contrarily, JA elicitation in Glycyrrhiza glabra effectively increased flavonoid production in callus cultures (Rani et al., 2021 ). JA treatment boosted the output of wogonoside, a flavonoid glucoside, in Scutellaria lateriflora (Wilczańska et al., 2023 ). 50 µM JA elevated flavonoid content by 72% over the control, and when combined with 10 mM CaCl₂, levels rose by 227% in Trifolium pratense (Kašparová & Siatka, 2014 ). The mode of action of both MeJA and jasmonic acid is similar. They bind to the COI1-JAZ receptor complex, destroying JAZ repressors, which release transcription factors like MYC2 that turn on flavonoid biosynthetic genes (Wasternack & Hause, 2013 ). MeJA enters cells quickly and triggers higher production of secondary metabolites. However, JA is less active or slow-acting because of its low stability and non-volatile nature (Ho et al., 2020 ). Like MeJA, SA also triggers phenolics and flavonoid accumulation. The SA elicitation is through NPR1-dependent signaling, triggering WRKY and TGA transcription factors, which boost the expression of key enzymes for flavonoid biosynthesis, such as Phenylalanine Ammonia Lyase, Chalcone Synthase, and Flavonol Synthase (Johnson et al., 2003 ). In the present study, 100µM SA worked best. The effective concentration of SA elicitation is 100–300 µM for enhancing flavonoid production. An elicitation with 100 µM SA enhanced phenolic production, including flavonoids, and improved biological activities in Momordica dioica cell suspension cultures (Chung et al., 2017 ). SA treatment at 300 µM significantly increased flavonoid content and antioxidant activity in cell suspension cultures of Thevetia peruviana (Mendoza et al., 2018 ). NaCl is perceived as an abiotic stress, creating osmotic and ionic stress and rapidly increasing ROS levels. ROS are secondary messengers that activate stress-responsive transcription factors such as MYB, WRKY, and DREB, which upregulate genes in the phenylpropanoid pathway (Gharibi et al., 2019 ). NaCl elicitation at 100–150 mM concentrations significantly increased the flavonoid rutin and upregulated genes involved in flavonoid biosynthesis in Haplophyllum virgatum suspension cultures (Abedi et al., 2023 ). Flavonoid content peaked at 129–172 mM NaCl concentrations, indicating enhanced catechin and epicatechin content in Rumex thyrsiflorus suspension cultures, though concentrations above 300 mM led to a waning in flavonoid content (Gozdur et al., 2024 ). CONCLUSION Medicinal plants treat diseases as they have countless pharmacological properties owing to the presence of various bioactive compounds. The limited availability of these compounds in intact plants warrants increasing production by many approaches, including eliciting secondary phyto-molecules in vitro . Apigenin and apigetrin are two important flavonoid medicinal compounds in J. gendarussa . In this work, we induced soft, friable calli to initiate cell suspension cultures of J. gendarussa. Various abiotic elicitors, MeJA, SA, JA, and NaCl, were tried at different concentrations and time intervals to enhance apigenin and apigetrin production in suspension cultures of J. gendarussa . 200 µM MeJA for 2 days and 200 mM NaCl for 6 days yielded the highest apigenin and apigetrin production. Among these, methyl jasmonate is the most reliable, effective, and fast-acting elicitor for producing apigenin and apigetrin in J. gendarussa in vitro cultures. Further investigations into the alternative culture systems for enhanced production of apigenin and apigetrin are the prospects. Declarations The authors have no relevant financial or non-financial interests to disclose. The authors have no competing interests to declare relevant to this article's content. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article. Author Contribution Statement Vinaya Chndran And Linu Mathew were involved in the conception and design; Vinaya Chandran, Maya Rajan, Shahena S, and Linu Mathew in the analysis and interpretation of the data; the drafting of the paper, revising it critically for intellectual content; and the final approval of the version to be published; and we agree to be accountable for all aspects of the work. Acknowledgments We acknowledge the instrumentation help rendered by DST-FIST, DST-PURSE, and DBT-BUILDER, School of Biosciences, MG University, Kottayam, Kerala, India; SAIF, MG University, Kottayam, Kerala, India, and SAIF, IIT Bombay, India. Data Availability Statement All data supporting the findings of this study are available within the paper and its Supplementary Information (Figure S1. S2 and S3). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6782131","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466040225,"identity":"fbc86561-1662-4cfc-a1ef-e90935426dba","order_by":0,"name":"Vinaya Chandran","email":"","orcid":"","institution":"Mahatma Gandhi University","correspondingAuthor":false,"prefix":"","firstName":"Vinaya","middleName":"","lastName":"Chandran","suffix":""},{"id":466040226,"identity":"93663e21-dace-47dc-97b3-72e10fca98ce","order_by":1,"name":"Maya Rajan","email":"","orcid":"","institution":"Mahatma Gandhi University","correspondingAuthor":false,"prefix":"","firstName":"Maya","middleName":"","lastName":"Rajan","suffix":""},{"id":466040227,"identity":"9903121d-f80c-47b9-b87b-9bfce62025c9","order_by":2,"name":"Shahena S","email":"","orcid":"","institution":"Mahatma Gandhi University","correspondingAuthor":false,"prefix":"","firstName":"Shahena","middleName":"","lastName":"S","suffix":""},{"id":466040228,"identity":"b7c04803-fcb8-45a0-be3e-1d25f360bba0","order_by":3,"name":"Linu Mathew","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACPmYIncDAwHwAKsbYgFcLG0SLAVALWwKRWhjgWngMiHMYGzvvMakbNX/y+CVyvm782sYgz9/A3PYAv8P40qRzjhkUS87I3XZbto3BcMYBxna89rEx85hJ57AZJG64DdQi2cbAuIGBsU2CsJZ/Bon7b+c8A2mxJ05LbhvQFqBdNz+2MSQSo8XYOrfPOHHG/WdmtxnOSSTPOExACz//GcPbOd/kEvt7Dj+7+aPMxra/vf0ZXi0ogJmHAaiYmWj1QMD4gxTVo2AUjIJRMGIAAPUcQcS2eOc+AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3727-6938","institution":"Mahatma Gandhi University","correspondingAuthor":true,"prefix":"","firstName":"Linu","middleName":"","lastName":"Mathew","suffix":""}],"badges":[],"createdAt":"2025-05-30 07:26:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6782131/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6782131/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-025-03185-1","type":"published","date":"2025-10-15T15:58:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84196194,"identity":"7baa0797-de70-46e2-9050-67cdc600333d","added_by":"auto","created_at":"2025-06-09 07:48:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11169,"visible":true,"origin":"","legend":"\u003cp\u003eCallus culture of \u003cem\u003eJ. gendarussa\u003c/em\u003e leaf on MS medium supplemented with 2 mg L\u003csup\u003e-1\u003c/sup\u003e 2,4-D and 0.2 mg L\u003csup\u003e-1\u003c/sup\u003e BA, incubated at 25 ± 2°C\u0026nbsp;\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6782131/v1/153d7d2ef75eba16ddc05904.jpg"},{"id":84195893,"identity":"4dae8920-7cee-4e90-b409-0052a204f0a2","added_by":"auto","created_at":"2025-06-09 07:40:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16634,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth curve of cell suspension cultures of \u003cem\u003eJ. gendarussa\u003c/em\u003e based on fresh and dry weight. Data represent mean ± SE of three experiments.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6782131/v1/d990ccff25d03e3cd26cf5e6.jpg"},{"id":84195888,"identity":"67b37a74-f554-447f-8ba8-5b5cd4fe84ba","added_by":"auto","created_at":"2025-06-09 07:40:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":293046,"visible":true,"origin":"","legend":"\u003cp\u003eTime course of Apigenin and Apigetrin production in cell suspension cultures of \u003cem\u003eJ. gendarussa\u003c/em\u003e Data represent the mean ± SE of three replicates, and the means with the same letter are not significantly different according to Duncan's multiple range test at \u0026nbsp;\u003cem\u003eP ≤ 0.0\u003c/em\u003e5\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6782131/v1/b294dacd4c062bc198bb91da.jpg"},{"id":84196196,"identity":"285dcca2-8433-4276-a699-f2346e729c20","added_by":"auto","created_at":"2025-06-09 07:48:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":323323,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Methyl Jasmonate elicitation on cell suspension cultures of \u003cem\u003eJ. gendarussa\u003c/em\u003eMeans ± SE of three replicates followed by the same letters are not significantly different according to Duncan's multiple range test at \u003cem\u003eP ≤ 0.0\u003c/em\u003e5\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6782131/v1/c26651d26a9fb4383a8b5299.jpg"},{"id":84195877,"identity":"3aa16cb8-53e0-4ea8-8dda-03e71b0c72f7","added_by":"auto","created_at":"2025-06-09 07:39:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":359615,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Jasmonic acid elicitation on cell suspension cultures of \u003cem\u003eJ. gendarussa\u003c/em\u003e Means ± SE of three replicates followed by the same letters are not significantly different according to Duncan's multiple range test at \u003cem\u003eP ≤ 0.05\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6782131/v1/5b145516094180c72105cdf0.jpg"},{"id":84195899,"identity":"89df429e-4dfc-4bef-820c-f17330a96fda","added_by":"auto","created_at":"2025-06-09 07:40:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":283686,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Salicylic acid elicitation on cell suspension cultures of \u003cem\u003eJ. gendarussa\u003c/em\u003eMeans ± SE of three replicates followed by the same letters are not significantly different according to Duncan's multiple range test at \u003cem\u003eP ≤ 0.05\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6782131/v1/49556a78454fdb81da306ec6.jpg"},{"id":84195878,"identity":"8ee9edd1-f021-4871-a34e-03ea89bf1ca5","added_by":"auto","created_at":"2025-06-09 07:39:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":302958,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Sodium chloride elicitation on cell suspension cultures of \u003cem\u003eJ. gendarussa\u003c/em\u003eMeans ± SE of three replicates followed by the same letters are not significantly different according to Duncan's multiple range test at \u003cem\u003eP ≤ 0.05\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6782131/v1/fdec1613890cb258ef294721.jpg"},{"id":93956765,"identity":"1ff0ee4f-aa33-48da-aa70-58eeaccd25f1","added_by":"auto","created_at":"2025-10-20 16:12:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2351514,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6782131/v1/6277ed6a-6028-40dd-b560-c02166d38b79.pdf"},{"id":84196195,"identity":"97793196-d863-413f-8bc1-2210c2634ed1","added_by":"auto","created_at":"2025-06-09 07:48:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":299690,"visible":true,"origin":"","legend":"","description":"","filename":"supplimentary.docx","url":"https://assets-eu.researchsquare.com/files/rs-6782131/v1/b2c9a56756cd17762313cea0.docx"}],"financialInterests":"","formattedTitle":"Elicitation using Jasmonates, Salicylic acid, and Sodium chloride in cell suspension cultures of Justicia gendarussa Burm f.: A route for enhanced and sustainable production of apigenin and apigetrin","fulltext":[{"header":"Key Message","content":"\u003cp\u003eMeJA, JA, SA, and NaCl elicitation boosts bioactive flavonoids, namely, apigenin and apigetrin, in \u003cem\u003eJ. gendarussa\u003c/em\u003e cell suspension cultures, with MeJA at 200 µM being the most effective.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cem\u003eJusticia gendarussa\u003c/em\u003e Burm f. is a perennial shrub of 1-1.5 meters growing in marshy areas across Southeast Asia. This plant has medicinal properties, with all parts utilized as medicines (Kavitha et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mnatsakanyan et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The plant contains bioactive substances like phenolic compounds, steroids, terpenoids, alkaloids, and flavonoids. The leaves treat diarrhea, fever, persistent cough, hypertension, and bone pain as they contain friedelin, lupeol, \u0026szlig;-sitosterol, and aromatic amines (Pal \u0026amp; Rahaman, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and possess anti-inflammatory, anti-bacterial, anti-fungal, antioxidant, and anti-angiogenic properties. Likewise, aerial parts of this plant showed HIV type-1 reverse transcriptase inhibition \u003cem\u003ein vitro\u003c/em\u003e (Woradulayapinij et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eApigenin (4\u0026prime;,5,7-trihydroxy flavone) is one of the key flavonoids present in fruits and vegetables, which is the aglycone of several natural glycosides with a chemical formula of C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e and a molecular weight of 270.24 (Salehi et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has three hydroxyl groups, the first and second in the C5 and C7 positions and the third at C4\u0026prime; of the B ring (Allemailem et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Apigenin treats diseases of the liver, lung, heart, and kidney, neurological diseases, and diabetes, and maintains good oral and skin health (Bi et al.,2023; Hu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Thomas et al.,2023; Siddique et al.,2025). This compound suppresses cancers by inducing apoptosis and cell-cycle arrest, inhibiting cell migration and angiogenesis, and reducing inflammation (Zhu et al.,2013; Naponelli et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Apigenin synergizes with other drugs, reduces the risk of side effects, and improves the chemotherapy response (Xu et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, due to its lipophilic nature, the acidic environment of the gastrointestinal tract may inactivate it, resulting in reduced bioavailability and limiting its potential use in medicines and nutraceuticals. The apigenin glycosides show greater aqueous solubility, biological activity, and binding affinity to specific targets (Kamel et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The common apigenin glycosides are apigetrin (apigenin-7-O-glucoside), vitexin (apigenin-8-C glucoside), isovitexin (apigenin-6-C-glucoside), and rhoifolin (apigenin-7-O-neohesperidoside) (Ali et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Among them, apigetrin and vitexin were reported in \u003cem\u003eJ. gendarussa\u003c/em\u003e (Raghu and Agarwal, 2016; Zhang et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Apigetrin or apigenin 7-O-glucoside is the most commonly found 'glucoside' in plants, has significant anti-inflammatory properties, and inhibits the pro-inflammatory cytokines such as TNF-α and IL-6 (Guo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Also, it prevents oxidative stress-related diseases, such as cardiovascular diseases and neurodegeneration, and protects the liver (Wojdyło et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It induces apoptosis and prevents cancer cell proliferation (Chahar et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e plant cell, organ, and tissue cultures are one of the most preferred systems for producing bioactive compounds. Elicitation in plant cells or tissue culture produces various secondary metabolites in large quantities in a limited time and space, continuously turning out commercially valuable and pharmaceutically active bio-compounds. The technique is sustainable and independent of geographical or seasonal variation, and modifying culture parameters can upscale product accumulation.\u003c/p\u003e \u003cp\u003eElicitors induce a specific biological response in plants by triggering various cellular processes, including defense responses, signal transduction pathways, and the production of secondary metabolites (Akula \u0026amp; Ravishankar, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In \u003cem\u003ein vitro\u003c/em\u003e medicinal plant cultures, biotic and abiotic elicitors alter or stimulate the metabolic pathways to produce novel/existing bioactive compounds (Namdeo, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Elicitors signal membrane-bound receptors, activate the signal transduction pathways, and change the expression of regulatory players, resulting in enhanced synthesis and accumulation of phyto-compounds (Petrova et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eJasmonates, including jasmonic acid (JA) and its methyl ester methyl jasmonate (MeJA), are a family of cyclopentanone compounds from linolenic acid, modulating a wide range of plant responses (Creelman \u0026amp; Mullet, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and act as effective elicitors to enhance secondary metabolites \u003cem\u003ein vitro\u003c/em\u003e (Sembdner \u0026amp; Parthier, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). MeJA, a volatile methyl ester, is an effective signaling molecule in abiotic and biotic stresses (Wang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) because it can permeate cell membranes easily and demethylates quickly to release free JA (Fattorini et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is a vital player in intra- and inter-communications in plant cells, increasing the antioxidant enzyme activity and the expression of defense genes (Ho et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Salicylic acid is a phenolic compound that has roles in various developmental and physiological processes, including ethylene production, stomatal movements, photosynthesis, membrane functions, pigment accumulation, enzyme activities, plant growth, and development (Ali, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hence, SA and its related precursors are classified as phytohormones (Koo et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). SA upregulates the genes for Phenylalanine Ammonia Lyase (PAL), Chalcone Synthase (CHS), and Flavonol Synthase (FLS), enhancing flavonoid accumulation (Ali et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It acts early in stress, sometimes with synergistic effects with other elicitors (Woch et al.,2023). Rajan et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that MeJA and SA enhance rhamnetin production in \u003cem\u003eVernonia anthelmintica\u003c/em\u003e cultures. Also, MeJA and SA stimulated the biosynthesis of alkaloids and flavonoids in \u003cem\u003ePiper cumanense\u003c/em\u003e, with MeJA producing a noticeable effect (Rodr\u0026iacute;guez et al., 2020). NaCl is a readily available and inexpensive abiotic elicitor with proven efficacy (Kitayama et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Salinity induces ionic and osmotic stress in plants. The intensity of the salt stress elicits a salinity response in plants with reversible or irreversible changes in plant function (Hawrylak-Nowak et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although excess salt can be toxic to plants, moderate salinity has a stimulatory effect on plant growth and the accumulation of health-promoting antioxidants and nutrients (Gupta \u0026amp; Huang, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Different concentrations of NaCl (50\u0026ndash;200 mM) enhanced the synthesis of soluble sugars, proteins, and bioactive phenolics in \u003cem\u003eInula crithmoides\u003c/em\u003e suspension cultures (Rodr\u0026iacute;guez et al., 2024). The present study explores the enhanced and sustainable production of apigenin and apigetrin by using abiotic elicitors in cell suspension cultures of \u003cem\u003eJ. gendarussa.\u003c/em\u003e\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Collection\u003c/h2\u003e \u003cp\u003eWe collected asymptomatic plants of \u003cem\u003eJ. gendarussa\u003c/em\u003e from the garden premises of Mahatma Gandhi University, Kottayam, Kerala. For the taxonomical identification, the herbarium was submitted with an accession number 7640 at the Regional Herbarium of Kerala, St Berchmans College, Changanassery, Kerala, India.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMedia preparation and Callus induction\u003c/h3\u003e\n\u003cp\u003eLeaf explants were disinfected using 70% (v/v) ethanol for 1 min, followed by 0.005% aqueous mercuric chloride solution for 1.5 min, repeatedly rinsed in sterile distilled water to remove excess mercuric chloride, and kept in sterile filter paper to eliminate excess water. We used MS media with 30 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e sucrose and 8 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e agar with different concentrations and combinations of plant hormones for the callus induction. The P\u003csup\u003eH\u003c/sup\u003e of the media was 5.6\u0026ndash;5.8, adjusted using 1 N NaOH before adding agar. After adding syringe-filtered hormones, 10 ml of autoclaved media was dispersed into boiling tubes and stored. Surface-sterilized explants were cut into small squares approximately 1 cm x 1 cm, inoculated into stored MS medium, and incubated under a 16/8 h photoperiod at a Photosynthetic Photon Flux Density ( PPFD) of 45\u0026ndash;50 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e provided by 40 W cool white fluorescent lamps, at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 70% relative humidity until callus induction. Friable calli generated from the leaf culture were maintained aseptically by continuous subculture with hormones in an MS medium.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of apigenin and apigetrin from\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003cb\u003e-grown callus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe suspended one gram of dried powdered callus in 10 ml of ethyl acetate for 48 hours. The supernatant was collected, evaporated to dryness, dissolved in HPLC-grade methanol (Merck, India), and filtered through a 0.22 \u0026micro;M nylon syringe filter to eliminate cellular debris. These cell extracts were subjected to High-resolution liquid chromatography-mass spectrometry (HR-LCMS) to identify the compounds. We performed HR- LCMS using the 1290 Infinity UHPLC System, 1260 Infinity Nano HPLC with Chipcube, 6550 iFunnel Q-TOFs (Agilent Technologies, USA), and a Hypersil GOLD C18 column (100 x 2.1 mm-3 MICRON). The method used is reverse-phase gradient UHPLC-MS, starting from aqueous to organic and returning to initial conditions for separating flavonoids. The mobile phase A comprised 0.1% formic acid in water, and B comprised 90% acetonitrile and 10% 0.1% formic acid in water. Ten microliters of the sample were injected with a flow rate of 0.3 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a pressure of 1200 bar throughout the process. We recorded the mass spectra in positive ion mode.\u003c/p\u003e \u003cp\u003eFor HPLC, we used a Shimadzu Prominence HPLC system equipped with an SPD M20 A PDA detector, a Prominence 20 AD pump, and a Phenomenex Gemini C18 column for the separation. The mobile phase was water: acetonitrile (92:8) with 2% orthophosphoric acid at a flow rate of 0.7 mL/ min and an injection volume of 20 \u0026micro;L, and apigenin and apigetrin were identified using their Reference standards (Sigma-Aldrich, Saint Louis, USA), by monitoring the peaks at 337 nm.\u003c/p\u003e\n\u003ch3\u003eEstablishment and determination of the growth curve of cell suspension cultures\u003c/h3\u003e\n\u003cp\u003eThe callus formed from the leaf explants was inoculated (2.5g) into a 250 ml flask containing 50 ml fresh medium supplemented with 2,4-D (1mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). They were kept on a rotary shaker at 80 rpm in complete darkness. The biomass was filtered using filter paper (Whatman No. 1), and fresh weight ( FW g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and dry weight (DW g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were measured every 8-day interval. Growth in cell suspension was monitored by weighing fresh and dry biomass, and growth curves were plotted. The filtered cells were washed with deionized water, and the FW was determined. Cells were dried below 60\u0026deg;C for 7\u0026ndash;8 hours till the water was removed entirely to calculate the dry weight and powdered using a mortar and pestle. At each 8-day interval, the dried biomass was extracted with ethyl acetate, and the compound production was estimated through HPLC analyses using the genuine apigenin and apigetrin reference standards.\u003c/p\u003e\n\u003ch3\u003ePreparation of elicitors\u003c/h3\u003e\n\u003cp\u003eWe used JA, MeJA, SA, and NaCl as elicitors for the present study. JA and MeJA were purchased from Sigma Aldrich, dissolved in 96% ethanol to prepare a stock solution, and sterilized by microfiltration. At the late-log phase, various elicitor concentrations were added individually to 40-day-old suspension cultures. For MeJA and JA elicitation studies, we selected concentrations of 20 \u0026micro;M, 50 \u0026micro;M, 100 \u0026micro;M, and 200 \u0026micro;M. We chose 50 \u0026micro;M, 100 \u0026micro;M, 200 \u0026micro;M, and 300 \u0026micro;M for SA elicitation, and 50 mM, 100 mM, 200 mM, and 300 mM concentrations for NaCl elicitation. We set different exposure times (2, 4, 6, and 8 days) for each set of treatments. After the specified exposure time, biomass was collected and extracted, and HPLC was performed. All the treatments were repeated thrice.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eWe did a one-way analysis of variance (one-way ANOVA) at a 0.05 significance level to check the influence of the above elicitors on apigenin and apigetrin accumulation in suspension cultures. A post-hoc test\u0026mdash;Duncan's Multiple Range Test- was performed to compare the means. We performed all statistical analyses using SPSS Ver.20 (SPSS Inc., Chicago, IL, USA). All experimental data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE of three independent replications.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eWe selected leaf explants for callus induction in \u003cem\u003eJ. gendarussa\u003c/em\u003e. Different growth regulators at various concentrations were applied to induce callus. We obtained the best calli on MS media enriched with 2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e 2,4-D and 0.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e BA, which were homogeneous, friable, and light green coloured (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We initiated cell suspension cultures from these calli. The growth-synchronized cells proliferated, and the growth parameters, such as viability and growth curve based on the fresh and dry weight of the filter-harvested cells, were measured for 64 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These data gave essential information for further culture management.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe monitored biomass accumulation at an 8-day interval for 64 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Fresh weight increased significantly from day 24 to day 48, reaching a peak of 13.78 g, indicating an active log-growth phase of \u003cem\u003eJ. gendarussa in vitro\u003c/em\u003e cultures. Dry weight increased modestly throughout, with the highest accumulation (0.8 g) observed on day 56.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIdentification and quantification of apigenin and apigetrin\u003c/h3\u003e\n\u003cp\u003eHR- LC-MS analysis of the callus extract identified apigenin and apigetrin based on retention time, molecular ion peak, and fragmentation pattern. The ESI-MS spectrum of callus in positive ionization mode (Fig. S1b) showed a prominent molecular ion peak at \u003cem\u003em/z\u003c/em\u003e 271.0605 at the retention time of 3.606 min( Fig. S1a), which matched the protonated molecular ion of apigenin [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e, with a molecular mass of 270.05 Da. In addition to the main ion peak, another notable peak was observed in the MS zoomed spectra at m/z 293.0622 [ M\u0026thinsp;+\u0026thinsp;Na] \u003csup\u003e+\u003c/sup\u003e, representing the sodium adduct. Water adducts were observed at m/z 275.0315 \u0026rarr; [M\u0026thinsp;+\u0026thinsp;H\u0026thinsp;+\u0026thinsp;H2O] \u003csup\u003e+\u003c/sup\u003e. The absence of fragment ions corresponding to the glycosidic moiety confirmed apigenin. The second compound (Fig. S1c) was detected at a retention time of 13.551 min. The compound exhibited a key molecular ion peak at \u003cem\u003em/z\u003c/em\u003e 433.1064 [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e, corresponding to the protonated form of apigetrin (C₂₁H₂₀O₁₀). An additional isotope peak at \u003cem\u003em/z\u003c/em\u003e 434.1181[M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e was detected, consistent with the natural ^13C abundance. In the fragmentation patterns (Fig. S1d), sodium adducts were observed at \u003cem\u003em/z\u003c/em\u003e 455.0852 [M\u0026thinsp;+\u0026thinsp;Na] \u003csup\u003e+,\u003c/sup\u003e and a dehydrated sodium adduct at \u003cem\u003em/z\u003c/em\u003e 437.0948 [M\u0026thinsp;+\u0026thinsp;Na\u0026thinsp;\u0026minus;\u0026thinsp;H2O] \u003csup\u003e+\u003c/sup\u003e, indicating the presence of an O-glycosidic moiety, confirming the compound as apigetrin. The HPLC chromatograms showed a single dominant peak at a retention time of 15.957 (Fig. S2a) and RT at 13.788 (Fig. S2 b), representing apigenin and apigetrin standards, respectively. Different plant parts, such as stem bark, leaves, and roots, were extracted with ethyl acetate to analyze and quantify apigenin and apigetrin. Apigenin and apigetrin accumulation was relatively high in leaves compared to the root and the stem bark (Fig. S2c, S2d, S2e). HPLC analysis of callus extracts divulged the presence of apigenin and apigetrin (Fig. S2g and S2h). Negligible amounts of these compounds were detected in the culture filtrate (Fig. S2f); hence, it was omitted from further studies. These compounds were quantified by plotting a calibration curve based on peak area (y-axis) versus standard concentration (x-axis) (Fig. S3a and S3 b).\u003c/p\u003e\n\u003ch3\u003eKinetics of Apigenin and Apigetrin accumulation in cell suspension cultures\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eApigenin recorded a concentration of approximately 9 \u0026micro;g g⁻\u0026sup1; DW, slightly higher than apigetrin (7 \u0026micro;g g⁻\u0026sup1; DW) on day 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). From days 8 to 32, both apigenin and apigetrin showed a consistent and slow increase in their concentration. During this initial phase, the production of apigenin was higher than apigetrin in suspension cultures. By day 32, both compounds had increased to 16\u0026ndash;17 \u0026micro;g g⁻\u0026sup1; DW. Thereafter, apigetrin accumulated more efficiently and in higher concentrations than apigenin, with the highest quantities observed on day 56. Apigetrin content peaked at 45 \u0026micro;g\u0026middot;g⁻\u0026sup1; DW, significantly higher than apigenin (23 \u0026micro;g\u0026middot;g⁻\u0026sup1; DW) at the same time point, showing a 2-fold increase in the production of apigenin glucoside compared to its aglycone. The overtaking of apigenin production by apigetrin happened on the 40th day. After the 56th day, there was no significant increase in apigenin and apigetrin production.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEnhancement of apigenin and apigetrin production by eliciting the suspension cultures with various abiotic elicitors\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eElicitation is one of the most efficient strategies for augmenting secondary metabolite content in medicinal plants. Here, we used methyl jasmonate, salicylic acid, jasmonic acid, and sodium chloride to test their relative abilities to enhance the production of apigenin and apigetrin in cell suspension cultures of \u003cem\u003eJ. gendarussa.\u003c/em\u003e We added 20\u0026ndash;200 \u0026micro;M of MeJA to 40-day-old cultures. We monitored the output of apigenin and apigetrin every 2-day interval for 8 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Across all days, apigenin and apigetrin accumulated in greater quantities in methyl jasmonate-elicited cultures than in control cultures. We noticed a significant dose and time-dependent increase up to 200 \u0026micro;M; beyond that, the effect decreased or led to browning and cell death. Maximum apigenin and apigetrin production occurred on day 2 at 200 \u0026micro;M MeJA (apigenin 119.25 \u0026micro;g\u0026middot;g⁻\u0026sup1; DW and apigetrin 342.87 \u0026micro;g\u0026middot;g⁻\u0026sup1; DW). Compared to the control cultures, 200 \u0026micro;M MeJA showed approximately a 6-fold increase in the case of apigenin and an 11-fold rise in apigetrin. Likewise, 100 \u0026micro;M MeJA effectively accumulated eapigenin ( 117.79\u0026micro;g\u0026middot;g⁻\u0026sup1; DW ) and apigetrin (332.72 \u0026micro;g\u0026middot;g⁻\u0026sup1; DW ) on the 4th day of elicitation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eJA elicitation influenced the accumulation of apigenin and apigetrin in \u003cem\u003eJ. gendarussa\u003c/em\u003e suspension cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Among the tested concentrations, 100 \u0026micro;M JA enhanced the production of both compounds, with maximal accumulation observed on day 6 (apigetrin 114.68 \u0026micro;g g-\u003csup\u003e1\u003c/sup\u003e DW; apigenin 73.93 \u0026micro;g g\u0026sup1; DW). Apigetrin levels were notably higher than apigenin across all treatments and time points. JA 100 \u0026micro;M, applied for 6 days, was optimal for secondary metabolite induction in the present study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn SA elicitation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e ), maximum concentrations of both compounds were recorded on day 4 at 100 \u0026micro;M SA, with apigetrin reaching 231.43\u0026micro;g g\u0026sup1; DW (7-fold) and apigenin 106.57 \u0026micro;g g\u0026sup1; DW (5-fold) compared to the control. Apigetrin levels were consistently higher than apigenin at all SA doses and time points. We noted a dose-dependent increase up to 100 \u0026micro;M, after which compound accumulation declined. 200\u0026micro;M SA induced the highest compound accumulation (apigenin \u0026ndash; 146.31 \u0026micro;g g\u0026sup1; DW and apigetrin 220.66 \u0026micro;g g\u0026sup1; DW) on day 4, slightly lower than 100 \u0026micro;M SA. Day 4 is the optimal harvesting time for maximum flavonoid production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNaCl significantly boosted the production of apigenin and apigetrin in a time- and dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The peak in concentration for both metabolites was observed on day 6, particularly under 200 mM treatment, where apigetrin reached 245.31 \u0026micro;g g⁻\u0026sup1; DW and apigenin 107.13 \u0026micro;g g⁻\u0026sup1; DW. We consider 200 mM NaCl for 6 days the most effective NaCl treatment. A decline in metabolite levels was observed by day 8, indicating that day 6 represents the optimal harvest point under NaCl elicitation. 300 mM NaCl did not enhance the accumulation of compounds, possibly due to the toxicity.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study overproduces apigenin and apigetrin in \u003cem\u003eJ. gendarussa in vitro\u003c/em\u003e cultures. Apigenin is a safe biomolecule with multiple pharmacological effects and does not cause severe toxicity, even at high doses. In an acute toxicity assessment of apigenin in rats or mice, no mortality or signs of toxicity were observed at oral dosages up to 5000 mg/kg (Shoubaky et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, \u003cem\u003ein vitro\u003c/em\u003e evaluation has shown that apigenin has no carcinogenic or mutagenic effects (Czeczot et al.,1990; Birt et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Nevertheless, at high doses, it can cause sedation and muscle relaxation (Ross \u0026amp; Kasum, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Apigetrin is an O-glucoside of apigenin at the 7-hydroxyl position, glycosylated by UDP-glucosyl transferases. The presence of this sugar group alters its chemical and metabolic properties (Jones \u0026amp; Vogt, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Usually, glycosylation improves solubility, stability, and transport within the plant tissues (Manach et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). So, this sugar moiety makes apigetrin more water soluble than apigenin, ensuring that apigetrin is better suited for aqueous formulations and oral delivery. Active transport can absorb glycosides more efficiently in the small intestine (Walle, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Moreover, the gut microflora or the enzymatic activity of β-glucosidases hydrolyze the weaker O-glycosidic bond of apigetrin to apigenin, making it biologically active (Del et al., 2010). Therefore, we can use apigetrin as a prodrug for the more biologically active apigenin.\u003c/p\u003e \u003cp\u003eThe selected plant, \u003cem\u003eJ. gendarussa\u003c/em\u003e, is recognized as a rich source of flavonoids, particularly apigenin. 6,8-di-C-α-Larabinosyl-apigenin, 6-C-α-L-arabinosyl-8-C-β-D-xylosyl-apigenin, and justidrusamides Apigenin and vitexin were isolated from methanolic extracts of \u003cem\u003eJ. gendarussa\u003c/em\u003e leaves ( Raghu and Agarwal, 2016). We find variations in metabolic content across different plant parts and their \u003cem\u003ein vitro\u003c/em\u003e cultures in several plant species. For example, an indole alkaloid, ajmalicine, was found at higher levels in shoot cultures of \u003cem\u003eCatharanthus roseus\u003c/em\u003e, whereas it was significantly lower or undetectable in root and callus cultures (Loyola \u0026amp; Ochoa, 2018). Similarly, diosgenin was present in plantlet and shoot cultures but absent in callus and root cultures of \u003cem\u003eCostus speciosus\u003c/em\u003e, indicating tissue-specific metabolite accumulation (Indrayanto et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHitherto, no reports identified apigenin and its glycoside apigetrin in \u003cem\u003eJ. gendarussa in vitro\u003c/em\u003e cultures. Therefore, we initiated friable calli from \u003cem\u003eJ. gendarussa\u003c/em\u003e leaf explants. We optimized callus induction using MS medium with 2,4-D and BA based on previous reports of callus induction in \u003cem\u003eJ. gendarussa\u003c/em\u003e leaf explants ( Wahyuni et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wahyuni et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The fresh and dry weight discrepancy in the growth of the callus suspension indicated water accumulation in the calli during culture. Fresh biomass reduced slightly after day 48, suggesting the onset of cell senescence and nutrient depletion. Subsequently, we confirmed the presence of apigenin and its glycoside in the callus through HR LC-MS. \u003cem\u003eJ. gendarussa\u003c/em\u003e suspension cultures (both stem and leaf) showed equal or slightly increased phenolic yields compared to plant tissues (leaf and stem) when extracted with methanol (Bhagya and Chandrasekhar (2013). Here, HPLC analyses of the leaf sample gave higher concentrations of apigenin and apigetrin than unelicited cell cultures. The decline of compound production on day 64 in suspension cultures suggests a possible metabolite conversion and degradation of the cells. Usually, seasonal variation, explant maturity, and soil composition significantly influence the biosynthesis of plant secondary metabolites. In \u003cem\u003eJ. gendarussa\u003c/em\u003e, collected from various locations in Indonesia, the metabolite profile was influenced by soil nutrient composition (Ningsih et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We observed that the glycoside form was higher than its aglycone apigenin in the leaf tissues and cell suspension cultures, in alignment with previous reports where glycosides were found to be more abundant than aglycones, with ratios ranging from 5:1 to 10:1 depending on the plant species (Fabjan et al., 2003). Many secondary metabolites like flavonoids, terpenoids, and phenolics are more biologically active in the aglycone form, so plants glycosylate them to regulate their activity (Xiao, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Also, plants frequently convert aglycones (reactive or toxic) into glucosides to detoxify and store them safely in vacuoles (Le Roy et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Glucosides are more water-soluble than aglycones, which makes them easier to store in aqueous compartments like vacuoles or cytosol (Song, 2018). Thus, the accumulation of apigetrin in cell suspension cultures may be an intrinsic mechanism to maintain metabolic stability and mitigate adverse effects like nutrient depletion.\u003c/p\u003e \u003cp\u003eElicitation upregulates flavonoid biosynthesis through transcriptional activation of key enzymes like Flavonol Synthase (FLS) and Chalcone Synthase (CHS). An early accumulation of aglycones by elicitation paves the way for improved glycoside formation as and when glycosyl transferase enzymes (UGTs) are activated. MeJA between 100\u0026ndash;200 \u0026micro;M concentrations effectively enhanced flavonoid production in various plant cell cultures. For instance, in \u003cem\u003eGlycyrrhiza inflata\u003c/em\u003e, 100 \u0026micro;M MeJA elicitation increased glycyrrhizin production in hairy root cultures (Li et al., 2013). In \u003cem\u003eHypericum perforatum\u003c/em\u003e, 100 \u0026micro;M MeJA treatment on day 15 resulted in the highest flavonoid production, i.e., a 2.7-fold increase compared to control cultures (Wang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Also, maximum quercetin content was observed at 100 \u0026micro;M MeJA concentration in \u003cem\u003eAllium cepa\u003c/em\u003e (Iqbal et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Likewise, 200 \u0026micro;M MeJA enhanced ginsenoside accumulation in \u003cem\u003ePanax ginseng\u003c/em\u003e suspension cultures (Yu et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In the present study, 200 \u0026micro;M MeJA for 2 days resulted in the highest accumulation of apigenin and apigetrin. Similarly, in \u003cem\u003eCardiospermum halicacabum\u003c/em\u003e, 200 \u0026micro;M MeJA effectively enhanced apigenin production (7.3-fold increase) in embryogenic suspension cultures (Harisaranra et al., 2008). Moreover, a considerable difference between apigenin and apigetrin content was observed after MeJA treatment, primarily due to the upregulation of glucosyl transferases triggered by MeJA. Similar effects were observed in \u003cem\u003eGlycyrrhiza uralensis\u003c/em\u003e, where MeJA significantly increased flavonoid aglycones initially; over time, its glucoside was dominated by the activation of glycosylation enzymes ( Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Likewise, MeJA initially increased aglycone baicalein and glycoside baicalin; later, baicalin accumulated over time in \u003cem\u003eScutellaria baicalensis\u003c/em\u003e (Zhao et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, Apigetrin levels vastly overtook apigenin across all treatments. However, the apigenin and apigetrin production varied depending on the type of elicitor, its concentration, and the exposure time. We gave the same concentrations and exposure time for MeJa and jasmonic acid elicitation. However, MeJA gave better results due to its volatile nature and efficient membrane permeability. Hence, we can surmise that it is an effective elicitor for the rapid induction of apigenin and apigetrin in the cell suspension culture of \u003cem\u003eJ. gendarussa\u003c/em\u003e compared to JA. Contrarily, JA elicitation in \u003cem\u003eGlycyrrhiza glabra\u003c/em\u003e effectively increased flavonoid production in callus cultures (Rani et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). JA treatment boosted the output of wogonoside, a flavonoid glucoside, in \u003cem\u003eScutellaria lateriflora\u003c/em\u003e (Wilczańska et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 50 \u0026micro;M JA elevated flavonoid content by 72% over the control, and when combined with 10 mM CaCl₂, levels rose by 227% in \u003cem\u003eTrifolium pratense\u003c/em\u003e (Kašparov\u0026aacute; \u0026amp; Siatka, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The mode of action of both MeJA and jasmonic acid is similar. They bind to the COI1-JAZ receptor complex, destroying JAZ repressors, which release transcription factors like MYC2 that turn on flavonoid biosynthetic genes (Wasternack \u0026amp; Hause, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). MeJA enters cells quickly and triggers higher production of secondary metabolites. However, JA is less active or slow-acting because of its low stability and non-volatile nature (Ho et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLike MeJA, SA also triggers phenolics and flavonoid accumulation. The SA elicitation is through NPR1-dependent signaling, triggering WRKY and TGA transcription factors, which boost the expression of key enzymes for flavonoid biosynthesis, such as Phenylalanine Ammonia Lyase, Chalcone Synthase, and Flavonol Synthase (Johnson et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In the present study, 100\u0026micro;M SA worked best. The effective concentration of SA elicitation is 100\u0026ndash;300 \u0026micro;M for enhancing flavonoid production. An elicitation with 100 \u0026micro;M SA enhanced phenolic production, including flavonoids, and improved biological activities in \u003cem\u003eMomordica dioica\u003c/em\u003e cell suspension cultures (Chung et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). SA treatment at 300 \u0026micro;M significantly increased flavonoid content and antioxidant activity in cell suspension cultures of \u003cem\u003eThevetia peruviana\u003c/em\u003e (Mendoza et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNaCl is perceived as an abiotic stress, creating osmotic and ionic stress and rapidly increasing ROS levels. ROS are secondary messengers that activate stress-responsive transcription factors such as MYB, WRKY, and DREB, which upregulate genes in the phenylpropanoid pathway (Gharibi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). NaCl elicitation at 100\u0026ndash;150 mM concentrations significantly increased the flavonoid rutin and upregulated genes involved in flavonoid biosynthesis in \u003cem\u003eHaplophyllum virgatum\u003c/em\u003e suspension cultures (Abedi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Flavonoid content peaked at 129\u0026ndash;172 mM NaCl concentrations, indicating enhanced catechin and epicatechin content in \u003cem\u003eRumex thyrsiflorus\u003c/em\u003e suspension cultures, though concentrations above 300 mM led to a waning in flavonoid content (Gozdur et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eMedicinal plants treat diseases as they have countless pharmacological properties owing to the presence of various bioactive compounds. The limited availability of these compounds in intact plants warrants increasing production by many approaches, including eliciting secondary phyto-molecules \u003cem\u003ein vitro\u003c/em\u003e. Apigenin and apigetrin are two important flavonoid medicinal compounds in \u003cem\u003eJ. gendarussa\u003c/em\u003e. In this work, we induced soft, friable calli to initiate cell suspension cultures of \u003cem\u003eJ. gendarussa.\u003c/em\u003e Various abiotic elicitors, MeJA, SA, JA, and NaCl, were tried at different concentrations and time intervals to enhance apigenin and apigetrin production in suspension cultures of \u003cem\u003eJ. gendarussa\u003c/em\u003e. 200 \u0026micro;M MeJA for 2 days and 200 mM NaCl for 6 days yielded the highest apigenin and apigetrin production. Among these, methyl jasmonate is the most reliable, effective, and fast-acting elicitor for producing apigenin and apigetrin in \u003cem\u003eJ. gendarussa in vitro\u003c/em\u003e cultures. Further investigations into the alternative culture systems for enhanced production of apigenin and apigetrin are the prospects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose. The authors have no competing interests to declare relevant to this article\u0026apos;s content. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVinaya Chndran And Linu Mathew were involved in the conception and design; Vinaya Chandran, Maya Rajan, Shahena S, and Linu Mathew in the analysis and interpretation of the data; the drafting of the paper, revising it critically for intellectual content; and the final approval of the version to be published; and we agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the instrumentation help rendered by DST-FIST, DST-PURSE, and DBT-BUILDER, School of Biosciences, MG University, Kottayam, Kerala, India; SAIF, MG University, Kottayam, Kerala, India, \u0026nbsp;and SAIF, IIT Bombay, India.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information (Figure S1. 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Cancer Cell Int 13:1\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1475-2867-13-54\u003c/span\u003e\u003cspan address=\"10.1186/1475-2867-13-54\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"apigenin, apigetrin, in vitro cultures, elicitation, J. gendarussa","lastPublishedDoi":"10.21203/rs.3.rs-6782131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6782131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study realized a high and sustained yield of apigenin and apigetrin in \u003cem\u003eJusticia gendarussa\u003c/em\u003e cell suspension cultures by using elicitors: methyl jasmonate (MeJA), jasmonic acid (JA), salicylic acid (SA), and sodium chloride (NaCl). We cultured the leaf segments on MS basal medium with 2,4-Dichlorophenoxy acetic acid (2,4-D) at 2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and Benzyl Adenine (BA) at 0.2 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for friable callus induction. We transferred the best-responding calli to liquid MS medium with 2,4-D at 1 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and elicited the 40th -day-old culture with varying doses of elicitors. Cultures treated with 200 \u0026micro;M MeJA recorded the highest accumulation of apigenin (123.98 \u0026micro;g g⁻\u0026sup1; DW) and apigetrin (342.87 \u0026micro;g g⁻\u0026sup1; DW) by the 2nd day of elicitation. Also, both compounds showed a significant increase upon JA elicitation, with the maximum accumulation (apigetrin 114.68 \u0026micro;g g⁻\u0026sup1; DW; apigenin 73.93 \u0026micro;g g\u0026sup1; DW) occurring at 100 \u0026micro;M JA by the 6th day. Likewise, SA at 100 \u0026micro;M boosted compound production (apigenin 106.57 \u0026micro;g g⁻\u0026sup1; DW and apigetrin 231.43\u0026micro;g g⁻\u0026sup1; DW) on day 4. Salt stress also promoted the accumulation of apigenin (107.13 \u0026micro;g g⁻\u0026sup1; DW) and apigetrin (245.31 \u0026micro;g g⁻\u0026sup1; DW), with the highest accumulation at 200 mM of NaCl. The elicitation capability of these four elicitors was in the order MeJA\u0026thinsp;\u0026gt;\u0026thinsp;NaCl\u0026thinsp;\u0026gt;\u0026thinsp;SA\u0026thinsp;\u0026gt;\u0026thinsp;JA. All elicitors produced higher concentrations of compounds than found in leaf extracts of field-grown plants (29.69 \u0026micro;g g⁻\u0026sup1; DW of apigenin and 122.42 \u0026micro;g g⁻\u0026sup1; DW of apigetrin).\u003c/p\u003e","manuscriptTitle":"Elicitation using Jasmonates, Salicylic acid, and Sodium chloride in cell suspension cultures of Justicia gendarussa Burm f.: A route for enhanced and sustainable production of apigenin and apigetrin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 07:39:11","doi":"10.21203/rs.3.rs-6782131/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-06-04T02:36:58+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-03T19:15:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-03T14:48:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-05-31T04:28:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5cea2589-f6f1-4398-adba-6fa3624843b1","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:09:30+00:00","versionOfRecord":{"articleIdentity":"rs-6782131","link":"https://doi.org/10.1007/s11240-025-03185-1","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2025-10-15 15:58:21","publishedOnDateReadable":"October 15th, 2025"},"versionCreatedAt":"2025-06-09 07:39:11","video":"","vorDoi":"10.1007/s11240-025-03185-1","vorDoiUrl":"https://doi.org/10.1007/s11240-025-03185-1","workflowStages":[]},"version":"v1","identity":"rs-6782131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6782131","identity":"rs-6782131","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}