Secondary Metabolite Enhancement of Pokeweed (Phytolacca americana L.) Calli Using Drought and Salinity Stress Under In Vitro Condition

Silybum marianum L. commonly known as milk thistle is a medicinally potent plant with a multitude of pharmacological applications. The present investigations demonstrated the effects of Zinc Oxide nanoparticles (ZnO NPs) on callus growth and biosynthesis of silymarin in milk thistle under various light conditions. The callus cultures developed on Murashige and Skoog (MS) basal media containing ZnO NPs (0.15 mg/L), under the dark condition maintained for two weeks, followed by transference into normal light produced the maximum callus fresh weight (2294 mg/L FW). Further, the metabolite profiling revealed that ZnO NPs significantly augmented the production of silymarin and upregulated the antioxidant system in the callus cultures. Maximum TPC (total phenolic content: 37 ± 0.20 mg/g DW), TFC (total flavonoid content: 8.9 ± 0.023), DPPH antioxidant activity (91.5 ± 1.75%), Superoxide dismutase activity (SOD: 4.1 ± 0.045 nM/min/mg FW) and the highest silymarin content (14.6 ± 0.023 mg/g DW) were recorded in the callus cultures developed on MS media supplemented with solitary ZnO NPs (0.15 mg/L). While the callus culture evolved in presence of only PGRs (2,4 D and BA: 2 mg/L, each) accumulated the lesser fresh weight (562 mg/L FW). A higher concentration of ZnO NPs (0.15 mg/L) enhanced the secondary metabolite accumulation and silymarin content in the callus of Silybum marianum. This is the first standardized protocol to be applied on the industrial level for the production of silymarin.

Efficient regeneration of shoots and roots in graphene oxide and carbon nanotubes mediated callus cultures: A qualitative and quantitative study

This study addresses the favourable effects of drought stress imposed by polyethylene glycol (PEG) 6000 on the micropropagated shoots of Stevia rebaudiana. Various concentrations, i.e., 0, 0.5, 1, 2, and 4% of PEG 6000 were applied to the nodal shoot explants for four weeks, and the influence produced on shoots growth parameters, bioactive steviol glycosides (rebaudioside A and stevioside), and nonenzymatic antioxidant activities (total phenolic content (TPC), total flavonoid content (TFC), total antioxidant capacity (TAC), total reducing power (TRP) and 1,1-diphenyl-2-picrylhydrazyl(DPPH)-free radical scavenging activity (FRSA)) was elucidated. The significantly highest yield (92.4% direct shoot organogenesis) and secondary metabolites (2.94% Reb A, 2.52% ST, 95.3% DPPH-FRSA, 15.0% TPC, 13.0 µg/mg TFC, 22.3 µg/mg TAC, and 19.8 µg/mg TRP) production in response to abiotic stress elicitors was obtained in Murashige and Skoog (MS) medium treatment provided with 4% of PEG 6000. The overall trend was significant enhancement of growth dynamics and pharmaceutical compounds from control to 4% of PEG 6000 concentration as a defensive response against reactive oxygen species (ROS) produced in excess by water deficit. This is a very promising study to be employed in bioreactors to get markedly enhanced content of compounds of medicinal importance in the pharmaceutical market.

Nowadays, phytochemicals and antioxidants in plants are raising interest in consumers for their roles in the maintenance of human health. Phenolics and flavonoids are known for their health-promoting properties due to protective effects against cardiovascular disease, cancers and other disease. In this study, total phenolic (TP), total flavonoids (TF) and antioxidant activities of the six different varieties of sweet potato (Ipomoea batatas) leaf extracts were determined. TP content varied from 4.47±1.88 to 8.11±2.11 mg/g DW in the leaf extracts and high content of TP was observed in Vardaman variety. TF content in the extracts ranged from 1.87±0.84 and 3.95±0.91 mg/g DW and Bush Porto Rico variety showed highest content. The antioxidant activity determined by the 1,1-diphenyl-2-picryl hydrazyl (DPPH) and ferric reduction antioxidant power (FRAP) showed high activities (IC50value of 184.3 µg/mL) in the leaves of Vardaman variety while the Centennial variety showed the lowest activity (IC50=450.46 µg/ml). Antioxidant activity was highly correlated with TP content (R2=0.827, P<0.001) while, no significant correlation was observed between TF content and antioxidant activity (R2=0.0448). Therefore, the total phenolic content could be served as a useful indicator for the antioxidant activities of sweet potatoes.This study validated the medicinal potential of the sweet potatoes leaf. Key words: Ipomoea batatas, total phenolic (TP), total flavonoids (TF), 1,1-diphenyl-2-picryl hydrazyl (DPPH) assays.

Polyalthia bullata is an endangered medicinal plant species. Hence, establishment of P. bullata callus culture is hoped to assist in mass production of secondary metabolites. Leaf and midrib were explants for callus induction. Both of them were cultured on Murashige and Skoog (MS) and Woody Plant Medium (WPM) containing different types and concentrations of auxins (2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), picloram, and dicamba). The callus produced was further multiplied on MS and WPM supplemented with different concentrations of 2,4-D, NAA, picloram, dicamba, indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA) media. The quantification of total phenolic content (TPC), total flavonoid content (TFC) and antioxidant capacity was further carried out on P. bullata callus, and the results were subjected to correlation analysis. Among the media, the WPM + 16.56 µM picloram (53.33 ± 22.06%) was the best for callus induction while MS + 30 µM dicamba was the best for callus multiplication. The TPC, TFC, and EC50 of DPPH scavenging activity were determined at 0.657 ± 0.07 mg GAE/g FW, 0.491 ± 0.03 mg QE/g, and 85.59 ± 6.09 µg/mL in P. bullata callus, respectively. The positive correlation between DPPH scavenging activity with TPC was determined at r = 0.869, and that of TFC was at r = 0.904. Hence, the P. bullata callus has an ability to accumulate antioxidants. It therefore can be a medium for secondary metabolites production.

This study investigated the effects of different concentrations of two elicitors, namely chitosan (CHT) (25 and 50 mg L−1) and salicylic acid (SA) (50 and 100 mg L−1) on the production of secondary metabolites (SMs) and antioxidant activity of safflower callus under salinity stress. The content of total phenolics, total flavonoids, total flavonols, anthocyanin and antioxidant activity with 1,1-diphenyl-2 picrylhydrazyl assay (DPPH), significantly increased under salinity stress including 1.5% (w/v) sodium chloride. Under salinity stress, the highest content of total phenolics and total flavonoids were observed under elicitation by 50 (mg L−1) of SA and 25 (mg L−1) of CHT, but the highest callus growth rate (0.048 mm day−1), highest content of total flavonols (4.2 mg RE g−1 FW) and the highest DPPH activity (61.48% of inhibition) were observed under elicitation by 50 (mg L−1) of SA under salinity stress. This indicated partial superiority elicitation of SA over CHT for callus growth and SMs production of safflower under salinity stress. This new elicitation opens new avenues for the selection and exploitation of the best elicitors and their dosages for the enhancement of commercially important SMs in safflower as an important medicinal plant at cellular level. Effects of chitosan and salicylic acid on elicitation of SMs of safflower under in vitro salinity stress.

Callus elicitation is advantageous for metabolite production due to its ability to increase yield, provide controllable conditions, and allow for genetic manipulation, offering a sustainable and scalable alternative to traditional plant-based extraction methods for the production of bioactive substances. In this research, in vitro callus cultures (CCs) of the wild medicinal plant Aerva sanguinolenta were used to evaluate the efficacy of various elicitation regimes of silver nanoparticles (AgNPs) and salicylic acid (SA) to evoke an increased production of secondary metabolites, such as aervine and antioxidant metabolites. Three concentrations of SA (i.e., 20, 50, and 100 µM) and three concentrations of AgNPs (i.e., 30, 60, and 90 µg/L) were used on shoot explant cultures using MS (Murashige and Skoog) media. All the SA and AgNP elicitation treatments significantly increased the production of antioxidant metabolites, total phenolic contents (TPCs), and total flavonoid contents (TFCs) compared to the control treatment experiments. The contents of aervine were increased significantly upon elicitation compared to the control trial. Furthermore, the antioxidant potential of the test extract was enhanced compared to the control treatment. Comparatively, the AgNPs were more beneficial as elicitors than the SA treatments. The elicitation treatments with about 90 µg/L AgNPs and 100 µM SA were the best among all elicitation regimes. Callus elicitation with SA and AgNPs can stimulate increased metabolite production and be used as a sustainable practice in the welfare and service industries for drug development and drug discovery.

The rising market interest for Nigella sativa (Black seeds) necessitates the development of cultivation strategies to enhance metabolites production. Zinc oxide nanoparticles (ZnO-NPs) have drawn global attention as efficient and bio safe elicitors for in vitro cultures, to enhance secondary metabolites production in medicinal plants. In this study, ZnO-NPs were utilized for establishment of callus and cell cultures in black seeds for the first time. Hypocotyl explants were cultured on Murashige and Skoog (MS) media with varying levels of ZnO-NPs, resulted in callus induction and biomass formation. Optimal response in callus growth parameters were observed when explants were grown on MS media supplemented with 60 mg/L ZnO-NPs, resulting in 71.2 % callus induction frequency, 28.2 g/L fresh biomass, 9.7 g/L dry biomass, and 63 % water content. A substantial increase in callus growth was observed when ZnO-NPs were combined with 6-Benzylaminopurine (BA) at ratio of 45:1.5 mg/L, resulting in 91.2 % callus induction frequency and 42.2 g/L fresh biomass. In cell suspension cultures, ZnO-NPs alone at 45 mg/L produced optimum callus biomass (60.9 g/L). However, in combination with BA, callus biomass did not increase significantly in cell cultures. Maximum accumulation of total phenolic content (TPC: 26.8 mg GAE/g DW; Gallic acid equivalent dry weight) and total flavonoid content (TFC: 19.5 mg QE/g DW; Quercetin equivalent dry weight) was observed in cell cultures treated with higher concentration (70 mg/L) of ZnO-NPs in the 5th week of the growth curve. Moreover, ZnO-NPs incremented substantially the Phenylalanine lyase (PAL), Superoxide dismutase (SOD) and Peroxidase (POD) enzyme activities in cell cultures. Nonetheless, Reverse Phase High Performance Liquid Chromatography (RP-HPLC) analysis indicated peak production of thymoquinone (168.5 mg/g FW) in cell cultures treated with 45 mg/L ZnO-NPs alone. This study offers a promising approach for commercial production of Nigella sativa biomass and bioactive metabolites.

Rhinacanthus nasutus is a medicinal plant valued for its antioxidant and pharmacological properties, largely attributed to phenolic and flavonoid contents.This study enhanced bioactive metabolite production in cultures of R. nasutus calli through abiotic elicitation using sodium chloride (NaCl) and polyethylene glycol (PEG 6000).Calli was produced from nodal on the Murashige and Skoog (MS) medium added with 1mg L -1 kinetin and 1mg L -1 2,4-D, and elicited with varying concentrations of NaCl (0-200mM) and PEG (0-4%) for 7 days, followed by a 2-week recovery period.Growth parameters, physiological traits, pigment content, total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity were evaluated.Results demonstrated that moderate stress conditions significantly improved biomass accumulation, with the highest fresh weight (1.820.21g)at 4% PEG combined with 100mM NaCl.Severe stress (4% PEG + 200mM NaCl) intensified oxidative stress, as indicated by increased malondialdehyde and electrolyte leakage.The highest TPC (26.960.69mgGAE g) occurred under 0 % PEG + 150mM NaCl, while the highest TFC (17.080.86mgQE g) was recorded at 0 % PEG + 100mM NaCl.The optimal condition for enhanced antioxidant activity was 1 % PEG + 100-150mM NaCl (FRAP = 33.400.53mgTE g; DPPH IC50 = 0.410.00mgmL; ABTS IC50 = 0.140.00mgmL).These findings demonstrated that controlled abiotic elicitation optimized phenolic and flavonoid biosynthesis, enhancing the antioxidant potential of R. nasutus calli for pharmaceutical applications.

Introduction: The growth, development, and biosynthesis of secondary metabolites in the plant are strongly affected by environmental stress. The pharmaceutical values of medicinal herbs depend on the presence of secondary metabolites that are influenced by environmental stress. Drought and salinity stress are the two most significant abiotic factors that restrict plant growth and productivity. As a memory enhancer, anti-inflammatory, analgesic, antipyretic, sedative, and antiepileptic, Bacopa monnieri has been used for a long time. The aim of the present work was to study the impact of simulated abiotic stress on Bacopa monnieri. Methodology: Potted plants of Bacopa monnieri were treated with various concentrations of NaCl and polyethylene glycol (PEG) solution to simulate salinity and drought stress. In order to evaluate the salinity and drought stress effect, several physiological parameters, such as cell membrane stability and relative water content (RWC), and biochemical parameters, such as proline, β-carotene, MDA, total phenolic, and total flavonoid content, were taken into consideration. Results: In the present study, significant changes in the physiological and biochemical parameters have been observed in NaCl and PEG 6000-induced salt- and drought-stressed plants about control. Stress decreases the relative RWC and Membrane stability index of the Bacopa leaves. However, it increases the plant’s proline, β-carotene, phenolic compounds, and total flavonoids content. Conclusion: The data obtained from the current study indicated that both salinity and drought stress influence the physiological and biochemical traits of Bacopa monnieri L. By increasing the synthesis of proline, β-carotene, phenolic compounds, and total flavonoids, the Bacopa monnieri plant defends itself against oxidative stress.

The plant tissue culture technique has enabled us to study the tolerance of cells, tissues, and plant organs and cultures to different stresses that plants suffer from. The in vitro culture facilitates the ability to stimulate the production of secondary metabolites. The present study investigated the influence of paclobutrazol (PBZ) pre-treatment on the growth, physiological, morphological, biochemical traits, and phenolic compounds production in callus cultures of Plantago major L. in a medium containing different concentrations of cadmium. The callus was cultured in a Murashige and Skoog (MS) medium with different concentrations of cadmium (0, 50, 100, 150, and 200 µM), with a PBZ concentration of 0 and 2 mg l-1. A factorial experiment based on a completely randomized design (CRD) was performed in triplicate. The results of the growth of the callus culture were obtained after 28 days. The increasing concentrations of cadmium were found to have a negative effect on some traits such as fresh weight (FW), dry weight (DW), browning intensity (BI), tolerance index (TI), electrolyte leakage (EL), and membrane stability index (MSI). The PBZ treatment reduced the effects of cadmium levels through the decrease in malondialdehyde (MDA), and hydrogen peroxide (H2O2). This was caused by a significant increase in superoxide dismutase (SOD) and ascorbate peroxidase (APX). Also, there was an increase in total phenol and flavonoids content in the callus grown on the tissue cultures supporting 200 µM cadmium compared to the other levels. The PBZ pre-treatment was a significant reducing factor to produce total phenolic and flavonoids content in P. major L. callus culture under the cadmium levels.

Salinity stress is one of the most important stress barriers to crop production worldwide. Developing and implementing new strategies against salinity stress is critical for increasing agricultural productivity and supporting sustainable farming. Elicitors such as nanoparticles and Salicylic acid have recently been used potentially for better product yield. Therefore, in our research the Salvia virgata plant was exposed to salinity (NaCl) stress, and zinc oxide nanoparticles (ZnONP), salicylic acid (SA), and the ZnONP + SA combination were applied to plants divided into different groups. While salinity stress decreased the amount of chlorophyll a, chlorophyll b, and carotenoid pigments, SA, ZnONP, and SA + ZnONP elicitors combined with salinity stress enhanced the content of all three pigments. While salt stress raised MDA, H2O2, total phenolic, total flavonoid, soluble sugar and proline content, elicitor applications enhanced proline, soluble sugar, total phenolic and total flavonoid content more. Additionally, the application of NaCl + SA + ZnONP increased proline content by 21.55% and sugar content by 15.73% compared to NaCl application, while decreasing MDA content by 42.28% and H2O2 levels by 42.34%, thereby alleviating the plant's salt stress. It was revealed that DPPH, ABTS, and CUPRAC antioxidant activity sequence used to determine the total antioxidant activity displayed similarities, and it was found as NaCI + ZnONP > NaCI + SA > NaCI + SA + ZnONP > NaCI > Control. Furthermore, all elicitor applications increased CAT, GR, APX, and SOD enzyme activities while reducing oxidative stress in S. virgata plants. When all the data were evaluated, it was confirmed that SA and ZnONP had a synergistic effect and that SA and ZnONP have the potential to support plant development and growth under salinity. SA and ZnONP applications may have the capacity to least the detrimental impacts of salinity stress on plants. However, further research is needed to investigate the effectiveness of SA and ZnONPs in ameliorating salinity or different stress factors in various other plants.

Pokeweed, Phytolacca americana L., is considered a widely spreading invasive plant, while saponin contents accumulated in the roots have pharmaceutical uses, such as rheumatism treatments and anti-inflammation. Adventitious root cultures are an important source of diverse secondary metabolites, which have significant applications in various fields. This study focused on the optimization of parameters for root induction using different medium states and treatments with auxins on a pokeweed leaf. Semisolid and liquid MS (Murashige and Skoog, 1962) media were supplemented with indole-3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) at 0.5, 1, 2, and 4 mg/L. Root growth parameters, e.g., induction percentage, root numbers, length, and weight, were measured to determine the adventitious root induction efficiency. Total phenolic content, total flavonoid content, total saponin content, and antioxidant activity were recorded. Results showed that adventitious roots induced in semisolid MS medium supplemented with 0.5 mg/L NAA exhibited a high density of lateral roots. Appropriate medium state and auxin for adventitious root induction in pokeweed were determined as semisolid medium supplemented with 2 mg/L NAA. Considering phytochemicals, adventitious roots induced in liquid medium containing 0.5–1 mg/L NAA had the highest yield extract percentage. Additionally, adventitious roots cultivated in a liquid medium enriched with 1 mg/L NAA exhibited the highest phenolic and saponin contents. A principal component analysis (PCA) biplot and hierarchical cluster analysis (HCA) heatmap demonstrated different response patterns between semisolid and liquid media applied with NAA. The results of the semisolid media were grouped together due to high expression levels of the root induction parameters, while elevated phytochemical values were observed in the liquid media treatments. The results suggested two different media that provide the highest adventitious root induction efficiency and the greatest phytochemical contents: semisolid medium with 2 mg/L NAA and liquid medium with 1 mg/L NAA, respectively. These culture media can be applied to optimize adventitious root culture of pokeweed and in vitro phytochemical production.

Kacip Fatimah (Labisia pumila Blume), one of the most famous and widely used herbs, especially in Southeast Asia, is found to have interesting bioactive compounds and displays health promoting properties. In this study, the antioxidant activities of the methanol extracts of leaves, stems and roots of three varieties of L. pumila (var. alata, pumila and lanceolata) were evaluated in an effort to compare and validate the medicinal potential of this indigenous Malaysian herb species. The antioxidant activity determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, as well as the total amount of phenolics and flavonoids were the highest in the leaves, followed by the stems and roots in all the varieties. A similar trend was displayed by the ferric reducing antioxidant potential (FRAP) activity, suggesting that the L. pumila varieties possess high foliar antioxidant properties. At low FRAP activity concentrations, the values of the leaves’ inhibition activity in the three varieties were significantly higher than those of the stems and roots, with var. alata exhibiting higher antioxidant activities and total contents of phenolics and flavonoids compared to the varieties pumila and lanceolata. The high production of secondary metabolites and antioxidant activities in var. alata were firmly related to low nitrogen content and high C/N ratio in plant parts. The study also demonstrated a positive correlation between secondary metabolite content and antioxidant activities, and revealed that the consumption of L. pumila could exert several beneficial effects by virtue of its antioxidant activity.

<i>Acmella</i><i> </i><i>oleracea</i><i>e</i> (L.) R.K. Jansen (Asteraceae) is a therapeutically important medicinal plant used in traditional systems of medicine. Present study was undertaken to compare the total phenolic contents (TPC), total flavonoid contents (TFC), total antioxidant capacity (TAC) and Thin Layer Chromatography (TLC) profiles of areal parts and callus extracts of <i>A. oleracea</i><i>e</i> grown under different growing systems (field grown, hydroponically grown and callus culture. Callus established in Murasige and Skoog (MS) medium, areal parts of field grown and hydroponically grown plants were extracted in 80% methanol. TPC, TFC and TAC were carried out by using colorimetric Folin-Ciocalteu method, aluminum nitrate method and Ferric Reducing Antioxidant Power (FRAP) assay respectively. TLC profiles were developed using established protocol. The best callus growth and the highest mean callus weight were observed in leaf explants established in MS medium supplemented with 2 mg/L BA and 1 mg/L IBA. Comparatively higher TPC (11.45 ± 0.17), TFC (12.33 ± 0.92) and TAC (10.27 ± 0.28) were observed in hydroponically grown plants. Order of TPC, TFC and TAC were increased as callus<field grown plants< hydroponically grown plants. The presence of higher TPC, TFC and TAC in hydroponically grown plants opens a new window for use of hydroponic system for growing of <i>A. oleraceae</i> for better secondary metabolites content.