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Enicostema axillare (Poir. ex Lam.) A. Raynal is an immense medicinal plant that has been extensively used to treat malaria, diabetes, stomachache, and fever in traditional medicine. This plant contains high amounts of secoiridoid glycosides (gentiopicroside and swertiamarin), which have numerous health benefits. However, no leaf callus or cell suspension culture has been established for E. axillare for the production of beneficial metabolites. As a result, the goal of this work was to standardize an efficient and robust method for callus induction, callus and cell biomass, as well as swertiamarin and gentiopicroside accumulation. The highest callus induction frequency (85.40 ± 0.51%) and formation of leaf into callus with a mean callus biomass (1.283 ± 0.02 g/explant fresh weight (FW) and 0.176 ± 0.00 g/explant dry weight (DW)) and metabolite contents (swertiamarin 0.53 ± 0.01 mg/g and gentiopicroside 0.64 ± 0.05 mg/g) were obtained on Murashige and Skoog (MS) medium augmented with 2,4-D (1.0 mg/L) + Kin (0.5 mg/L) after 12 weeks of culture. The induced leaf callus was studied for cell suspension growth kinetics (biomass) and metabolite production. After 60th days of culture, the maximum of the leaf cell suspension (5.25 ± 0.05 g FW (50 mL medium) and 0.39 ± 0.00 g DW (50 mL medium) and secoiridoid glycosides (gentiopicroside, 1.44 ± 0.05 mg/g, and swertiamarin, 0.88 ± 0.01 mg/g DW) were obtained, which corresponded to 4.19 and 36 times greater than the lag phase (after 10th days). Cultured leaf cell suspensions were treated with methyl jasmonate (MJ) and salicylic acid (SA) at 50, 100 and 200 μM after 60th day of cell growth and evaluated for swertiamarin and gentiopicroside contents after 24 h of treatment. The greatest swertiamarin (2.43 ± 0.05 mg/g DW, 2.76-fold) and gentiopicroside (2.41 ± 0.09 mg/g DW, 1.67-fold) levels were observed in the treatments with 200 and 100 μM SA elicitor, respectively. These findings can lead to the use of this plant as an alternative source to improve the commercial production of secoiridoid glycosides in large-scale cultures.Key messageThis is the first report on leaf callus induction in Enicostema axillare and enhanced production of swertiamarin and gentiopicroside in leaf cell suspension culture elicited by methyl jasmonate and salicylic acid.

Green synthesis of selenium nanoparticles (SeNPs) was achieved by a simple biological procedure using the reducing power of E. axillare leaf extract. This method is capable of producing SeNPs in a size range of around 56.23–98.18 nm, under ambient lab condition. The synthesized nanoparticles were characterized by UV–Vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM) and Energy-dispersive X-Ray (EDX). To check the potential hazards of nanotoxicity median lethal concentration (LC50) and Na + /K + -ATPase activity was assessed on zebra fish which showed the LC50 of 258.72 ppm for 96 h and Na + /K + -ATPase seems to significantly decrease with increase in SeNPs concentration. Antibactericidal activity explored the use of SeNPs against wide range of pathogens. Cytotoxicity of SeNPs was assayed against human lung cancer cells (A549) confirmed that SeNPs are able to inhibit the cell growth by dose-dependent manner. In conclusion, green synthesized selenium nanoparticles are less toxic and harmless. This is the first report on green synthesized selenium nanoparticles using E. axillare possessing anti-bacterial and anti-cancer activity.

Plants have their own defense mechanisms such as induced systemic resistance (ISR) and systemic-acquired resistance. Bacillus spp. are familiar biocontrol agents that trigger ISR against various phytopathogens by eliciting various metabolites and producing defense enzyme in the host plant. In this study, B. paralicheniformis (strain EAL) was isolated from the medicinal plant Enicostema axillare. Butanol extract of B. paralicheniformis showed potential antagonism against Fusarium oxysporum compared to control well (sterile distilled water) A liquid chromatography mass spectrometry analysis showed 80 different compounds. Among the 80 compounds, we selected citrulline, carnitine, and indole-3-ethanol based on mass-to-charge ratio, database difference, and resolution of mass spectrum. The synthetic form of the above compounds showed biocontrol activity against F. oxysporum under in vitro condition in combination, not as individual compounds. However, the PCR amplification of 11 antimicrobial peptide genes showed that none of the genes amplified in the strain. B. paralicheniformis inoculation challenged with F. oxysporum on tomato plants enhanced production of defense enzymes such as peroxidase (POD), superoxide dismutase (SOD), phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and proline compared to control plants (without inoculation of B. paralicheniformis) at significant level (p < 0.005). Stem of tomato plants expressed higher POD (2.2-fold), SOD (2.2-fold), PPO (1.9-fold), and PAL (1.3-fold) contents followed by the leaf and root. Elevated proline accumulation was observed in the leaf (1.8-fold) of tomato plants. Thus, results clearly showed potentiality of B. paralicheniformis (EAL) in activation of antioxidant defense enzyme against F. oxysporum-infected tomato plants and prevention of oxidative damage though hydroxyl radicals scavenging activities that suppress the occurrence of wilt diseases.

As a progressive neurological disease with increased morbidity and mortality, Alzheimer Disease (AD) is characterized by neuron damage that controls memory and mental functions.  Enicostema axillare  (EA), an herb with a history of combativeness and effectiveness in treating Rheumatoid Arthritis, Cancer, and Diabetes, is used in Indian folk medicine from a holistic point of view. Though the herb is used for many illnesses, the molecular mechanism of its bioactive on AD has not been deciphered by intricate research. A unique pharmacology approach based on ADME drug screening and targeting, pathway enrichment (GO and KEGG), and network pharmacology, was established to explore the molecular mechanisms of  E. axillare  (EA) bioactive compounds for the treatment of AD. In brief, we bring to light the three active compounds of EA and seven potential molecular targets of AD, which are mainly implicated in four signaling pathways, i.e., MAPK, Apoptosis, neurodegeneration, and the TNF pathway. Moreover, the network analysis of the active compounds, molecular targets, and their pathways reveals the pharmacological nature of the compounds. Further, molecular docking studies were carried out to explore the interactions between the EA bioactive compounds and the targets and examine the binding affinity. The outcome of the work reflects the potential therapeutic effects of the compounds for treating AD through the modulation of the key proteins, which further corroborates the reliability of our network pharmacology analysis. This study not only helps in understanding the molecular mechanism of the drugs but also helps in finding and sorting new drugs for the treatment of AD, and other complex diseases through modern medicine.

An efficient protocol has been developed for in vitro propagation of Enicostema axillare using shoot tip explants. The shoot tip explants were cultured on MS medium supplemented with various combinations of (BAP, KIN) and (NAA/IAA & IBA) in different concentrations between 0.5 and 2.0 mg/l for multiple shoot bud induction. The highest percent of (98.51 %) was observed at 1.0 mg/l BAP in combination with 0.2 mg/l KIN while maximum number of shoot buds (8.41 shoots/explant) was noticed on MS medium containing 1.0 mg/l BAP and 0.2 mg/l KIN combination. The highest frequency (90.82 %) of multiple shoot bud regeneration was observed at 1.0 mg/l BAP and 0.5 mg/l IBA with 15.12 ± 2.12 shoots/explants. The regenerated multiple shoots were transferred to half-strength MS medium augmented with different concentration of 0.5–2.5 mg/l IBA for rooting. Among the different concentrations of IBA tested, maximum percentage of rooting (100 %) was observed in MS medium augmented with 1.5 mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing soil and sand in the ratio of 1:1. Subsequently established in the field conditions with 90 % of survival rate. The protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species. The described method can be successfully employed for large-scale multiplication and in vitro conservation as well as production of secondary metabolites of E. axillare .

Rice striped stem borer, Chilo suppressalis (Walker, 1863), is considered as a major destructive pest in paddy fields in the most regions of Iran. In this study, the impact of the rice cultivars, Tarom and Shiroudi, and the insecticides, cypermethrin, tebufenozide and the emulsion and granule formulations of diazinon were examined on the damage indices, percentages of dead heart and whitehead. The study was performed as factorial randomized complete block design using artificial infestation in semi-field conditions in Amol County. No significant interaction was found between rice cultivar and insecticide for all indices in both vegetative and reproductive stages (P>0.05). The results of main effects in both phenological stages indicated that the weight of live larvae and damage indices were significantly higher on Tarom than those of Shiroudi (P?0.05), while no significant difference was detected in larval mortality on the cultivars (P>0.05). The main effects of insecticide were significant for all biological and damage indices (P?0.05). The efficacies of tebufenozide and cypermethrin in terms of larval mortality were 47.57 and 74.90 % at vegetative stage reaching to 79.15 and 92.08% at reproductive stage, respectively. The lowest efficacy in terms of larval mortality (73.33%) was obtained on plants treated by diazinon emulsion. At reproductive, the survived larvae weighed 170.80 mg.seedlingi and the corrected whitehead index compared to the control reached 50.04% on plants treated by diazinon emulsion. According to the results, tebufenozide, regarding its relatively high efficacy and environmental compatibility, is suggested to be assessed further for integrated C. suppressalis management in the field conditions.

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux. Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide. These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.

Objective and design Rheumatoid arthritis is a chronic inflammatory and autoimmune disease that leads to aggressive joint cartilage and bone destruction. Swertiamarin is a secoiridoid glycoside found in Enicostema axillare (Lam) A. Raynal, a medicinal plant used in the Indian system of traditional medicine. In the present study, the potential of swertiamarin was evaluated in IL-1β induced fibroblast-like synoviocytes (FLS). Methods The FLS were isolated from Freund’s Complete Adjuvant induced arthritic (AA) rats and cultured with IL-1β. The normal FLS and AA-FLS were cultured and used for subsequent experiment in fibroblastic morphology form. The efficacy of swertiamarin (10–50 μg/ml) was evaluated on mRNA and protein expression levels of inflammatory and osteoclastogenesis mediators. The efficacy was also evaluated on p38 MAPKα levels with time course studies (2, 4, 6, 8 and 12 h). Results IL-1β induced cell proliferation (149.46 ± 13.73 %) and NO production (162.03 ± 11.03 %) in AA-FLS; treatment with swertiamarin controlled proliferation (82.77 ± 4.22 %) and NO production (82.06 ± 3.91 % at 50 μg/ml) in a dose-dependent manner. It also significantly ( P  < 0.05) modulated the expression of apoptotic marker (caspase 3), proinflammation mediators (TNFα, IL-6, PGE2, COX-2, iNOS, MMPs) and osteoclastogenic mediator (RANKL) at both the mRNA and protein levels. Treatment with swertiamarin inhibited the levels of p38 MAPKα in a dose-dependent manner and also significantly ( P  < 0.05) attenuated the release of the same in time dependent mode. Conclusion These findings suggest that treatment with swertiamarin attenuated IL-1β induced FLS, and it revealed anti-inflammatory potential by attenuating aggressive FLS.

Background: Oral candidiasis is caused by Candida albicans, which is most prevalent in immunocompromised patients. Aim: The study aimed to investigate the antifungal activity of plant species used for oral candidiasis against C. albicans. Setting: The study was conducted in Aganang Local Municipality, Capricorn District, Limpopo province, South Africa. Methods: A survey was conducted using a semi-structured questionnaire supplemented with guided field walks with traditional health practitioners to gather information on medicinal plants used to treat oral candidiasis. Nine plant species (Artemisia afra Jacq. ex Willd., Blepharis subvolubilis subsp. subvolubilis C.B. Clarke, Enicostemma axillare [Lam.], Helichrysum caespititium [DC.] Harv., Solanum incanum L., Waltheria indica L., Ximenia caffra Sond. var. caffra, Ximenia caffra Sond. var. natalensis and Ziziphus mucronata Willd.) were investigated for antifungal activity. The plant material were extracted with solvents of varying polarities: acetone, dichloromethane, ethyl acetate, ethanol, hexane, methanol, and water. The Micro-dilution and bioautography assays were used to determine the antifungal activity of the plant extracts. Results: Leaf extracts of A. afra and S. incanum were more active against C. albicans with MIC values of 0.02 mg/mL. Bioautography assay demonstrated active compounds in S. incanum, W. indica and X. caffra var. caffra extracts developed in Benzene: Ethanol: Ammonia hydroxide (BEA). Conclusion: An ethnobotanical survey is a worthy starting point in selecting potential plant species for ethnopharmacological studies. Contribution: The effectiveness of oral administrations of the medicinal plants was confirmed by the excellent antifungal activity of the aqueous extracts.

Multiple sclerosis (MS) is a degenerative autoimmune disease attacks the myelin sheath of the central nervous system (CNS) neurons causing different disabilities. According to recent evidence, the main bioactive component in Enicostema axillare , the Swrtiamarin (SM) has been found to exert anti-inflammatory and antioxidant activities against several diseases. However, SM activities in treatment of autoimmune diseases remain to be explored. Herein, we used a murine model of MS, to show that SM treatment ameliorates the severity of experimental autoimmune encephalomyelitis (EAE). This occurs through reducing the levels of pro-inflammatory cytokines and infiltration of CD4+CD45+ cells into CNS. That was associated with a reduction in the expression of STAT3 and NFkB in CD4+ T cells under Th17 and LPS-stimulated macrophages. Furthermore, in silico studies revealed that SM interacts with NF-E2-related factor 2 (NRF2), and therefore, suppressed oxidative stress by inducing formation NRF2-antioxidant response element (ARE) complex. We found that SM is an agonist of NRF2 complex regulating the total CD4 population and antioxidant markers in EAE mice. Molecular docking analysis showed a stable and higher binding affinity between SM and NRF2. Results revealed that SM treatment increased the complex formation between ARE and NRF2 where immunoprecipitation methods showed a higher binding affinity of ARE to NRF2 in SM treated animals. Complex formation triggered ARE cascade of antioxidant gene clusters and reduces the MS pathological alterations in EAE mice model. Current data proposed SM as an effective biomolecule in treatment of MS and controlling neuronal damage through inhibiting oxidative stress markers and targeting NRF2.