Discovery of Anti-NRLP3 Inflammasome, Immunomodulatory Phytochemicals from the Extract of Habenaria intermediaD. Don: An Unexplored Plant Species.

The present study describes the isolation, identification, and quantification of biomarker compounds in plant extracts of Habenaria intermedia D. Don (Orchidaceae). The isolation of the compounds was carried out from H. intermedia D. Don by repeated column chromatography of petroleum ether and ethanol fractions of extract of tubers. These compounds were characterized by 1H and 13C NMR and mass spectral data. A new quantitative method was established by using high-performance liquid chromatography (HPLC)-PDA. As a result, seven compounds were isolated and characterized. This is the first report of isolation of these compounds from this plant species H. intermedia D.Don. Out of seven isolated compounds, five were used for the quantitative study. A reliable and suitable HPLC method was developed for the well-resolved chromatogram of compounds. The proposed method was applied successfully to the detection and quantification of compounds. This study also represents the immunomodulatory and anti-inflammasome biological studies of isolated natural products. Loroglossol (HBR-4) has been reported to possess immunomodulatory activity. The immunostimulating assay indicated that HBR-4 could significantly promote the cell proliferation, especially via IL-2, TNF-α, and IFN-γ secretion from spleen cells. These results suggested the potential utilization of HBR-4 as an attractive functional health supplement candidate for hypoimmunity population. Additionally, cyclophosphamide-induced immunosuppression was counteracted by treatment with HBR-4, revealing significant increase in hemagglutinating antibody responses and hemolytic antibody responses. The current work revealed the potential anti-inflammasome and immunomodulatory activities of H. intermedia D. Don compounds and validates the usage of this prominent Rasayna plant.

Dereplication approach for the first time isolation of tatarinowin a and pentadecanoic acid from Acorus calamus L. by using GC-MS.

In the present study, we report herein the isolation of cadinane-type sesquiterpenoid, tatarinowin A (ACH-6), and pentadecanoic acid (ACH-8) from petroleum ether extract of rhizome of Acorus calamus L. (Acoraceae) along with 6 other known compounds in this species. It is pertinent to mention here that this is the first report to stain these compounds in which dereplication approach based on GC-MS was applied to target unknown compounds ACH-6 and ACH-8 in A. calamus L. Derelpication approaches based on GC-MS is very useful technique in the area of drug discovery and have eminence potential to identify known and unknown compounds present in extracts of medicinal important plants. This technique can be used to expedite the process of purification of unknown compounds from different matrixes. The isolated compounds were identified with the help of inbuilt library search which reveals the presence of 17 known and 4 unknown compounds. Further, the structure elucidation of all isolated compounds was done using spectroscopy techniques. Also, the structure of ACH-6 was further confirmed by using the single-crystal X-ray diffraction technique.

Dereplication Based Strategy for Rapid Identification and Isolation of a Novel Anti-inflammatory Flavonoid by LCMS/MS from Colebrookea oppositifolia.

Colebrookea oppositifolia is a folkloric medicinal plant, well known for its tremendous medicinal properties such as curing epilepsy, ulcers, and urinary problems. The aim of the present study was to apply the dereplication strategy on the ethanol extract of C. oppositifolia with potent anti-inflammatory activity for the rapid identification and isolation of novel bioactive molecules to aid the drug discovery process. An integrated approach using liquid chromatography-mass spectrometry (LCMS) followed by preparative high-performance liquid chromatography (HPLC) was used for the isolation of potent molecules from the anti-inflammatory extract of C. oppositifolia . Purity of the compounds (>98.5%) was established by HPLC, and identification was carried out by NMR and ESI-MS. 5,6,7-Trihydroxyflavone-3-O-glucuronide methyl ester (compound III) isolated from C. oppositifolia was extensively studied for anti-inflammatory potential in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the mice model. Compound III significantly repressed various proinflammatory mediators and upregulated the release of anti-inflammatory cytokine IL-10. Compound III reduced inflammation when studied for parameters such as the phagocytic index, carrageenan-induced paw edema in mice, and effect on organ weight. It reduced inflammation in a dose-dependent manner both in vitro and in vivo. Further molecular insights into the study revealed that compound III blocks the phosphorylation of I kappa b kinase α/ß (IKKα/ß), IκBα, and nuclear factor kB p65 (NF-κBp65) which is a key controller of inflammation, thereby showing anti-inflammatory potential. Hence, this study permits further investigation to develop compound III as an anti-inflammatory drug.

Investigating 11 Withanosides and Withanolides by UHPLC-PDA and Mass Fragmentation Studies from Ashwagandha (Withania somnifera).

Withania somnifera (WS), also known as ashwagandha or Indian ginseng, is known for its pharmacological significance in neurodegenerative diseases, stress, cancer, immunomodulatory, and antiviral activity. In this study, the WS extract (WSE) from the root was subjected to ultrahigh-performance liquid chromatography with photodiode array detection (UHPLC-PDA) analysis to separate 11 withanoside and withanolide compounds. The quantification validation was carried out as per ICHQ2R1 guidelines in a single methodology. The calibration curves were linear (r 2 > 0.99) for all 11 compounds within the tested concentration ranges. The limits of detection and quantification were in the range of 0.213-0.362 and 0.646-1.098 µg/mL, respectively. The results were precise (relative standard deviation, <5.0%) and accurate (relative error, 0.01-0.76). All compounds showed good recoveries of 84.77-100.11%. For the first time, withanoside VII, 27-hydroxywithanone, dihydrowithaferin A, and viscosalactone B were quantified and validated along with bioactive compounds withanoside IV, withanoside V, withaferin A, 12-deoxywithastramonolide, withanolide A, withanone, and withanolide B simultaneously in WS. This UHPLC-PDA method has practical adaptability for ashwagandha raw material, extract, and product manufacturers, along with basic and applied science researchers. The method has been developed on UHPLC for routine analysis. The 11 withanosides and withanolides were confirmed using the fragmentation pattern obtained by the combined use of electrospray ionization and collision-induced dissociation in triple-quadrupole tandem mass spectrometry (TQ-MS/MS) in the WSE.

Attenuation of Glutamate-Induced Excitotoxicity by Withanolide-A in Neuron-Like Cells: Role for PI3K/Akt/MAPK Signaling Pathway.

Glutamate-induced excitotoxicity is one of the major underlying mechanisms for neurodegenerative diseases. Efforts are being made to treat such conditions with an array of natural compounds that can modulate the release of glutamate or the underlying mechanisms associated with it. Withania somnifera extract has potent pharmacologic activity similar to that of Korean Ginseng tea and is used to treat several neuronal disorders. However, to date, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to investigate withanolide-A, one of the active constituents of Withania somnifera against glutamate-induced excitotoxicity in retinoic acid differentiated Neuro2a neuroblastoma cells. The results indicated that glutamate treatment for 2 h induced death in cells that was significantly attenuated by pre-treatment with MK-801 (specific NMDA receptor antagonist) and different concentrations of withanolide-A. Withanolide-A abated the glutamate-induced influx of intracellular calcium and excessive ROS production significantly. Further on, glutamate treatment resulted in increased levels of pro-apoptotic and decreased levels of anti-apoptotic proteins, and these protein levels were normalized by various doses of withanolide-A. All of these protective effects were partly due to inhibition of MAPK family proteins and activation of PI3K/Akt signaling. Thus, our results suggest that withanolide-A may serve as potential neuroprotective agent.

Withanone, an Active Constituent from Withania somnifera, Affords Protection Against NMDA-Induced Excitotoxicity in Neuron-Like Cells.

Withania somnifera has immense pharmacologic and clinical uses. Owing to its similar pharmacologic activity as that of Korean Ginseng tea, it is popularly called as Indian ginseng. In most cases, extracts of this plant have been evaluated against various diseases or models of disease. However, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to evaluate Withanone, one of the active constituents of Withania somnifera against NMDA-induced excitotoxicity in retinoic acid, differentiated Neuro2a cells. Cells were pre-treated with 5, 10 and 20 µM doses of Withanone and then exposed to 3-mM NMDA for 1 h. MK801, a specific NMDA receptor antagonist, was used as positive control. The results indicated that NMDA induces significant death of cells by accumulation of intracellular Ca2+, generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, crashing of Bax/Bcl-2 ratio, release of cytochrome c, increased caspase expression, induction of lipid peroxidation as measured by malondialdehyde levels and cleavage of poly(ADP-ribose) polymerase-1 (Parp-1), which is indicative of DNA damage. All these parameters were attenuated with various doses of Withanone pre-treatment. These results suggest that Withanone may serve as potential neuroprotective agent.

Hydroxychavicol: A phytochemical targeting cutaneous fungal infections.

The present study was designed to investigate the potency of hydroxychavicol on selected cutaneous human pathogenic fungi by the use of in vitro and in vivo assays and mechanistic characterization along with toxicological effects. Hydroxychavicol consistently displayed a fungicidal effect against all fungal species tested. Inoculum concentrations over the range of 104 to 107 CFU/ml did not significantly alter its antifungal potential and time-kill curve results revealed concentration-dependent killing. It also inhibited the growth of biofilm generated by Trichophyton mentagrophytes and Candida parapsilosis and reduced the preformed biofilms. Hydroxychavicol was highly effective in the treatment, and mycological eradication of an experimentally induced topical infection model of dermatophytosis (tinea corporis) and cutaneous candidiasis in guinea pigs, respectively. The mode of action of hydroxychavicol appears to originate from the disruption of cell membrane integrity. Administration of hydroxychavicol in mice at 500 mg per kg of body weight by orally produced no overt toxicity. The retention capacity of hydroxychavicol in vitro, in the presence of keratin has attributed to its in vivo effectiveness in the guinea pig model of topical infections. Furthermore, it is suggestive of its potential use as phytochemical for topical use in cutaneous fungal infections.

Therapeutic effects of R8, a semi-synthetic analogue of Vasicine, on murine model of allergic airway inflammation via STAT6 inhibition.

This is a follow-up study of our previous work in which we screened a series of Vasicine analogues for their anti-inflammatory activity in a preventive OVA induced murine model of asthma. The study demonstrated that R8, one of the analogues, significantly suppressed the Th2 cytokine production and eosinophil recruitment to the airways. In the present study, we have been using two standard experimental murine models of asthma, where the mice were treated with R8 either during (preventive use) or after (therapeutic use) the development of asthma features. In the preventive model, R8 reduced inflammatory cell infiltration to the airways, OVA specific IgE and Th2 cytokine production. Also, the R8 treatment in the therapeutic model decreased methacholine induced AHR, Th2 cytokine release, serum IgE levels, infiltration of inflammatory cells into the airways, phosphorylation of STAT6 and expression of GATA3. Moreover, R8 not only reduced goblet cell metaplasia in asthmatic mice but also reduced IL-4 induced Muc5AC gene expression in human alveolar basal epithelial cells. Further, R8 attenuated IL-4 induced differentiation of murine splenocytes into Th2 cells in vitro. So, we may deduce that R8 treatment profoundly reduced asthma features by attenuating the differentiation of T cells into Th2 cells by interfering with the binding of IL-4 to its receptor in turn decreasing the phosphorylation of STAT6 and expression of GATA3 in murine model of asthma. These preclinical findings suggest a possible therapeutic role of R8 in allergic asthma.

Ethanolic extract of Alternanthera sessilis (AS-1) inhibits IgE-mediated allergic response in RBL-2H3 cells.

The present study was designed to investigate the anti-allergic effects of ethanolic extract of Alternanthera sessilis (AS-1) in rat basophilic leukemia (RBL-2H3) cells. It significantly reduced the ß-hexosaminidase release from anti-DNP-IgE sensitized RBL-2H3 cells. AS-1also inhibited the IgE antibody-induced increase in Interleukin-6 (IL-6), TNF-α, IL-13 and IL-4 production in these cells. The inhibitory effect of AS-1 on these cytokine was found to be nuclear factor-KB (NF-kB) dependent, as it attenuated the degradation of IKBa and nuclear translocation of NFkB. In addition, AS-1 significantly attenuated the DNP HAS-induced intracellular Ca(2+) release from these cells, which makes us speculate strongly that the decreased intracellular Ca(2+) is involved in the inhibitory effect of AS-1 on ß-hexoaminidase release. Taken together, anti-allergic effects of AS-1 suggest possible therapeutic application of this extract in allergic diseases.

Anti-arthritic activity of agnuside mediated through the down-regulation of inflammatory mediators and cytokines.

OBJECTIVE AND DESIGN: The purpose of this study was to elucidate the probable mechanism for the anti-arthritic activity of agnuside (AGN), a compound isolated from the leaf extract of Vitex negundo. METHODOLOGY: The anti-inflammatory activity of AGN within a dose range of 1.56-12.50 mg/kg in normal and adrenalectomized rats was evaluated against different inflammagens. An array of pro-inflammatory mediators (PGE(2) and LTB(4)) and T-cell-mediated cytokines (IL-2, TNF-α, IFN-γ, IL-4, IL-10, IL-17) was assayed using flow cytometry, in arthritic paw tissue homogenate and splenocytes of treated animals. RESULTS: Significant anti-arthritic activity was observed in the polyarthritis test in rats and this was associated with significant suppression of inflammatory mediators and T-cell-mediated cytokines (Th1/Th2). The anti-inflammatory activity in adrenalectomized rats confirmed that the effect of AGN is not mediated by the pituitary-adrenal axis. AGN also showed inhibition of vascular permeability and leukocyte migration in vivo. CONCLUSION: The study suggests the possible development of AGN as a therapeutic agent in the treatment of arthritis by the modulation of the host immune response.

Differential effects of chlorogenic acid on various immunological parameters relevant to rheumatoid arthritis.

Despite chlorogenic acid (CGA) being widely present in nature, particularly in the human diet, there is very little information regarding its pharmacological activities. The present investigation was carried out to investigate the antiarthritic activities of this compound in adjuvant induced-arthritis in male Wistar rats, and to explore the underlying mechanisms of actions in view of immunological responses. We observed that CGA effectively controlled the total (CD3) and differentiated (CD4 and CD8) T cells count at the dose of 40 mg/kg. We also assessed the effect on co-stimulatory molecules (CD28, CD80/86) and found that CGA efficiently suppressed CD80/86 but failed to bring any changes in the CD28 count, whereas ibuprofen (standard drug) resulted in highly significant inhibition of both. We next examined the effect on CD4⁺ T cells specific Th1/Th2 cytokines by flow cytometry and observed that CGA suppressed the Th1 cytokines in a highly significant manner but elevated Th2 cytokines with dose dependence. Results of the present investigation suggest that CGA is a potent antiarthritic agent.

In vitro antifungal activities of amphotericin B in combination with acteoside, a phenylethanoid glycoside from Colebrookea oppositifolia.

This study was undertaken to investigate the synergistic interaction between amphotericin B (AmB) and acteoside, isolated from the aerial parts of the shrub Colebrookea oppositifolia (Lamiaceae). Acteoside alone exhibited no intrinsic antifungal activity but showed a potent synergism in combination with AmB against selected pathogenic species, with fractional inhibitory concentration indices in the range of 0.0312-0.1562. The combination of acteoside at 3.12 and 12.5 µg ml(-1) with subinhibitory concentrations of AmB resulted in a potent fungicidal effect and also exhibited a significantly extended post-antifungal effect. Furthermore, the combination also reduced the minimum biofilm reduction concentration values of AmB (2-16-fold) in preformed biofilms of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. There was decreased viability of the cells, increased uptake of propidium iodide and enhanced leakage of 260 nm-absorbing material by Candida albicans cells when exposed to AmB in the presence of acteoside. The reason for potentiation is likely to be that the subinhibitory concentrations of AmB facilitated the uptake of acteoside, which resulted in increased killing of the fungal cells. Administration of acteoside in mice at up to 2000 mg (kg body weight)(-1) by the intraperitoneal or oral route produced no overt toxicity. The data presented here support synergism between acteoside and AmB, and it is therefore proposed that a prospective new management strategy for therapeutic application of this combination should be explored.

Stimulatory effects of Cuminum cyminum and flavonoid glycoside on Cyclosporine-A and restraint stress induced immune-suppression in Swiss albino mice.

Many herbs and spices are known to modulate the immune system and have been shown to restore the immunity in immuno-compromised individuals. Spices generally used to increase the taste and flavor of food also has the history of usage as an ayurvedic medicine. Therefore to explore the health modulating effects of Cuminum cyminum and to identify the active compound, immunomodulatory properties were evaluated using flowcytometry and ELISA in normal and immune-suppressed animals. C. cyminum and compound 1 stimulated the T cells and Th1 cytokines expression in normal animals. Swiss albino mice subjected to Cyclosporine-A induced immune-suppression were dosed orally with C. cyminum (25, 50, 100 and 200 mg/kg) on consecutive days. The results showed that administration significantly increased T cells (CD4 and CD8) count and Th1 predominant immune response in a dose dependent manner thereby suggesting immunomodulatory activity through modulation of T lymphocytes expression. In restraint stress induced immune-suppressed animals, compound 1 countered the depleted T lymphocytes, decreased the elevated corticosterone levels and size of adrenal glands and increased the weight of thymus and spleen. Based on the data we may conclude that C. cyminum is a potent immunomodulator and may develop as a lead to recover the immunity of immuno-compromised individuals.

Amelioration of adjuvant induced arthritis by apocynin.

The premise of the study was to investigate the antiarthritic potential of apocynin (APO) in Balb/c mice (in vivo). The experiment showed a dose-dependent decrease in oedema and showed a suppression of proinflammatory cytokines such as TNF-alpha and IL-1beta and mediators such as prostaglandin E(2) (PGE(2)) and LTB(4). At oral doses of 0.5, 1.0, 2.0 and 4.0 mg/kg once daily during the course of the experiment, APO induced an inhibition of T cell mediated immune response causing suppression of CD4+ and CD8+ T cells and of intracellular interferon-gamma (IFN-gamma) by flow cytometry in arthritic mice. In parallel there was a dose-dependent inhibition in vascular permeability causing an inhibition in the migration of leucocytes and exudate volume at the site of the inflammatory reaction. These observations validate the immunoregulatory potential of apocynin.

Evaluation of the antimicrobial, antioxidant, and anti-inflammatory activities of hydroxychavicol for its potential use as an oral care agent.

Hydroxychavicol isolated from the chloroform extraction of aqueous extract of Piper betle leaves showed inhibitory activity against oral cavity pathogens. It exhibited an inhibitory effect on all of the oral cavity pathogens tested (MICs of 62.5 to 500 microg/ml) with a minimal bactericidal concentration that was twofold greater than the inhibitory concentration. Hydroxychavicol exhibited concentration-dependent killing of Streptococcus mutans ATCC 25175 up to 4x MIC and also prevented the formation of water-insoluble glucan. Interestingly, hydroxychavicol exhibited an extended postantibiotic effect of 6 to 7 h and prevented the emergence of mutants of S. mutans ATCC 25175 and Actinomyces viscosus ATCC 15987 at 2x MIC. Furthermore, it also inhibited the growth of biofilms generated by S. mutans and A. viscosus and reduced the preformed biofilms by these bacteria. Increased uptake of propidium iodide by hydroxychavicol-treated cells of S. mutans and A. viscosus indicated that hydroxychavicol probably works through the disruption of the permeability barrier of microbial membrane structures. Hydroxychavicol also exhibited potent antioxidant and anti-inflammatory activities. This was evident from its concentration-dependent inhibition of lipid peroxidation and significant suppression of tumor necrosis factor alpha expression in human neutrophils. Its efficacy against adherent cells of S. mutans in water-insoluble glucan in the presence of sucrose suggests that hydroxychavicol would be a useful compound for the development of antibacterial agents against oral pathogens and that it has great potential for use in mouthwash for preventing and treating oral infections.

Immunomodulation exhibited by piperinic acid through suppression of proinflammatory cytokines.

Piper longum (PL) has been reported for its varied pharmacological activities including bio-enhancer and anti-inflammatory activities in traditional medicine. Here the premise of the study was to investigate the immunoregulatory potential of PL and piperinic acid, one of its active constituent, in Balb/C mice (in vivo) and human PBMCs (in vitro) models. Piperinic acid moderated the proinflammatory mediators and cytokines in our experiments. At doses of 10, 20, 40 and 80 mg/kg p.o. PL showed a dose dependent decrease of lymphocytes (CD4+ and CD8+ T cells) and cytokine levels in sensitized Balb/C mice with a marked inhibition at 40 mg/kg. At an in vitro dose of 20 mug/ml of PL and 5 mug/ml of piperinic acid, there was a significant inhibition of mitogen induced human PBMC proliferation, mRNA transcripts of IL-2 (ConA) and TNFalpha, IL-1beta and iNOS (LPS) respectively under stimulated conditions in time dependent (6 h, 12 h and 24 h respectively) expression studies. In parallel, induced nitric oxide production was also reduced by stimulated macrophages. Our observations rationalize the traditional use of PL and also validate the immunoregulatory potential of piperinic acid.

Anti-arthritic activity of a biopolymeric fraction from Euphorbia tirucalli.

The present study was undertaken to investigate the anti-arthritic activity of a biopolymeric fraction (BET) from plant Euphorbia tirucalli Boiss (Euphorbiaceae). The fraction showed dose dependent anti-arthritic activity and also showed in vivo immunomodulatory capacity being a major component in inhibiting arthritis. It caused suppression of CD4(+) and CD8(+) T cells, inhibition of intracellular Interleukin-2 (IL-2) and Interferon-gamma (IFN-gamma) by flowcytometry. It inhibited vascular permeability and the migration of leucocytes at the site of the insult. The oral LD(0) in both rats and mice was more than 2000 mg/kg.