Evaluation of an ethnomedicinal combination containing Semecarpus kurzii and Hernandia peltata used for the management of inflammation.

CONTEXT: Scientific validation of an ethnomedicinal combination consisting of Semecarpus kurzii Engler (Anacardeaceae) leaves (SKL) and Hernandia peltata Meisn (Hernandeaceae) stem-bark (HPB), traditionally used in ailments related to inflammation, pain and fever. OBJECTIVE: To validate in vivo and in vitro analgesic and antiinflammatory activities of methanol extract of SKL, HPB and their combination. MATERIALS AND METHODS: Analgesic activity was tested by acetic acid induced writhing reflex and tail flick in Swiss albino mice, while the anti-inflammatory activity was studied in acute, subacute and chronic model on Wistar rats. The vascular permeability, membrane stabilization and protein denaturation were examined to know the possible mode of action. RESULTS: Significant (p < 0.01) analgesic (78.04% inhibition of writhing) and antiinflammatory (72.54% inhibition of paw edema) activity was observed in combination of SKL and HPB extracts at 250 mg/kg each. The SKL extract alone inhibits acetic acid-induced vascular permeability (64.4%) at 500 mg/kg, while in combination at 250 mg/kg each, the inhibition was 69.49% (p < 0.01). Furthermore, SKL in combination with HPB (0.25 mg/mL each) prevent RBC hemolysis (61.91%) and inhibition of protein denaturation (76.52%)-like indomethacin. DISCUSSION AND CONCLUSION: The SKL and HPB extract, alone (500 mg/kg) and in combination, (250 mg/kg each) had significant analgesic and antiinflammatory activity, probably by inhibiting the release of certain inflammatory mediators and membrane stabilization, due to the presence of triterpenes, tannins and related phytochemicals in the extracts. Thus, our results demonstrated that this combination provide the scientific rationale of its folk use.

Correction: Anti-Inflammatory Activity of Odina wodier Roxb, an Indian Folk Remedy, through Inhibition of Toll-Like Receptor 4 Signaling Pathway.

[This corrects the article DOI: 10.1371/journal.pone.0104939.].

Anti-herpes virus activities of bioactive fraction and isolated pure constituent of Mallotus peltatus: an ethnomedicine from Andaman Islands.

BACKGROUND: Viral infections, particularly the infections caused by herpes simplex virus (HSV), represent one of the most serious public health concerns globally because of their devastating impact. The aim of this study was to evaluate the antiviral potential of methanolic crude extract of an ethnomedicine Mallotus peltatus, its active fraction and pure compound, against HSV-1 F and HSV-2 G. RESULT: The cytotoxicity (CC(50), the concentration of 50% cellular toxicity), antiviral effective concentration (EC(50), the concentration required to achieve 50% protection against virus-induced cytopathic effect), plaque reduction and the selectivity index (SI, the ratio of CC(50) and EC(50)) was determined. Results showed that the crude methanolic extract of M. peltatus possessed weak anti-HSV activity. In contrast, the active fraction A and isolated ursolic acid from fraction A exhibited potent antiherpesvirus activity against both HSV-1 (EC(50)= 7.8 and 5.5 µg/ml; SI = 22.3 and 20) and HSV-2 (EC(50)= 8.2 and 5.8 µg/ml, and SI = 21.2 and 18.97). The fraction A and isolated ursolic acid (10 µg/ml) inhibited plaque formation of HSV-1 and HSV-2 at more than 80% levels, with a dose dependent antiviral activity, compared to acyclovir. The time response study revealed that the anti-HSV activity of fraction A and isolated ursolic acid is highest at 2-5 h post-infection. Moreover, the time kinetics study by indirect immunofluorescence assay showed a characteristic pattern of small foci of single fluorescent cells in fraction A- treated virus infected cells at 2 h and 4 h post-infection, suggesting drug inhibited viral dissemination. Further, the PCR study with infected cell cultures treated with fraction A and isolated ursolic acid at various time intervals, failed to show amplification at 48-72 h, like acyclovir treated HSV-infected cells. Moreover, fraction A or isolated ursolic acid showed no interaction in combination with acyclovir. CONCLUSION: This study revealed that bioactive fraction A and isolated ursolic acid of M. peltatus has good anti-HSV activity, probably by inhibiting the early stage of multiplication (post-infection of 0-5 h), with SI value of 20, suggesting its potential use as anti-HSV agents.

Validation of a traditional preparation against multi-drug resistant Salmonella Typhi and its protective efficacy in S. Typhimurium infected mice.

Shorea robusta Gaertn has been used for skin and intestinal ailments in Indian Traditional medicine; while two tribal communities used its tender leaves in 'Meyadi-bukhar' or long-term fever. This prompted us to validate the aqueous and methanol extracts of Shorea robusta tender leaves against wild- and multidrug-resistant clinical isolates of Salmonella enterica Serovar Typhi (S. Typhi), the causative agent of typhoid fever. The antibacterial activity, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and growth inhibition were determined using disc diffusion, agar-and-broth dilution, dose- and time-response assays, along with the safety and protective efficacy in Balb/C mice, infected with S. Typhimurium. The MIC of the extract was 256-450 µg/ml against S. Typhi isolates, and 700 µg/ml for mouse virulent S. Typhimurium, while MBC was ≤512-1024 µg/ml. The growth curve revealed that the extract was bactericidal at 4-6 h of exposure. Toxicity study showed that the extract was safe up to 3000 mg/kg (p.o.). Moreover, it significantly (p > 0.01) protect the challenged (1.4 × 108 cfu/ml) mice at 93.75 (i.p.) and 300 mg/kg (p.o.) dose, compared to the infection control (distilled water treatment group). Collectively, our results confirmed the antibacterial potential of the test extracts against MDR-isolates of S. Typhi.

Anti-inflammatory activity of Odina wodier Roxb, an Indian folk remedy, through inhibition of toll-like receptor 4 signaling pathway.

Inflammation is part of self-limiting non-specific immune response, which occurs during bodily injury. In some disorders the inflammatory process becomes continuous, leading to the development of chronic inflammatory diseases including cardiovascular diseases, diabetes, cancer etc. Several Indian tribes used the bark of Odina wodier (OWB) for treating inflammatory disorders. Thus, we have evaluated the immunotherapeutic potential of OWB methanol extract and its major constituent chlorogenic acid (CA), using three popular in vivo antiinflammatory models: Carrageenan- and Dextran-induced paw edema, Cotton pellet granuloma, and Acetic acid-induced vascular permeability. To elucidate the possible anti-inflammatory mechanism of action we determine the level of major inflammatory mediators (NO, iNOS, COX-2-dependent prostaglandin E2 or PGE2), and pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-12). Further, we determine the toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), c-Jun N-terminal kinases (JNK), nuclear factor kappa-B cells (NF-κB), and NF-kB inhibitor alpha (IK-Bα) by protein and mRNA expression, and Western blot analysis in drug treated LPS-induced murine macrophage model. Moreover, we determined the acute and sub-acute toxicity of OWB extract in BALB/c mice. Our study demonstrated a significant anti-inflammatory activity of OWB extract and CA along with the inhibition of TNF-α, IL-1ß, IL-6 and IL-12 expressions. Further, the expression of TLR4, NF-κBp65, MyD88, iNOS and COX-2 molecules were reduced in drug-treated groups, but not in the LPS-stimulated untreated or control groups, Thus, our results collectively indicated that the OWB extract and CA can efficiently inhibit inflammation through the down regulation of TLR4/MyD88/NF-kB signaling pathway.

A dihydro-pyrido-indole potently inhibits HSV-1 infection by interfering the viral immediate early transcriptional events.

In our continued quest for identifying novel molecules from ethnomedicinal source we have isolated an alkaloid 7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole, also known as Harmaline (HM), from an ethnomedicinal herb Ophiorrhiza nicobarica. The compound exhibited a potent anti-HSV-1 activity against both wild type and clinical isolates of HSV-1. Further we demonstrated that HM did not interfere in viral entry but the recruitment of lysine-specific demethylase-1 (LSD1) and the binding of immediate-early (IE) complex on ICP0 promoter. This leads to the suppression of viral IE gene synthesis and thereby the reduced expression of ICP4 and ICP27. Moreover, HM at its virucidal concentration is nontoxic and reduced virus yields in cutaneously infected Balb/C mice. Thus, the interference in the binding of IE complex, a decisive factor for HSV lytic cycle or latency by HM reveals an interesting target for developing non-nucleotide antiherpetic agent with different mode of action than Acyclovir.

Anti-herpes virus activities of Achyranthes aspera: an indian ethnomedicine, and its triterpene acid.

The aim of this study was to evaluate the antiviral potential of methanolic extract (ME) of Achyranthes aspera, an Indian folk medicine and one of its pure compound oleanolic acid (OA) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The ME possessed weak anti-herpes virus activity (EC50 64.4µg/ml for HSV-1 and 72.8µg/ml for HSV-2). While OA exhibited potent antiherpesvirus activity against both HSV-1 (EC50 6.8µg/ml) and HSV-2 (EC50 7.8µg/ml). The time response study revealed that the antiviral activity of ME and OA is highest at 2-6h post infection. The infected and drug-treated peritoneal macrophage at specific time showed increased level of pro-inflammatory cytokines (IL6 and IL12). Further, the PCR of DNA from infected cultures treated with ME and OA, at various time intervals, failed to show amplification at 48-72h, similar to that of HSV infected cells treated with acyclovir, indicating that the ME and OA probably inhibit the early stage of multiplication (post infection of 2-6h). Thus, our study demonstrated that ME and OA have good anti-HSV activity, with SI values of 12, suggesting the potential use of this plant.

Evaluation of the wound healing activity of Shorea robusta, an Indian ethnomedicine, and its isolated constituent(s) in topical formulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Different parts of Indian ethnomedicinal plant Shorea robusta is traditionally used for several ailments including wounds and burn by different tribal groups, since ages. Here we have validated, for the first time, the effectiveness and the possible mechanism of action of young leaf extracts of Shorea robusta, used by two distinct tribes of India, and its isolated compounds as a topical formulation in three wound models in rats. MATERIALS AND METHODS: Bioactivity-guided study of the active extract resulted in the isolation of two known compounds. The prepared ointment containing extracts (2.5 and 5%, w/w), fractions (5% w/w) and isolated compounds (0.25% w/w) were evaluated on excision, incision and dead space wound models in rats by the rate of wound closure, period of epithelialisation, tensile strength, granulation tissue weight, hydroxyproline content and histopathology. RESULTS: The animals treated with the extracts and fractions (5%) showed significant reduction in wound area 96.55 and 96.41% with faster epithelialisation (17.50 and 17.86), while the isolated compounds bergenin and ursolic acid heal the wound faster, but complete epithelialisation with 100% wound contraction was evident with 5% povidone-iodine group on 18th post-wounding day. Moreover, the tensile strength of incision wound, granuloma tissue weight, and hydroxyproline content was significantly increased in both the extract and compound(s) treated animals. Furthermore, the tissue histology of animals treated with the isolated compound(s) showed complete epithelialisation with increased collagenation, similar to povidone-iodine group. CONCLUSION: Thus, our results validated the traditional use of Shorea robusta young leaves in wound management.

An indole alkaloid from a tribal folklore inhibits immediate early event in HSV-2 infected cells with therapeutic efficacy in vaginally infected mice.

Herpes genitalis, caused by HSV-2, is an incurable genital ulcerative disease transmitted by sexual intercourse. The virus establishes life-long latency in sacral root ganglia and reported to have synergistic relationship with HIV-1 transmission. Till date no effective vaccine is available, while the existing therapy frequently yielded drug resistance, toxicity and treatment failure. Thus, there is a pressing need for non-nucleotide antiviral agent from traditional source. Based on ethnomedicinal use we have isolated a compound 7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole (HM) from the traditional herb Ophiorrhiza nicobarica Balkr, and evaluated its efficacy on isolates of HSV-2 in vitro and in vivo. The cytotoxicity (CC50), effective concentrations (EC50) and the mode of action of HM was determined by MTT, plaque reduction, time-of-addition, immunofluorescence (IFA), Western blot, qRT-PCR, EMSA, supershift and co-immunoprecipitation assays; while the in vivo toxicity and efficacy was evaluated in BALB/c mice. The results revealed that HM possesses significant anti-HSV-2 activity with EC50 of 1.1-2.8 µg/ml, and selectivity index of >20. The time kinetics and IFA demonstrated that HM dose dependently inhibited 50-99% of HSV-2 infection at 1.5-5.0 µg/ml at 2-4 h post-infection. Further, HM was unable to inhibit viral attachment or penetration and had no synergistic interaction with acyclovir. Moreover, Western blot and qRT-PCR assays demonstrated that HM suppressed viral IE gene expression, while the EMSA and co-immunoprecipitation studies showed that HM interfered with the recruitment of LSD-1 by HCF-1. The in vivo studies revealed that HM at its virucidal concentration was nontoxic and reduced virus yield in the brain of HSV-2 infected mice in a concentration dependent manner, compared to vaginal tissues. Thus, our results suggest that HM can serve as a prototype to develop non-nucleotide antiviral lead targeting the viral IE transcription for the management of HSV-2 infections.