Durbadal Ojha

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

BackgroundViral 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.ResultThe cytotoxicity (CC50, the concentration of 50% cellular toxicity), antiviral effective concentration (EC50, the concentration required to achieve 50% protection against virus-induced cytopathic effect), plaque reduction and the selectivity index (SI, the ratio of CC50 and EC50) 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 (EC50 = 7.8 and 5.5 μg/ml; SI = 22.3 and 20) and HSV-2 (EC50 = 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.ConclusionThis 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.

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.

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.

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.

Fluorescence sensing and intracellular imaging of Al3+ ions by using naphthalene based sulfonamide chemosensor: structure, computation and biological studies

A naphthalene based sulfonamide Schiff base, (E)-4-(((2-hydroxynaphthalen-1-yl)methylene)amino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (HL) has been found to be a flourescence turn-on probe for selective detection of Al3+ in aqueous system. Structure of the probe has been established by FTIR, 1H NMR, mass spectra and X-ray single crystal study. The probe has shown 24 times flourescence enhancement in presence of Al3+. The limit of detection (LOD) obtained by 3σ method is 33.2 nM. The probable co-ordination environment of L-Al2+ complex has been supported by mass spectral data and DFT computational study. By TD-DFT calculation UV-Vis spectra of HL and L-Al2+ complex has been predicted and that has well correlated with experimental data. Cell imaging study reveals that the probe can be used for the intracellular detection of Al3+ in cultured Vero cell. Antimicrobial activity of HL has also been evaluated and probable mode of binding inside the DHPS cavity has been predicted by docking study. This sensor is unique with reference to two other predecessors because of its bio-compatibility of sulfonamide derivative and nonmutagenicity.

An efficient vanillinyl Schiff base as a turn on fluorescent probe for zinc(II) and cell imaging

6,6′-((1Z,1′Z)-(((Ethane-1,2-diylbis(oxy))bis(2,1-phenylene))bis(azanylylidene))bis(methanyl ylidene))bis(3-methoxyphenol), a vanillinyl Schiff base, shows unprecedented fluorescent zinc sensing properties and has been examined for zinc bioimaging. Zinc induced turn-on fluorescence enhancement is observed at 472 nm and steady state photophysical studies establish the metal promoted deprotonation and chelation enhancement of emission. Formation of the 1:1 metal-to-ligand complex has been ascertained by 1H NMR, mass spectral analysis and Jobs plot. Limit of detection (LOD) is the lowest, 0.018 μM in the family of fluorogenic Zn2+-sensors. Apart from this, theoretical interpretation of the experimental outcome has also been obtained by applying density functional theory (DFT) to the ligand and the complex. The practical applicability of the ligand has been examined in living cells (African Monkey Vero Cells). MTT assay proves the no toxicity of probe upto 300 μg ml−1.

Antibacterial and antiviral evaluation of sulfonoquinovosyldiacylglyceride: a glycolipid isolated from Azadirachta indica leaves

Assessment of antibacterial as well as antiherpes virus activity of sulfonoquinovosyldiacylglyceride (SQDG), a glycolipid, isolated from the leaves of Azadirachta indica has been described. Antimicrobial activity was evaluated against Gram‐positive, Gram‐negative bacteria and herpes simplex virus. SQDG showed significant inhibitory activity against Salmonella typhi and two isolates of Shigella dysenteriae with MIC values 32 μg ml−1, while three isolates of Salm. typhi, Escherichia coli and Vibrio cholerae were inhibited at 64 μg ml−1 and have shown zone diameter ranging from 6·2 to 12·3 mm. The growth kinetics study of SQDG on Salm. typhi and Sh. dysenteriae revealed that the growths were completely inhibited at their MIC values within 24 h of exposure. Interestingly, SQDG inhibits herpes simplex virus (HSV) type 1 and 2 with the EC50 of 9·1 and 8·5 μg ml−1, compared with acyclovir (2·2 and 2·8 μg ml−1 against HSV‐1 and HSV‐2). The selectivity index (SI) was found to be 12·4 against HSV‐1 and 13·41 with HSV‐2. Furthermore, the expression of proinflammatory cytokines of HSV‐infected and SQDG‐treated macrophages using ELISA kit revealed that SQDG significantly downregulated the production of TNF‐α, IL‐1β, IL‐12 and IL‐6.

Imine-functionalized thioether Zn(II) turn-on fluorescent sensor and its selective sequential logic operations with H2PO4−, DFT computation and live cell imaging

The diformyl thioether Schiff base (H2L) exhibits fluorescent sensing behaviour towards Zn2+ with a limit of detection (LOD) of 0.050 μM, which is far below the WHO guideline (76 μM) in drinking water. The formation of a 1 : 1 metal-to-ligand complex, [ZnL], has been ascertained by X-ray crystallography, Jobs plot and mass spectra. The fluorogenic complex, [ZnL] (either in situ or a solution of the isolated complex) has shown selective ON–OFF emission toward H2PO4−. A two-input, one-output sequential INHIBIT logic circuit has been constructed from H2L, Zn2+ and H2PO4−. The practical applicability of H2L has been examined by the identification of Zn2+ and H2PO4− in the intracellular fluid of African monkey Vero cells.

Anti-HSV activity and mode of action study of α-pyrone carboxamides

The clinical management of herpes virus diseases is limited due to ineffective clearance of virus particles and frequent emergence of drug-resistant viruses, particularly in immunocompromised patients, pregnant women and neonates. In our continued quest for new antiviral leads, α-pyrone carboxamide propanol derivatives were synthesized and evaluated in HSV infected Vero cells. Compound 3d showed potent antiviral activity against HSV-IF (EC50 = 9.8 μg ml−1 and EC99 = 18.0 μg ml−1) and HSV-2G (EC50 = 12.4 μg ml−1 and EC99 = 24.0 μg ml−1) at 4–6 h post-infection. The mode of action studies demonstrated that 3d did not interfere in viral attachment or penetration, however, it reduced the expression of ICP4 and ICP27 (immediate-early gene products) as well as the HSV DNA polymerase.