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.

A natural small molecule inhibitor corilagin blocks HCV replication and modulates oxidative stress to reduce liver damage.

Hepatitis C virus (HCV) infection causes chronic liver disease, which often leads to hepatocellular carcinoma. Earlier, we have demonstrated anti-HCV property of the methanolic extract of Phyllanthus amarus, an age-old folk-medicine against viral hepatitis. Here, we report identification of a principal bioactive component 'corilagin', which showed significant inhibition of the HCV key enzymes, NS3 protease and NS5B RNA-dependent-RNA-polymerase. This pure compound could effectively inhibit viral replication in the infectious cell culture system, displayed strong antioxidant activity by blocking HCV induced generation of reactive oxygen species and suppressed up-regulation of NOX4 and TGF-ß mRNA levels. Oral administration of corilagin in BALB/c mice demonstrated its better tolerability and systemic bioavailability. More importantly, corilagin could restrict serum HCV RNA levels, decrease collagen deposition and hepatic cell denaturation in HCV infected chimeric mice harbouring human hepatocytes. Taken together, results provide a basis towards developing a pure natural drug as an alternate therapeutic strategy for restricting viral replication and prevent liver damage towards better management of HCV induced pathogenesis.

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.

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.

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.

Structure based molecular design, synthesis and biological evaluation of α-pyrone analogs as anti-HSV agent.

Several options for treating Herpes Simplex Virus type 1 and type 2 are available. However, non-specific inhibition and drug resistance warrants the discovery of new anti-herpetic compounds with better therapeutic profile or different mode of action. The non-nucleoside inhibitors of HSV DNA polymerase target the site that is less important for the binding of a natural nucleoside or nucleoside inhibitors. In the present study, we have explored the possibility to find a new lead molecule based on α-pyrone analogs as non-nucleoside inhibitors using structure based modeling approach. The designed molecules were synthesized and evaluated for anti-HSV activity using MTT assay. The compound 5h with EC(50) 7.4µg/ml and CC(50) 52.5µg/ml was moderately active against HSV when compared to acyclovir. A plaque reduction assay was also carried out and results reveal that 5h is more effective against HSV-1 with better selective index of 12.8 than against HSV-2 (SI=3.6). The synthesized compounds were also evaluated for anti-HIV activity, but none were active.

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.

Recent advancements for the evaluation of anti-viral activities of natural products.

Significant progress has been achieved for the development of novel anti-viral drugs in the recent years. Large numbers of these newly developed drugs belong to three groups of compounds, nucleoside analogues, thymidine kinase-dependent nucleotide analogues and specific viral enzyme inhibitors. It has been found that the natural products, like plant extract, plant-derived compounds (phytochemicals) and so on, as well as traditional medicines, like Ayurvedic, traditional Chinese medicine (TCM), Chakma medicines and so on, are the potential sources for potential and novel anti-viral drugs based on different in vitro and in vivo approaches. In this chapter some of these important approaches utilised in the drug discovery process of potential candidate(s) for anti-viral agents are being discussed. The key conclusion is that natural products are one of the most important sources of novel anti-viral agents.