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.].

Effective inhibition of HCoV-OC43 and SARS-CoV-2 by phytochemicals in vitro and in vivo.

OBJECTIVE: Several coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus OC43 (HCoV-OC43), can cause respiratory infections in humans. To address the need for reliable anti-coronavirus therapeutics, we screened 16 active phytochemicals selected from medicinal plants used in traditional applications for respiratory-related illnesses. METHODS: An initial screen was completed using HCoV-OC43 to identify compounds that inhibit virus-induced cytopathic effect (CPE) and cell death inhibition. Then the top hits were validated in vitro against both HCoV-OC43 and SARS-CoV-2 by determining virus titer in cell supernatant and virus-induced cell death. Finally, the most active phytochemical was validated in vivo in the SARS-CoV-2-infected B6.Cg-Tg(K18-ACE2)2Prlmn/J mouse model. RESULTS: The phytochemicals lycorine (LYC), capsaicin, rottlerin (RTL), piperine and chebulinic acid (CHU) inhibited HCoV-OC43-induced cytopathic effect and reduced viral titres by up to 4 log. LYC, RTL and CHU also suppressed virus replication and cell death following SARS-CoV-2 infection. In vivo, RTL significantly reduced SARS-CoV-2-induced mortality by ∼40% in human angiotensin-converting enzyme 2 (ACE2)-expressing K18 mice. CONCLUSION: Collectively, these studies indicate that RTL and other phytochemicals have therapeutic potential to reduce SARS-CoV-2 and HCoV-OC43 infections.

Drug-Screening Strategies for Inhibition of Virus-Induced Neuronal Cell Death.

A number of viruses, including Herpes Simplex Virus (HSV), West Nile Virus (WNV), La Crosse Virus (LACV), Zika virus (ZIKV) and Tick-borne encephalitis virus (TBEV), have the ability to gain access to the central nervous system (CNS) and cause severe neurological disease or death. Although encephalitis cases caused by these viruses are generally rare, there are relatively few treatment options available for patients with viral encephalitis other than palliative care. Many of these viruses directly infect neurons and can cause neuronal death. Thus, there is the need for the identification of useful therapeutic compounds that can inhibit virus replication in neurons or inhibit virus-induced neuronal cell death. In this paper, we describe the methodology to test compounds for their ability to inhibit virus-induced neuronal cell death. These protocols include the isolation and culturing of primary neurons; the culturing of neuroblastoma and neuronal stem cell lines; infection of these cells with viruses; treatment of these cells with selected drugs; measuring virus-induced cell death using MTT or XTT reagents; analysis of virus production from these cells; as well as the basic understanding in mode of action. We further show direct evidence of the effectiveness of these protocols by utilizing them to test the effectiveness of the polyphenol drug, Rottlerin, at inhibiting Zika virus infection and death of neuronal cell lines.

Boswellia serrata oleo-gum-resin and ß-boswellic acid inhibits HSV-1 infection in vitro through modulation of NF-кB and p38 MAP kinase signaling.

BACKGROUND: Herpes Simplex Virus (HSV), a highly contagious pathogen, is responsible for causing lifelong oral to genital infection in human. Boswellia serrata oleo-gum-resin possesses a strong traditional background of treating diverse skin ailments including infection but its effect on HSV-1 has not been examined yet. PURPOSE: To exploit its potential, we aimed to explore the antiviral activity of methanol extract of B. serrata oleo-gum-resin (BSE) and one of its major constituent ß-boswellic acid (BA) against HSV-1 along with the underlying mechanism of action involved. METHODS: BSE was subjected to RP-HPLC analysis to quantify the active constituent. Cytotoxicity (CC50) and antiviral activity were evaluated by MTT and plaque reduction assay, followed by the determination of median effective concentration (EC50). The mode of antiviral activity was assessed by time-of-addition assay and confirmed by reverse transcriptase-PCR (RT-PCR). Further, the expressions of various cytokines were measured by RT-PCR, while the proteins by Western blot. RESULTS: BSE and BA potently inhibited wild-type and a clinical isolate of HSV-1 (EC50 5.2-6.2 and 12.1-14.63 µg/ml), with nearly-complete inhibition (EC99) at 10 and 30 µg/ml, respectively. The inhibitory effect was significant at 1 h post-infection and effective up to 4 h. Based on target analysis we examined the inhibition of NF-κB, essential for virus replication, and observed significant down-regulation of NF-κB, and p38 MAP-kinase activation, with reduced expression of tumor necrosis factor (TNF)-α, Interleukin (IL)-1ß and IL-6, involved in scheming NF-κB signaling. CONCLUSION: Thus, our results support the ethnomedicinal use of BSE in skin infection by inhibiting HSV-1 through the modulation of NF-κB and p38 MAPK pathway.

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.

The Phytochemical Bergenin Enhances T Helper 1 Responses and Anti-Mycobacterial Immunity by Activating the MAP Kinase Pathway in Macrophages.

Tuberculosis (TB) remains one of the greatest health concerns worldwide, which has hindered socioeconomic development in certain parts of the world for many centuries. Although current TB therapy, "Directly Observed Treatment Short-course," is effective, it is associated with unwanted side effects and the risk for the generation of drug-resistant organisms. The majority of infected individuals successfully confine the mycobacterial organisms and remain asymptotic unless immune responses are perturbed. Thus, host immunity can protect against TB and immunomodulation is therefore an attractive therapeutic option. Previous studies have shown that TNF-α and Nitric Oxide (NO) in conjunction with IFN-γ-producing T helper 1 (Th1) cells play critical roles in host protection against TB. Here, we show that bergenin, a phytochemical isolated from tender leaves of Shorea robusta, activates the MAP kinase and ERK pathways and induces TNF-α, NO and IL-12 production in infected macrophages. We further show that bergenin induces Th1 immune responses and potently inhibits bacillary growth in a murine model of Mycobacterium tuberculosis infection. These findings identify bergenin as a potential adjunct to TB therapy.

Inositol hexa phosphoric acid (phytic acid), a nutraceuticals, attenuates iron-induced oxidative stress and alleviates liver injury in iron overloaded mice.

Inositol hexa phosphoric acid (IP6) or Phytic acid, a natural antioxidant of some leguminous plants, known to act as a protective agent for seed storage in plants by suppressing iron catalyzed oxidative process. Following the same mechanism, we have tested the effect of IP6 on iron overloaded in vitro oxidative stress, and studied it's in vivo hepatoprotective ability in iron-dextran (injection)-induced iron overloaded liver injury in mice (intraperitoneal). Our results showed that IP6 had in vitro iron chelation (IC50 38.4µg/ml) activity, with the inhibition of iron-induced lipid peroxidation (IC50 552µg/ml), and deoxyribose sugar degrading hydroxyl radicals (IC50 448.6µg/ml). Oral administration of IP6 (0-200mg/kg) revealed significant decrease in biochemical markers such as serum iron, total iron binding, serum ferritin and serum enzymes. Histopathology of liver stained with hematoxylin-eosin and Prussian blue showed reduced hepatocellular necrosis, ballooning and inflammation, indicating the restoration of normal cellular integrity. Interestingly, the IP6 was found to down-regulate the mRNA expression of tumor necrosis factor (TNF)-α, Interleukin (IL)-1ß, and IL-6 in iron overloaded liver tissues. Thus, we provide an insight that IP6, a natural food component, can serve as an iron chelator against iron overload diseases like Thalassemia, and also as a dietary hepatoprotective supplement.

Pedilanthus tithymaloides Inhibits HSV Infection by Modulating NF-κB Signaling.

Pedilanthus tithymaloides (PT), a widely used ethnomedicinal plant, has been employed to treat a number of skin conditions. To extend its utility and to fully exploit its medicinal potential, we have evaluated the in vitro antiviral activity of a methanolic extract of PT leaves and its isolated compounds against Herpes Simplex Virus type 2 (HSV-2). Bioactivity-guided studies revealed that the extract and one of its constituents, luteolin, had potent antiviral activity against wild-type and clinical isolates of HSV-2 (EC50 48.5-52.6 and 22.4-27.5 µg/ml, respectively), with nearly complete inhibition at 86.5-101.8 and 40.2-49.6 µg/ml, respectively. The inhibitory effect was significant (p<0.001) when the drug was added 2 h prior to infection, and was effective up to 4 h post-infection. As viral replication requires NF-κB activation, we examined whether the observed extract-induced inhibition of HSV-2 was related to NF-κB inhibition. Interestingly, we observed that treatment of HSV-2-infected cells with extract or luteolin suppressed NF-κB activation. Although NF-κB, JNK and MAPK activation was compromised during HSV replication, neither the extract nor luteolin affected HSV-2-induced JNK1/2 and MAPK activation. Moreover, the PT leaf extract and luteolin potently down-regulated the expression of tumor necrosis factor (TNF)-α, Interleukin (IL)-1ß, IL-6, NO and iNOS and the production of gamma interferon (IFN-γ), which are directly involved in controlling the NF-κB signaling pathway. Thus, our results indicate that both PT leaf extract and luteolin modulate the NF-κB signaling pathway, resulting in the inhibition of HSV-2 replication.

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.

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.

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.

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.

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.