Antileptospiral Activity Research Articles

A New Beginning to the Existing Medicines; Repurposing FDA-Approved Drugs for the Neglected Re-Emerging Disease Leptospirosis.

Leptospirosis is one of the re-emerging zoonotic diseases, especially in tropical regions. Many antibiotics are used to treat leptospirosis, but there are no scientific evidence-based guidelines or systematic clinical trials for using these drugs. A bioinformatics approach was made to shortlist some Food and Drug Administration (FDA) of the United States of America-approved and currently used drugs for leptospirosis. The existing drugs from the Drug Bank database, which are currently not used for leptospirosis, were selected to identify their target proteins and binding sites using bioinformatics methods. Orthologues of these target proteins were selected from the proteome database of Leptospira. The similar sites and their interactions with the drugs were validated and recommended for use in leptospirosis. Further, the sensitivity of recommended drugs was also validated in vitro. The sequences and structures of these proteins were compared under strictly controlled parameters and shortlisted Gatifloxacin, Imipenem, Latamoxef, Doripenem, Tigecycline, and Lactams as repurposable drugs for leptospirosis. An in vitro validation of the drugs showed significant antileptospiral activity in 12 serovars with low IC50 concentrations and also showed that the IC50 values varied across Leptospira serovars. Further, suitable proteins under the concept of "One Target, Many Drugs" identified DNA gyrase subunit A (Q72WD1), 30S ribosomal protein S9 (Q72U99), and 30S ribosomal protein S12 (Q72UA6), and these proteins were found across the pathogenic, saprophytic, and intermediate species of Leptospira. We describe a method to find repurposable drugs from the approved list that are not currently used to treat leptospirosis and validate them to be taken forward for systematic clinical trials specific to leptospirosis for recommendations in clinical use.

Natural products, deforestation, and wildlife tourism could be a potential threat due to leptospirosis outbreak in Gashaka Gumti national park, and the in vitro antileptospiral activity of Annona senegalensis, its synergistic effects with commonly prescri

Introduction: The research on biodiversity as regards natural products, deforestation, and wildlife tourism has witnessed a tremendous growth as several scholars from different jurisdictions have directed their attention toward this subject matter. This growth shows the importance to academia in addressing the critical issues of health of biodiversity conservation. Leptospirosis is a worldwide infectious and zoonotic disease. The incidence of this disease is high in temperate regions, the plant Annona senegalensis, also known as wild custard apple and wild soursop is a member of Annonaceae family. It is a fruit tree native to Senegal and found in semi-arid to sub humid regions of Gashaka Gumti National Park, with a long history of traditional use. Numerous ethnomedicinal uses have been attributed to different parts of A. senegalensis, as well as its use as food and food additives. The aim of this study was to evaluate the natural products, deforestation, and wildlife tourism a potential threat due to Leptospirosis outbreak in Gashaka Gumti National Park and investigate the effects of plant extract found in Gashaka gumti national park plant extract on pathogenic antileptospiral properties of Annona senegalensis against pathogenic Leptospira species (spp.) and to study its synergistic effects with commonly prescribed antibiotics. Materials and Methods: The tested Leptospira serovars were Australis, Bataviae, Canicola and Javanica. Solvent extract of hexane, dichloromethane, chloroform, ethyl acetate and methanol extracts were used. Broth dilution methods were used to determine the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and the synergistic effects between the Solvent extract and the tested antibiotics. The synergistic effects was evaluated by using the fractional inhibitory concentration (FIC) index. Morphological changes of the treated Leptospira were observed under a Scanning Electron Microscope (SEM). Results: The ASH, ASD, ASC, ASEt, ASM and ASEtol were found to have antileptospiral properties against the tested Leptospira spp. The synergy result showed that only combination of ASM ASEt and ASEtol and penicillin G against serovar Australis has demonstrated synergistic effect with the FIC index of 0.38. Morphological study using SEM showed significant structural changes of the treated Leptospira spp. Conclusions: The result suggests that Annona senegalensis extracts could potentially be used as either a complimentary or an alternative therapeutic agent against pathogenic Leptospira spp which will help in the curtailing natural products, deforestation, and wildlife tourism threat due to Leptospirosis.

Zingiber zerumbet (L.) Smith Hexane Crude Extract Caused DNA Damage on Leptospira spp.

Numerous attempts have been made to control leptospirosis by using chemoprophylaxis, but with limited success. The present study was done to investigate the antileptospiral potential of hexane, ethyl acetate and methanol extracts of Zingiber zerumbet rhizomes. The extracts were assayed for antileptospiral activity using broth microdilution method against Leptospira interrogans (serovar Batavie, Canicola, Australis) and Leptospira biflexa serovar Patoc. The Z. zerumbet hexane extract exhibited antileptospiral activity, with IC50 values of 248 μg/mL against L. interrogans serovar Canicola, IC50 of 125 μg/mL against L. interrogans serovar Australis, IC50 of 15.63 μg/mL against L. interrogans serovar Batavie and IC50 of 109 μg/mL against L. biflexa serovar Patoc. However, both ethyl acetate and methanol extracts did not show any distinct antileptospiral activity. Since the hexane extract of Z. zerumbet showed antileptospiral activity, the DNA-damaging properties of this extract were tested according to their IC50 and IC25 values that were specific to each serovars. The DNA-damaging properties were determined by treating the selected Leptospira spp. with the hexane extract and subjecting its DNA to electrophoresis and analysis on agarose gels. The results demonstrated that the hexane extract had DNA-damaging properties towards L. biflexa serovar Patoc and L. interrogans serovar Australis, as proven by the appearance of fragmented DNA on the gels. We conclude that the Z. zerumbet hexane extract could inhibit the growth of Leptospira spp. serovar Patoc and Australis through DNA-damaging activity and thus, could be a potential antileptospiral agent. Further studies are needed to investigate the potential of this hexane extract as an antileptospiral agent using in vivo rat models of leptospirosis.

In Vitro Anti-Leptospiral Activity of Phyllanthus amarus Extracts and Their Combinations with Antibiotics.

Despite modern medicine, there is an increasing trend for cases of the bacterial infection leptospirosis, and this has led to the exploration of alternative medicines from various sources including plants. The aim of this study was to investigate the in vitro anti-leptospiral activity of Phyllanthus amarus extracts alone and combined with penicillin G, ceftriaxone, and doxycycline. Antimicrobial susceptibility testing was performed using the microdilution broth technique upon methanol extract (ME), aqueous extract (AE), and antibiotics against the Leptospira interrogans serovars Australis, Bataviae, Canicola, and Javanica, to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The results were analyzed using an ELISA microplate reader combined with microscopic analysis. Synergy testing using a checkerboard assay was performed to determine the fractional inhibitory concentration index values of extracts combined with antibiotics against leptospires. Scanning electron microscopy (SEM) was used to investigate morphological changes of leptospires caused by potential anti-leptospiral agents alone and combined with antibiotics. The MICs and MBCs for P. amarus extracts ranged from 100 to 400 µg/mL for AEs and from 400 to 800 µg/mL for MEs. Penicillin G was the most effective anti-leptospiral drug, with MICs and MBCs ranging from <0.01 to 0.78 and <0.01 to 3.13 µg/mL, respectively, followed by ceftriaxone, with both MICs and MBCs ranging from 0.05 to 0.78 µg/mL, and doxycycline, with MICs and MBCs ranging from 0.39 to 3.13 µg/mL and 12.5 to 25 µg/mL, respectively. Combinations of P. amarus extracts and antibiotics did not show synergistic effects on all tested Leptospira serovars, with some combinations demonstrating antagonistic effects. SEM analysis, however, showed distorted Leptospira surfaces. P. amarus AE performed better anti-leptospiral activity than P. amarus ME. The morphological effects of P. amarus extract alone and its combination with antibiotic on Leptospira cells revealed promising anti-leptospiral properties.

Callus induction and shoot regeneration from the immature flower bud of Caesalpinia bonducella and its antileptospiral potential by in vitro and in silico analysis

Background: An economical plant regeneration was established for Caesalpinia bonducella by culturing immature flower buds and phytoconstituents such as β-Sitosterol (LC3) and methyl (4E)-5-(2-[(1E)-buta-1,3-dien-1-yl]-4,6-dihydroxyphenyl) pent-4-enoate (SC2) were isolated from its extracts and the isolated phytoconstituents were tested against Leptospira interrogans. Objectives: The aim of the study is to establish the C. bonducella through tissue culture technique and to investigate the antileptospiral activity through computational and in vitro screening. Materials and Methods: Morphogenic calli were initiated from 96% of immature flower buds on MS medium supplemented with 2, 4 D (2, 4-Dichlorophenoxyacetic acid) 2.0 mg/l and 6-Benzylaminopurine (BAP) 0.5 mg/l. The calli formed were excised and subcultured on MS medium. Extreme percentage organogenesis (84%) and average shoots per culture were determined on MS medium fortified with 3.0 mg/l BAP and 0.5 mg/l indole-3-butyric acid (IBA). The addition of IBA in ½ MS medium favored the development of recovered shoots. Out of 30 shoots transferred to soil, 27 survived once acclimated. The isolated compounds were selected for in vitro and in silico screening. The primary pharmacological assay for leptospirosis was carried out by test tube dilution and microdilution technique and the computational screening was done using molecular docking. Results: The phytoconstituents obtained from the medicinal plant showed promising results in both in vitro and in silico antileptospiral activity. Conclusion: The LC3 and SC2 compounds isolated from C. bonducella were evaluated on Gram-negative bacteria L. interrogans. The assay and molecular docking studies revealed the efficacy of phytocomponents of C. bonducella as traditional medicine which has an ability to cure bacterial diseases.

Optimized Tube Dilution Technique and Sole Carbon Utilization Assay for Anti-leptospiral In Vitro Screening of Plant Extracts

Introduction: Leptospirosis is one of the neglected reemerging zoonoses that is of public health concern globally. The need to discover novel therapeutic alternatives for leptospirosis through screening for and elucidating the mechanism/s of the anti-leptospiral activity of plant extracts is therefore necessary. This study analyzes the optimized tube dilution technique and the BiologTM sole carbon utilization phenotype microarray as screening tool for anti-leptospiral activity of plant extracts. Methods: The suitability of the optimized tube dilution technique was evaluated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and motility inhibition property of a plant extract and an antimicrobial control (pen G) against 4 dominantly circulating Leptospira serovars/serogroup in the Philippines. Likewise, the suitability of the BiologTM sole carbon utilization assay was evaluated using a plant extract and selected antimicrobials against L. interrogans serovar Manilae strain K64 and L. interrogans serovar Losbanos strain K37. Results: The MIC, MBC, and motility inhibition property of a plant extract and the antibiotic controls as well as its effect on the carbon utilization phenome of the Leptospira serovars gave consistent results, within and between several runs. With standard deviation = 0 for all serovars. The MIC and MBC of the antimicrobial control (pen G), the positive control, was 10 ug/ml. The growth control (leptospires without treatment), the negative control, showed presence of motile leptospires. The MIC and the MBC of the test plant extract was 250 ug/ml – 500 ug/ml. Results of the carbon utilization phenome or pattern of carbon utilization were consistent within the 3 replicates and between two runs. Conclusion: The optimized tube dilution technique and the BiologTM sole carbon utilization assay is a potential in vitro screening tool for determining anti-leptospiral activity of plant extracts.

Potential antileptospiral constituents from Phyllanthus amarus

Background: Phyllanthus amarus (PA) is a well-known herb for its medicinal properties and widely used worldwide. PA has a significant role in Indian Ayurveda system of medicine for treating various ailments such as gonorrhea, menorrhagia, and other genital infections. Objectives: The aim of the study is to investigate antileptospiral activity and isolate the potential antileptospiral constituents of the methanol extract of PA (MPA). Materials and Methods: The primary pharmacological tests for leptospirosis were investigated by test tube dilution technique and microdilution technique. To examine the morphogenesis of experimental leptospirosis by morphologic and histological methods, albino mice were inoculated intraperitoneally with Leptospira interrogans sero group Icterohaemorrhagiae strains. Results: The activity-guided repeated fractionation for MPA through silica gel column chromatography yielded three compounds that exhibited antioxidant and in vitro, in vivo, and in silico antileptospiral activities. Based on diverse physicochemical and spectroscopic analyses (viz.,13C NMR,1H NMR, ultraviolet [UV], IR, and mass spectroscopy), the potential constituents were elucidated as 5-(3-(3,4-dimethoxybenzyl)-4-methoxy-2-(methoxymethyl)butyl)-4,7-dimethoxybenzo[d][1,3]dioxole(C1),1-(3-(3,4-dimethoxybenzyl)-4-methoxy-2-(methoxymethyl)butyl)-2,3,4,5tetramethoxybenzene(C2), and 4-(3-(3,4dimethoxybenzyl)-4-methoxy-2-(methoxymethyl)butyl)-3,6-dimethoxybenzene-1,2-diol (C3). The histopathological examinations of both kidney and liver showed promising activity with C3 at 75 and 100 μg/mL, respectively. Conclusion: The in vitro , in vivo, and in silico studies revealed that benzo methoxy class of compounds has great potential as antileptospiral agents.

2D QSAR Analysis of Substituted Quinoxalines for their Antitubercular and Antileptospiral Activities.

The Quantitative structure activity relationship for thirty two novel substituted quinoxalines was performed for their antitubercular (Mycobacterium tuberculosis H37Rv) and antileptospiral (Leptospirainterrogans) activities. The quinoxalines were substituted with azetidinones, thiazolidinones and fluoroquinolones. Several compounds exhibited good activity against both the infections and they all possess fluoroquinolone moiety with the quinoxaline. The models developed showed good linear relationship (r2 = 0.71-0.88), with an internal predictive ability (q2> 0.61) and good external predictive ability (pred_r2>0.71). The compounds were separated into a training set on which regression was performed and a test set on which the predictive ability of the model was tested. Other statistical parameters including Ro2, Ro'2, k, k' and Z- score were in the acceptable range. The descriptors obtained explained the necessity of spatial orientation of atoms including branching and adjacency, presence of electronegative groups, balance between lipophilic elements and their binding strengths.

Anti-leptospiral potential of Zingiber zerumbet (L.) Smith crude extracts against Leptospira spp.

Event Abstract Back to Event Anti-leptospiral potential of Zingiber zerumbet (L.) Smith crude extracts against Leptospira spp. Asmah Hamid1*, Nurul A. Abdul Aziz1, Latifah Ibrahim2, Farah W. Ibrahim1 and Nurul F. Jufri1 1 School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, National University of Malaysia, Malaysia 2 Institute for Medical Research, Ministry of Health (Malasiya), Malaysia Background Leptospirosis is one of the emerging infectious diseases that the number of cases had been increasing progressively in Malaysia. Numerous attempts have been made to control the disease by using chemoprophylaxis but only showed limited success. The present study investigated the anti-leptospiral potential of hexane, ethyl acetate and methanol extracts of Zingiber zerumbet. Methods The extracts were assayed for anti-leptospiral activity using broth microdilution method towards Leptospira interrogans (serovar Batavie, Canicola, Australis) and Leptospira biflexa serovar Patoc. The DNA damaging properties of Z. zerumbet on DNA of Leptospira sp. was assessed by incubating the DNA of Leptospira sp. in the presence or absence of Z. zerumbet crude extracts and subsequently analyzed by electrophoresis. Results The anti-leptospiral activity of Z. zerumbet hexane extracts showed an IC₅₀ value of 248 μg/ml towards L. interrogans serovar Canicola, IC₅₀ value 125 μg/ml towards L. interrogans serovar Australis, IC₂₅ value 15.63 μg/ml towards L. interrogans serovar Batavie and IC₅₀ value 109 μg/ml towards L. biflexa serovar Patoc. However, ethyl acetate and methanol extracts exhibited a weak/mild anti-leptospiral activity. Since the hexane extract of Z. zerumbet displayed strong anti-leptospiral activity, the DNA damaging properties of this extract was tested at their IC₅₀ and IC25 values that were specific to each serovars. The result showed that hexane extract do not possess DNA damaging properties towards L. interrogans serovar Canicola and Bataviae as there was no smearing or fragmentation of DNA band. For L. biflexa serovar Patoc and L. interrogans serovar Australis there were DNA damage which was evidenced by the appearance of fragmented DNA on agarose gel. Conclusion The present study showed that Z. zerumbet hexane extract has the potential to inhibit the growth of Leptospira spp. by damaging the bacterial DNA. Keywords: Anti-leptospiral, Zingiber zerumbet, Leptospira, DNA Damage, extract Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Poster Presentation Topic: Infectious diseases Citation: Hamid A, Abdul Aziz NA, Ibrahim L, Ibrahim FW and Jufri NF (2019). Anti-leptospiral potential of Zingiber zerumbet (L.) Smith crude extracts against Leptospira spp.. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00117 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Asmah Hamid, School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, National University of Malaysia, Kuala Lumpur, Malaysia, asmah0901@ukm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Asmah Hamid Nurul A Abdul Aziz Latifah Ibrahim Farah W Ibrahim Nurul F Jufri Google Asmah Hamid Nurul A Abdul Aziz Latifah Ibrahim Farah W Ibrahim Nurul F Jufri Google Scholar Asmah Hamid Nurul A Abdul Aziz Latifah Ibrahim Farah W Ibrahim Nurul F Jufri PubMed Asmah Hamid Nurul A Abdul Aziz Latifah Ibrahim Farah W Ibrahim Nurul F Jufri Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Potential anti-leptospiral compound, leptomycin B from marine Streptomyces indiaensis MSU5: taxonomy, fermentation, compound isolation, in vitro and in vivo efficacy

Leptospirosis is a worldwide reemerging tropical zoonotic disease with symptoms of mild febrile illness to more severe multiple organ failure caused by pathogenic leptospiral strains. There was no effective antibiotic for treating leptospirosis. Here, the anti-leptospiral potential of marine actinobacterial compound from Streptomyces indiaensis MSU5 isolated from Manakudy marine sediment, Tamil Nadu, India was evaluated. The potential actinobacterial strain was identified by phenotypic, cell wall, 16S rRNA gene sequence and phylogenetic analysis. In vitro anti-leptospiral activity of the actinobacterial compound was determined using broth microdilution test against various serovars of Leptospira with different concentration ranging from 15.625 to 500µg/ml. Mass production of anti-leptospiral compound was carried out in agar surface fermentation with optimized condition and purified by preparative TLC. The purified fraction of anti-leptospiral compound named as MSU5-1, and it was confirmed by microdilution test. Remarkably, the compound MSU5-1 showed minimum inhibitory concentration of 62.5µg/ml and minimum bactericidal concentration of 125µg/ml against human pathogenic leptospiral isolate strain N2. The structural elucidation of purified compound was carried out using UV, FT-IR, NMR and LC-MS analysis. The compound MSU5-1 was tentatively identified as leptomycin B (C33H48O6) with molecular weight 541.1g/mol. Anti-leptospiral activity of compound MSU5-1 exhibited 80% of survival rate in mice model, further it was confirmed by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) analysis. From the available literature, this is the first report on the marine actinobacterial compound for evaluating both in vitro and in vivo leptospiricidal activity.

Microwave-assisted synthesis, structural activity relationship and biological activity of some new quinoxaline Schiff base derivatives as highly potent spirochete bactericidal agents

The main aim of this work was to synthesise a new (E)-3-(4-or3-Aminophenylimino) quinoxaline-2(3H)-one oxime Schiff base derivatives and evaluate the anti-leptospiral activity against Leptospira icterohaemorrhagiae. The mentioned derivatives were prepared by performing microwave-assisted condensation reactions of (E)-3-(4-or3-Aminophenyl imino)quinoxaline-2(3H)-one and aromatic aldehydes. The structures of these interesting compounds were characterised by FT-IR, 1H NMR and mass spectroscopy. Furthermore, these compounds were screened for spirocidal activity against Leptospira icterohaemorrhagiae by using in vitro and in vivo method. The anti-leptospiral activity result reveals that most of the compounds were exhibiting considerable activity against Leptospira icterohaemorrhagiae. Compound 6c demonstrated remarkable activity at low concentration against the Leptospira icterohaemorrhagiae as compared to the standard drugs.

Discovery of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones as a new class of pathogen specific anti-leptospiral agents

A simple and efficient method for the synthesis of a series of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one derivatives starting from 5-carboalkoxy-2,3-dihydropyranone (5-CDHPs) has been developed. Pyranoisoxazolones 10a-j, dihydronaphthopyran-4-one (DHNPs) class of natural product 12b and 12c and its analogues 12a and 13a-c were preliminarily screened against pathogenic leptospiral serovar Autumnalis strain N2 at various concentrations. Six pyranoisoxazolones, 10b, 10d, 10f, 10g, 10i and 10j which displayed very good anti-leptospiral activity was taken for secondary screening against twelve strains of pathogenic and one non-pathogenic leptospiral serovars. While all the compounds displayed significant anti-leptospiral activity against the pathogenic serovars at MIC of 62.5–500 μg/mL. Compounds 10d, 10g and 10j did not show any significant effect on non-pathogenic serovar. Inhibition of leptospires at a significant level by pyranoisoxazolone 10g was confirmed using RT-qPCR assay. In vivo treatment of BALB/c mice with compound 10g revealed that, it has 95% survivability against the pathogenic strain Canicola and also showed inhibition of renal colonization of leptospires. Compound 10g was found to show cytotoxicity against THP-1 cells only at higher concentration (≥75 μg/mL). Effective binding of compound 10g with leptospiral outer membrane protein LipL32 observed via in silico molecular docking provided a suitable explanation for pathogen specificity of compound 10g. Antibiotics acting against leptospirosis in human are very few. The results obtained from in vitro, in vivo and in silico study reveals that 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones class of compounds are lead molecules for further development as pathogen specific anti-leptospiral agents.

In-Vitro Screening of Phyllanthus Amarus and Eclipta Alba Against Leptospira Autumnalis

Background and Study Aims: Multi-drug resistance is one of the major public health problems especially in developing countries where relatively easy availability and higher consumption of medicines have lead to disproportionately higher incidence of inappropriate use of antibiotics and greater levels of resistance compared to developed countries. Leptospirosis is the most wide spread zoonoses in the world and recognized as an important public health problem. Aim and Methods: To extract and identify some phytochemicals from the commonly available and widely studied Indian medicinal plants (Phyllanthus amarus and Eclipta alba) with anti-bacterial, hepatoprotective and renoprotective properties and also to conduct an anti-leptospiral susceptibility study on MIC of a most common Leptospira interrogans serovar autumnalis following the standard TDT. Results: The present study revealed the presence of tannins, alkaloids, saponins, flavonoids, terpenes and anthraquinones in the leaves of these plants extracts thereby exhibiting anti-leptospiral activity. The drug dose concentration the extracts of P. amarus was found to be having a better active principle (160 μg/ml) than the extracts of E. alba (320 μg/ml).

GC/MS profiling, in vitro anti-leptospiral and haemolytic activities of Boesenbergia rotunda (L.) Mansf. used as a medicinal plant by Nicobarese of Andaman and Nicobar Islands

Leaves of the plant Boesenbergia rotunda are used by the Nicobarese tribe of Andaman and Nicobar Islands, India, to prepare traditional medicine for treating fever, headache and body ache. In the present investigation, methanol fraction of these leaves were analysed by GC/MS that revealed the presence of 25 compounds. The anti-leptospiral activity of methanol crude extract was determined by both microdilution and macrodilution methods. The MICs of the extract were tested against 24 pathogenic leptospiral strains and ranged between 62.5–125 μg/mL in both microdilution and macrodilution. The range of MBCs was 250 and 500 μg/mL in macrodilution and microdilution respectively. The crude extract was subjected to cytotoxic studies and found to have negligible or no haemolytic activity, exhibiting IC50 values of greater than 4 mg/mL. Further in vivo studies are needed to investigate the pharmacological and toxicological properties of Boesenbergia rotunda, before it can be considered as a new anti-leptospiral agent.

Anti-leptospiral activities of an endemic plant Glyptopetalum calocarpum (Kurz.) Prain used as a medicinal plant by Nicobarese of Andaman and Nicobar Islands

Leaves of an endemic plant Glyptopetalum calocarpum are used by Nicobarese tribes of Andaman and Nicobar Islands, India, to prepare traditional medicine for treating fever. In the present investigation, pharmacologically active compounds were isolated from this plant and their antimicrobial efficacy was evaluated against the leptospiral strains. The anti-leptospiral activity of six plant-derived compounds was determined by both microdilution and macrodilution methods. Two out of six compounds, namely lupenone and stigmasterol, showed anti-leptospiral activity. The minimum inhibitory concentrations of the two compounds tested against pathogenic leptospiral strains belonging to 10 serovars were in the range of 100–200 μg/mL. The range of minimum bactericidal concentrations was 400–800 μg/mL. Compounds lupenone, stigmasterol, lupeol, β-amyrin and β-amyrin acetate had negligible or no haemolytic activity, exhibiting IC50 values of greater than 5 mg/mL. Further in vivo studies are needed to investigate the pharmacological and toxicological properties of G. calocarpum before it can be considered as a new anti-leptospiral agent.

Antileptospiral activity of xanthones from Garcinia mangostanaand synergy of gamma-mangostin with penicillin G

BackgroundLeptospirosis, one of the most widespread zoonotic infectious diseases worldwide, is caused by spirochetes bacteria of the genus Leptospira. The present study examined inhibitory activity of purified xanthones and crude extracts from Garcinia mangostana against both non-pathogenic and pathogenic leptospira. Synergy between γ-mangostin and penicillin G against leptospires was also determined.MethodsMinimal inhibitory concentrations (MIC) of crude extracts and purified xanthones from G. mangostana and penicillin G for a non-pathogenic (L. biflexa serovar Patoc) and pathogenic (L. interrogans serovar Bataviae, Autumnalis, Javanica and Saigon) leptospires were determined by using broth microdilution method and alamar blue. The synergy was evaluated by calculating the fractional inhibitory concentration (FIC) index.ResultsThe results of broth microdilution test demonstrated that the crude extract and purified xanthones from mangosteen possessed antileptospiral activities. The crude extracts were active against all five serovars of test leptospira with MICs ranging from 200 to ≥ 800 μg/ml. Among the crude extracts and purified xanthones, garcinone C was the most active compound against both of pathogenic (MIC =100 μg/ml) and non-pathogenic leptospira (MIC = 200 μg/ml). However, these MIC values were higher than those of traditional antibiotics. Combinations of γ-mangostin with penicillin G generated synergistic effect against L. interrogans serovars Bataviae, Autumnalis and Javanica (FIC = 0.52, 0.50, and 0.04, respectively) and no interaction against L. biflexa serovar Patoc (FIC =0.75). However, antagonistic activity (FIC = 4.03) was observed in L. interrogans serovar Saigon.ConclusionsCrude extracts and purified xanthones from fruit pericarp of G. mangostana with significant antibacterial activity may be used to control leptospirosis. The combination of xanthone with antibiotic enhances the antileptospiral efficacy.

Cytomorphological Changes and Inhibition of Inclusion Body Formation in Leptospira interrogans on Treatment with the Extracts of Adhatoda vasica

Leptospirosis is most common bacterial zoonosis worldwide, caused by spirochetes of the genus Leptospira that are transmitted from animals to humans. To treat leptospirosis, traditional healers use phytoremedy. In the present study, a scientific study was made on the plants used by the traditional healers to cure liver problems with symptoms of leptospirosis. Methanolic and ethanolic extracts of four plants were screened for antiLeptospiral activity. Of the four plants, Adhatoda vasica extracts exhibited a good anti-Leptospiral activity. On treatment of Leptospira interrogans culture with leaf extracts of the plant Adhatoda vasica, the cellular architechure of lengthy spirochaete L. interrogans was severely damaged. An electron microscopic observation showed breaks at different locations in the lengthy cell, and inhibition of the development of inclusion body responsible for virulence. The minimum inhibitory concentration of extracts was observed to be 5 mg/ml. The antiLeptospiral effect of A.vasica was compared with that of penicillin, and it was found that the medicine used by traditional healers to cure liver disorders has very effective inhibition activity over the microbe responsible for Leptospirosis. Hence, an alternate phytotheraphy can be recommended and the bioactive compound present in leaves of A.vasica has to be identified.

Antileptospiral, Antioxidant and DNA damaging properties of Eclipta alba and Phyllanthus amarus

Leptospirosis is globally important disease affecting almost all mammals including human being. Several cases of this disease occur in India every year and studies have made to trace the epidemiology, pathology of this disease. It occurs in urban environments of industrialized and developing countries, as well as in rural regions worldwide. The disease is found mainly wherever human come in contact with the urine of infected animals and urine contaminated environment. The unsuccessful attempts to control this disease by chemo therapeutics prompted us to go for alternative control strategies. The present study comprises the use of traditional medicines to overcome the side effects caused by chemotherapeutics. In the present study an attempt has been made to use the herbal medicine to cure leptospirosis either by direct killing or by inhibiting the growth of leptospira. Inhibition of growth has been realized as unique approach to stop the growth. A methonolic and aqueous extract of whole plants of Eclipta alba and Phyllanthus amarus assessed to determine the mechanism(s) of its antileptospiral and antioxidant activity. In this study the antioxidant activity and radical scavenging activity of methanolic and aqueous extracts of selected plant materials were evaluated against 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical and 2,2’azinobis(3-ethylbenzthiazoline- 6-sulfonic acid) (ABTS) activity. DNA damaging studies on leptospira were also investigated. Finally, extracts were tested in different concentration in their respective solvent systems by Tube Dilution Technique (TDT) and Micro Dilution Technique (MDT) tests. The antileptospiral activity of Eclipta alba and P. amarus were studied by both tube dilution and micro dilution techniques and the results showed better inhibitory action against various serogroups of Leptospira. DPPH free radical scavenging effect of extract was compared with standard antioxidant ascorbic acid. IC 50 value was found 75µg/ml for extract and 45µg /ml for ascorbic acid and the methanol extracts of both plants had good activity and IC 50 value was found 55µg/ml and 30µg/ml in ABTS assay. The plant extracts were also found to be very effective against leptospirosis. The possible use of E.alba and P.amarus extracts to control Leptospirosis are discussed in the present studies.

In vitro Evaluation of Eclipta alba against Serogroups of Leptospira interrogans.

Leptospirosis is now acknowledged as the most widespread zoonoses in the world. Hundreds of cases occur in India every year accounting for considerable morbidity and sizable mortality. Several studies have delineated the epidemiology, pathology and variable clinical features of this condition. The present study comprises the importance and utilization of traditional based medicines to overcome the adverse reaction by conventional drugs and standardize the technology. The antileptospiral activity of Eclipta alba L. was well studied by both tube dilution and micro dilution techniques and the result showed better inhibitory action against various serogroups of Leptospira interrogans. L. australis, L. autumnalis and L. grippotyphosa are inhibited by both water and ethanol extract by tube dilution technique. The MIC level observed are 50 μg and 100 μg respectively. Similarly acetone extract, Icterohaemorrhagiae was responded to 200 μg/ml as MIC whereas in petroleum ether extract, no inhibition was observed. In the case of micro dilution technique, the entire inhibition rates are supported to the tube dilution technique. It showed that the micro dilution technique is the best method where we obtained the results within 30 minutes; at the same time tube dilution technique takes minimum of 7 days to provide the result.

SUSCEPTIBILITY OF LEPTOSPIRA SEROVARS TO ANTIMALARIAL AGENTS

Leptospirosis has recently been described to cause concomitant infection with malaria. Only doxycycline has proven to have chemoprophylactic and therapeutic efficacy for both malaria and leptospirosis. To assess whether other traditional antimalarial agents have antileptospiral activity, we performed broth microdilution susceptibility testing of 16 Leptospira serovars (6 species/14 serogroups) to various agents. Artemisinin, atovaquone, chloroquine, mefloquine, primaquine, proguanil, pyrimethamine, sulfadoxine, quinine, quinidine, and combinations of atovaquone/proguanil and pyrimethamine/sulfadoxine all had a 90% minimum inhibitory concentration (MIC(90)) > 25 microg/mL (the upper limit of testing). The only agents identified with the potential to treat both infections other than doxycycline (MIC(90) = 1.56 microg/mL) were azithromycin (MIC(90) = 0.002 microg/mL) and clindamycin (MIC(90) = 0.2 microg/mL).