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.

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.

In-vitro antileptospiral activity of Canarium odontophyllum Miq. (Dabai) leaves extract

Aims@#To evaluate the anti-leptospiral activity of Canarium odontophyllum leaves against Leptospira interrogans serovar Bataviae and Leptospira borgpetersenii serovar Javanica. @*Methodology and results@#The extracts (hexane, acetone, methanol and aqueous) used in this study were tested at concentration ranging from 0.049 mg/mL to 25 mg/mL using broth microdilution method. Percentage inhibition (%) was obtained through OD reading at 400 nm. Only methanol extract was incubated with Leptospira to observe population changes under dark field microscope prior to subjected for DNA damaging studies through gel electrophoresis of genomic DNA. Methanol extract showed the highest percentage inhibition of 66% against L.interrogans serovar Bataviae and 74% against L. borgpetersenii serovar Javanica. The IC50 value of methanol extract was 4.60 mg/mL and 2.25 mg/mL against serovar Bataviae and serovar Javanica, respectively. Both Leptospira culture which was treated with IC50 value of methanol extract showed drastic decrease in population compared to untreated Leptospira for both serovar. There was no DNA damage towards serovar Bataviae. However, serovar Javanica exhibited DNA damage as observed from the presence of DNA fragmentation on the gel electrophoresis. @*Conclusion, significance and impact of study@#These findings confirmed that methanol leaves extract from of Canarium odontophyllum has a potential to control leptospirosis.

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.

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, ß-amyrin and ß-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.

Pseudo-peptides as novel antileptospiral agents: synthesis and spectral characterization.

In this paper, we describe the synthesis of novel class of pseudo-peptides derived by coupling an amino acid with a heterocyclic moiety containing free amine group using suitable coupling agents. The synthesized compounds were characterized using spectral ((1)H NMR, (13)C NMR and MS) techniques. Preliminary pharmacological assays for Leptospirosis were studied by test tube dilution (TDT) and micro dilution technique (MDT). In particular, all the analyses led to the conclusion that the synthesized compound inhibiting the Leptospira a causal organism of Leptospirosis.