Preeti Chandra

Discovery of a New Class of Natural Product-Inspired Quinazolinone Hybrid as Potent Antileishmanial agents

The high potential of quinazolinone containing natural products and their derivatives in medicinal chemistry led us to discover four novel series of 53 compounds of quinazolinone based on the concept of molecular hybridization. Most of the synthesized analogues exhibited potent leishmanicidal activity against intracellular amastigotes (IC50 from 0.65 ± 0.2 to 7.76 ± 2.1 μM) as compared to miltefosine (IC50 = 8.4 ± 2.1 μM) and nontoxic toward the J-774A.1 cell line and Vero cells. Moreover, activation of Th1 type and suppression of Th2 type immune responses and induction in nitric oxide generation proved that 8a and 8g induce murine macrophages to prevent survival of parasites. Compounds 8a and 8g exhibited significant in vivo inhibition of parasite 73.15 ± 12.69% and 80.93 ± 10.50% against Leishmania donovani /hamster model. Our results indicate that compounds 8a, 8g, and 9f represent a new structural lead for this serious and neglected disease.

Detection of biosynthetic gene and phytohormone production by endophytic actinobacteria associated with Solanum lycopersicum and their plant-growth-promoting effect

In the present study, fifteen endophytic actinobacterial isolates recovered from Solanum lycopersicum were studied for their antagonistic potential and plant-growth-promoting (PGP) traits. Among them, eight isolates showed significant antagonistic and PGP traits, identified by amplification of the 16S rRNA gene. Isolate number DBT204, identified as Streptomyces sp., showed multiple PGP traits tested in planta and improved a range of growth parameters in seedlings of chili (Capsicum annuum L.) and tomato (S. lycopersicum L.). Further, genes of indole acetic acid (iaaM) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS) were successively amplified from five strains. Six antibiotics (trimethoprim, fluconazole, chloramphenicol, nalidixic acid, rifampicin and streptomycin) and two phytohormones [indole acetic acid (IAA) and kinetin (KI)] were detected and quantified in Streptomyces sp. strain DBT204 using UPLC-ESI-MS/MS. The study indicates the potential of these PGP strains for production of phytohormones and shows the presence of biosynthetic genes responsible for production of secondary metabolites. It is the first report showing production of phytohormones (IAA and KI) by endophytic actinobacteria having PGP and biosynthetic potential. We propose Streptomyces sp. strain DBT204 for inoculums production and development of biofertilizers for enhancing growth of chili and tomato seedlings.

Analysis of phytochemical variations in dioecious Tinospora cordifolia stems using HPLC/QTOF MS/MS and UPLC/QqQLIT-MS/MS

INTRODUCTION The stem of dioecious Tinospora cordifolia (Menispermaceae) is a commonly used traditional Ayurvedic medicine in India having several therapeutic properties. OBJECTIVE To develop and validate LC-MS methods for the identification and simultaneous quantitation of various secondary metabolites and to study metabolomic variations in the stem of male and female plants. METHODS Ethanolic extract of stems were analysed by HPLC/ESI-QTOF-MS/MS for rapid screening of bioactive phytochemicals. High resolution MS and MS/MS in positive ESI mode were used for structural investigation of secondary metabolites. An UPLC/ESI-QqQ(LIT) -MS/MS method in MRM mode was developed and validated for the simultaneous quantitation of five bioactive alkaloids. RESULTS Identification and characterisation of 36 metabolites including alkaloids, sesquiterpenes and phytoecdysteroids were performed using LC-MS and MS/MS techniques. The bioactive alkaloids such as jatrorrhizine, magnoflorine, isocorydine, palmatine and tetrahydropalmatine were successfully quantified in male and female plants. The mean abundances of magnoflorine jatrorrhizine, and oblongine were significantly (P < 0.05) higher in male plants while mean abundances of tetrahydropalmatine, norcoclaurine, and reticuline were significantly (P < 0.05) higher in female plants. CONCLUSIONS Phytochemicals in the stem of male and female Tinospora cordifolia showed significant qualitative and quantitative variations. LC-MS and MS/MS methods can be used to differentiate between male and female plants based on their chemical profiles and quantities of the marker bioactive alkaloids. This chemical composition difference was also evident during vegetative stage when there were no male and female flowers.

Production of Potent Antimicrobial Compounds from Streptomyces cyaneofuscatus Associated with Fresh Water Sediment

The genus Streptomyces under phylum actinobacteria has been recognized as a prolific source for the production of bioactive secondary metabolites. An actinobacterial strain designated as DST103 isolated from a wetland fresh water sediment of Tamdil Lake, Mizoram, Northeast, India was identified as Streptomyces cyaneofuscatus (KY287599) using 16SrRNA gene sequencing which shares 99.87% sequence similarity with Streptomyces cyaneofuscatus NRRL B-2570T. The strain showed broad spectrum antimicrobial activities against Gram negative bacteria (Escherichia coli MTCC 739 and Pseudomonas aeruginosa MTCC 2453), Gram positive bacteria (Micrococcus luteus NCIM 2170 and Staphylococcus aureus MTCC 96) and yeast pathogen Candida albicans MTCC 3017). The methanolic extract of the strain DST103 exhibited highest antimicrobial activity against E. coli (IC50 = 2.10 μg/mL) and minimum activity against S. aureus (IC50 = 43.63 μg/mL). Five antibiotics [trimethoprim (18 μg/g), fluconazole (6 μg/g), ketoconazole (18 μg/g), nalidixic acid (135 μg/g), and rifampicin (56 μg/g)] were detected and quantified using ultra-performance liquid chromatography (UPLC-ESI-MS/MS). Further, biosynthetic potential genes [polyketide synthases type II, non-ribosomal peptide synthetases, and aminodeoxyisochorismate synthase (phzE)] were also detected in strain DST103 which may possibly be responsible for the production of antimicrobial compounds. Additionally, gas chromatography-mass spectrometry analysis showed the presence of four volatile compounds which might be responsible for their diverse biological activity. The present study revealed the presence of bioactive compounds in strain DST103, which may be a promising resource for the discovery of novel bioactive metabolites against wide range of pathogens.

Metabolic profiling of Piper species by direct analysis using real time mass spectrometry combined with principal component analysis

Piper nigrum (black pepper), Piper longum (Indian tipali) and Piper chaba (Bangla tipali) are widely used medicinal herbs in traditional Indian systems of medicine. P. longum as well as P. chaba are considered to be inferior substitutes of P. nigrum. A rapid analytical method for metabolic profiling was developed to generate chemical fingerprints as an alternative means to the only available morphological basis for the identification of these species. Direct analysis using real time mass spectrometry was applied to generate the chemical fingerprints of various parts viz. fruit, leaf and root of these Piper species followed by multivariate analysis for discrimination. Principal component analysis of DARTMS data served as a reliable method for species identification. Phytochemical analysis showed the presence mainly of alkaloids in various parts of Piper species. The results showed that different Piper species could be successfully differentiated using DARTMS fingerprinting together with principal component analysis and this method is promising for the rapid identification and quality control of Piper species.

Simultaneous quantitative determination of multiple bioactive markers in Ocimum sanctum obtained from different locations and its marketed herbal formulations using UPLC-ESI-MS/MS combined with principal component analysis.

INTRODUCTION Ocimum sanctum L., with phenolic acids, flavonoids, propenyl phenols and terpenoids as active pharmacological constituents, is a popular medicinal herb and is present as an ingredient in many herbal formulations. Therefore, development of a reliable analytical method for simultaneous determination of the pharmacologically active constituents of O. sanctum is of high importance. OBJECTIVE To develop and validate a new, rapid, sensitive and selective UPLC-ESI/MS/MS method for simultaneous determination of 23 bioactive markers including phenolic acids, flavonoids, propenyl phenol and terpenoid in the leaf extract and marketed herbal formulations of O. sanctum. METHODS An UPLC-ESI/MS/MS method using negative electrospray ionisation (ESI) in multiple-reaction-monitoring (MRM) mode was used for simultaneous determination. Chromatographic separation was achieved on an Acquity UPLC BEH C18 -column using a gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Principal component analysis (PCA) was applied to correlate and discriminate eight geographical collections of O. sanctum based on quantitative data of the analytes. RESULTS The developed method was validated as per International Conference on Harmonization guidelines and found to be accurate, with overall recovery in the range 95.09-104.84% (RSD ≤ 1.85%), precise (RSD ≤ 1.98%) and linear (r(2)  ≥ 0.9971) over the concentration range of 0.5-1000 ng/mL. Ursolic acid was found to be the most abundant marker in all the samples investigated, except for the marketed tablet. CONCLUSION The method established is simple, rapid and sensitive, hence it can be reliably utilised for the quality control of O. sanctum and derived herbal formulations.

Quality control assessment of polyherbal formulation based on a quantitative determination multimarker approach by ultra high performance liquid chromatography with tandem mass spectrometry using polarity switching combined with multivariate analysis.

An ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry method has been developed and validated for the simultaneous quantification of 28 major bioactive compounds in Mentat tablet, a complex Indian herbal medicine used in the treatment of neurological disorder and improvement of mental health. Multiple-reaction monitoring scanning was employed for quantification in positive and negative ion switching mode. The analysis was accomplished on Waters ACQUITY UPLC BEH C18 column with linear gradient elution of water/formic acid (0.1%) and acetonitrile/formic acid (0.1%) at a flow rate of 0.3 mL/min. The proposed method was validated with acceptable linearity (r2 , 0.9984-0.9999), precision (RSD, 0.22-2.11%), stability (RSD, 0.16-1.78%), and recovery (RSD ≤ 3.74%), under optimum conditions. The limits of quantitation ranged from 0.28 to 3.88 ng/mL. The method was successfully applied for simultaneous determination of 28 compounds in 20 batches of Mentat tablet. Hierarchical cluster analysis and principal component analysis were performed to evaluate the similarity and variation of the 20 samples based on the characteristics of 28 bioactive compounds. Results indicated that this method is rapid, sensitive, and reliable to show the quality of the Mentat tablets composition, hence may be used for quality control of polyherbal formulations having similar markers/raw herbs.

A rapid analytical method for characterization and simultaneous quantitative determination of phytoconstituents in Piper betle landraces using UPLC-ESI-MS/MS

A rapid ultra performance liquid chromatography electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for identification and characterization of phytoconstituents with simultaneous quantitative determination of three major bioactive phenolics namely allylpyrocatechol-3,4-diacetate, eugenyl acetate and eugenol from thirteen landraces of Piper betle leaf extracts. Among the 29 phytoconstituents detected, 19 were identified and characterized based on their fragmentation pattern obtained via MS/MS by means of collision-induced-dissociation (CID) and by comparison of their retention time with authentic standards or by previously reported literature. The proposed method was fully validated in terms of linearity, LOD, LOQ, precision, stability and recovery. All calibration curves showed a good linear relationship (r2 ≥ 0.9981). The precision evaluated by an intra- and inter-day study showed RSD ≤ 1.0% and good accuracy with overall recovery in the range from 96.14–98.46% (RSD ≤ 1.6%) for all analytes. The quantitative results showed that there were remarkable differences in the contents of the major bioactive phenolics in all the thirteen landraces of P. betle. The developed UPLC-ESI-MS/MS method was found to be simple, precise, sensitive and accurate. Hence, it can be reliably utilized as a quality control method for P. betle or derived herbal formulations. In vitro antimicrobial activity of the crude extract of P. betle landraces was also evaluated, and all extracts tested exhibited antifungal activity but none of the extracts were found to be active against bacteria even at 500 μg mL−1 concentration.

A rapid and highly sensitive method for simultaneous determination of bioactive constituents in leaf extracts of six Ocimum species using ultra high performance liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry

Ocimum species have tremendous value in pharmaceutical, perfumery, food processing and cosmetic industries, also in traditional rituals and medicines. These are a rich source of terpenoids and phenolic compounds. Therefore, determination of these bioactive constituents is significant for quality evaluation of Ocimum species. In this study, we have developed and validated a rapid and highly sensitive method for simultaneous determination of sixteen bioactive constituents in the leaf extracts of six Ocimum species using ultra high performance liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry (UHPLC-QqQLIT-MS/MS). The developed method is applied in the leaf extracts of six Ocimum species to investigate variations in the content of sixteen bioactive constituents. Quantitative analysis was performed by UHPLC-QqQLIT-MS/MS operating under multiple reaction monitoring mode in negative electrospray ionization. Chromatographic separation was accomplished on an Acquity UPLC BEH C18 column using a gradient elution of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The calibration curves of all sixteen analytes showed good linearity (r2 ≥ 0.9989) over the concentration range of 0.5–500 ng mL−1. The intra- and inter-day precisions and accuracy were within RSDs ≤ 1.95% and ≤ 1.68%, respectively. The results indicated that ursolic acid and rosmarinic acid were the major constituents in almost all the investigated Ocimum species except for O. americanum. All the results obtained from this study demonstrated that the developed method is rapid, sensitive and efficient for the quantification of multiple constituents. Therefore, it could be reliably utilized for the quality control and authenticity establishment of Ocimum species.

Bioprospection of actinobacteria derived from freshwater sediments for their potential to produce antimicrobial compounds

BackgroundActinobacteria from freshwater habitats have been explored less than from other habitats in the search for compounds of pharmaceutical value. This study highlighted the abundance of actinobacteria from freshwater sediments of two rivers and one lake, and the isolates were studied for their ability to produce antimicrobial bioactive compounds.Results16S rRNA gene sequencing led to the identification of 84 actinobacterial isolates separated into a common genus (Streptomyces) and eight rare genera (Nocardiopsis, Saccharopolyspora, Rhodococcus, Prauserella, Amycolatopsis, Promicromonospora, Kocuria and Micrococcus). All strains that showed significant inhibition potentials were found against Gram-positive, Gram-negative and yeast pathogens. Further, three biosynthetic genes, polyketide synthases type II (PKS II), nonribosomal peptide synthetases (NRPS) and aminodeoxyisochorismate synthase (phzE), were detected in 38, 71 and 29% of the strains, respectively. Six isolates based on their antimicrobial potentials were selected for the detection and quantification of standard antibiotics using ultra performance liquid chromatography (UPLC–ESI–MS/MS) and volatile organic compounds (VOCs) using gas chromatography mass spectrometry (GC/MS). Four antibiotics (fluconazole, trimethoprim, ketoconazole and rifampicin) and 35 VOCs were quantified and determined from the methanolic crude extract of six selected Streptomyces strains.ConclusionInfectious diseases still remain one of the leading causes of death globally and bacterial infections caused millions of deaths annually. Culturable actinobacteria associated with freshwater lake and river sediments has the prospects for the production of bioactive secondary metabolites.