Assessment of Genetic Diversity, Micromorphology and Antimicrobial Activity in Nepeta cataria L.

The mosquito repellent Nepetalactone rich Nepeta cataria L. (catmint) plant has a variety of therapeutic and industrial potential. Reports on the genetic diversity of N. cataria germplasm are minimal globally and need attention for adding a new variety into commercial cultivation. The present study, therefore, assessed the genetic diversity among thirteen half-sib genotypes of N. cataria using agro economic and phytochemical traits. The experimental set has shown substantial variation for agro economic traits studied. Among all the studied populations, fresh herb-based essential oil content ranged from 0.1 % to 0.3 %, with a grand mean of 1.67 %. However, the estimated oil yield ranged from 44.4 kg/h to 120.73 kg/h with an average of 71.34 kg/h. Among the eleven phytochemical constituents detected in different concentrations in the essential oil of experimental sets, 4aα,7α,7aα-Nepetalactone (67.9-87.5 %) constituted the significant proportion of essential oil. Altogether, based on mean comparison, the population NC8 was found to be promising for estimated oil yield and 4aα,7α,7aα-Nepetalactone content. The greater heritability estimates (h2 bs) and genetic advance as percent of mean (GAM) were observed for important economic parameters, i. e., oil content, herb yield, and oil yield. The cluster analysis revealed the least interactions between various agro economic and phytochemical variables. The microscopic study of trichome showed a positive correlation of abaxial leaf surface with essential oil content. The promising antimicrobial potential of catmint oil was also observed against human health-related pathogens. The results infer from our study provide valuable insight for genetic improvement and product development in the catmint germplasm.

Membrane-damaging potential of natural L-(-)-usnic acid in Staphylococcus aureus.

The purpose of this investigation was to try to understand the antibacterial mechanism of L-(-)-usnic acid isolated for the first time from fruticose lichen Usnea subfloridana using clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration (MIC) of L-(-)-usnic acid against the clinical isolates of MRSA and reference strain S. aureus MTCC-96 (SA-96) was in the range 25-50 µg/ml. Treatment of both reference and clinical strains (MRSA-ST 2071) with four-fold MIC concentrations (100-200 µg/ml) of L-(-)-usnic acid reduced the viability of cells without damaging the cell wall. However, the loss of 260 nm absorbing material and increase in propidium iodide uptake was observed in both of the strains. Similarly, a combined effect of L-(-)-usnic acid (25-50 µg/ml) and 7.5 % NaCl resulted in a reduced number of viable cells within 24 h in comparison to the control. These observations clearly indicate that L-(-)-usnic acid exerts its action by disruption of the bacterial membrane. Further, in vivo efficacy showed that L-(-)-usnic acid significantly (p < 0.001) lowered the microbial load of spleen at doses ranging from 1 to 5 mg/kg. Further, toxicity studies in infected mice at doses 20 times higher than the efficacious dose indicated L-(-)usnic acid to be safe. Paradoxically, L-(-)usnic acid exhibited changes in serum triglycerides, alkaline phosphatase (ALKP) and liver organ weight in the healthy mice administered with only 25 mg/kg body weight. The results obtained in this study showed that natural L-(-)-usnic acid exerts its antibacterial activity against MRSA by disruption of the cell membrane. Further, the natural L-(-)-usnic acid was found to be safe up to 100 mg/kg body weight, thereby, making it a probable candidate for treating S. aureus infections.

Comparative genetic analysis of trichome-less and normal pod genotypes of Mucuna pruriens (Fabaceae).

Velvet bean (Mucuna pruriens) seeds contain the catecholic amino acid L-DoPA (L-3,4-dihydroxyphenylalanine), which is a neurotransmitter precursor and used for the treatment of Parkinson's disease and mental disorders. The great demand for L-DoPA is largely met by the pharmaceutical industry through extraction of the compound from wild populations of this plant; commercial exploitation of this compound is hampered because of its limited availability. The trichomes present on the pods can cause severe itching, blisters and dermatitis, discouraging cultivation. We screened genetic stocks of velvet bean for the trichome-less trait, along with high seed yield and L-DoPA content. The highest yielding trichome-less elite strain was selected and indentified on the basis of a PCR-based DNA fingerprinting method (RAPD), using deca-nucleotide primers. A genetic similarity index matrix was obtained through multivariant analysis using Nei and Li's coefficient. The similarity coefficients were used to generate a tree for cluster analysis using the UPGMA method. Analysis of amplification spectra of 408 bands obtained with 56 primers allowed us to distinguish a trichome-less elite strain of M. pruriens.

Endophytic bacteria from Ocimum sanctum and their yield enhancing capabilities.

Endophytes are beneficial microbes that reside intercellularly inside the plants. Interaction of endophytes with the host plants and their function within their host are important to address ecological relevance of endophyte. Four endophytic bacteria OS-9, OS-10, OS-11, and OS-12 were isolated from healthy leaves of Ocimum sanctum. These isolated microbes were screened in dual culture against various phytopathogenic fungi viz. Rhizoctonia solani, Sclerotium rolfsii, Fusarium solani, Alternaria solani, and Colletotrichum lindemuthianum. Of these, strain OS-9 was found to be antagonistic to R. solani, A. solani, F. solani, and C. lindemuthianum while OS-11 was found antagonistic against A. solani only. The growth-promoting benefits of the endophytes were initially evaluated in the glasshouse by inoculated seeds of O. sanctum. Treatment with endophytes OS-10 and OS-11 resulted in significant enhancement of growth as revealed by increase in fresh as well as dry weight. Further, field trials involving two genotypes OS Purple and CIM-Angana were conducted with strains OS-10 and OS-11. The growth-promoting effect was visible on both the genotypes tested as the endophytes significantly enhanced fresh herbage yield (t/ha). Interestingly, these endophytes increased the content of essential oil particularly in cultivar OS Purple and thereby increasing the total oil yields. Molecular characterization of strain OS-11 indicated the strain to be highly related to the type strain of Bacillus subtilis.

AFLP markers for identification of Swertia species (Gentianaceae).

The genus Swertia is well known for its medicinal properties, as described in the Indian pharmacopoeia. Different members of this genus, although somewhat similar in morphology, differ widely in their pharmacological and therapeutic properties. The most important species of this genus, with maximal therapeutic properties, is S. chirayita, which is often adulterated with other less-potent Swertia spp. There is an existing demand in the herbal drug industry for an authentication system for Swertia spp, in order to enable their commercial use as genuine phytoceuticals. To this end, we used amplified fragment length polymorphism (AFLP) to produce DNA fingerprints for six Swertia species. Nineteen accessions (2 of S. chirayita, 3 of S. angustifolia, 2 of S. bimaculata, 5 of S. ciliata, 5 of S. cordata, and 2 of S. alata) were used in the study, which employed 64 AFLP selective primer pairs. Only 46 selective primer pairs were found to be useful for all the accessions. A total of 5312 fragments were produced by these 46 primer pairs. Species-specific markers were identified for all six Swertia species (131 for S. chirayita, 19 for S. angustifolia, 181 for S. bimaculata, 47 for S. ciliata, 94 for S. cordata, and 272 for S. alata). These AFLP fingerprints of the Swertia species could be used to authenticate drugs made with Swertia spp and to resolve adulteration-related problems faced by the commercial users of these herbs.

Molecular investigation against the resistant mechanism of PncA mutated pyrazinamide resistance and insight into the role of pH environment for pyrazinamide activation.

Pyrazinamide (PZA), a crucial component of anti-TB therapy, is a prodrug. PZA interacts with PncA protein to be converted into its functional form i.e. pyrazinoic acid (POA). It has unique feature to kill dormant tubercle bacilli of acidic environment. Although significance of pH environment in PZA activation has been investigated in several of previous studies, insight into the significant atomistic variations in the interaction pattern of PZA with PncA, at different pH environments, are still required to be explored. On the other hand, continuously emerging PncA mutants, associated with PZA resistance, have also become a serious threat for global TB control program. Therefore, the current study was designed to understand the role of pH environment in the PZA activation and to explore the PZA resistance mechanism in various PncA mutants. The study included various in silico experiments like molecular docking, MD simulation, binding free energy estimation, PCA and FEL. In our study, we have found pH-3 and pH-5 environment as a highly significant environment for PZA activation. It was found that protonation or deprotonation of PZA activation site (PAS) residues, majorly K48, D56, K96 and E107, resulted in rearrangement of the PAS according to the pH conditions. It has also been observed that positioning of PZA binding near to Fe2+ and residues of catalytic triad (i.e. D8, K96 and C138) also play a very crucial role in the activation of PZA. The overall insight from the current study may help to develop new therapeutics against PncA mutated PZA resistance.Communicated by Ramaswamy H. Sarma.

Antifungal activity of Glycyrrhiza glabra extracts and its active constituent glabridin.

Glabridin, an active constituent of Glycyrrhiza glabra roots, was found to be active against both yeast and filamentous fungi. Glabridin also showed resistance modifying activity against drug resistant mutants of Candida albicans at a minimum inhibitory concentration of 31.25-250 microg/mL. Although the compound was reported earlier to be active against Candida albicans, but this is the first report of its activity against drug resistant mutants.

Gallic acid-based indanone derivatives as anticancer agents.

Gallic acid-based indanone derivatives have been synthesised. Some of the indanones showed very good anticancer activity in MTT assay. Compounds 10, 11, 12 and 14 possessed potent anticancer activity against various human cancer cell lines. The most potent indanone (10, IC(50)=2.2 microM), against MCF-7, that is, hormone-dependent breast cancer cell line, showed no toxicity to human erythrocytes even at higher concentrations (100 microg/ml, 258 microM). While, indanones 11, 12 and 14 showed toxicities to erythrocytes at higher concentrations.

Antimicrobial potential of Glycyrrhiza glabra roots.

The present study was aimed to investigate antimicrobial potential of Glycyrrhiza glabra roots. Antimycobacterial activity of Glycyrrhiza glabra was found at 500 microg/mL concentration. Bioactivity guided phytochemical analysis identified glabridin as potentially active against both Mycobacterium tuberculosis H(37)Ra and H(37)Rv strains at 29.16 microg/mL concentration. It exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Our results indicate potential use of licorice as antitubercular agent through systemic experiments and sophisticated anti-TB assay.

Synthesis of chalcone derivatives on steroidal framework and their anticancer activities.

Chalcone derivatives on estradiol framework have been synthesized. Some of the derivatives showed potent anticancer activity against some human cancer cell lines. Compounds 9 and 19 showed potent activity against MCF-7, a hormone dependent breast cancer cell line. Chalcone 7 was further modified to the corresponding indanone derivative (19) using the Nazarov reaction, which showed better activity than the parent compound against the MCF-7 breast cancer cell line. Active anticancer derivatives were also evaluated for osmotic hemolysis using the erythrocyte as a model system. It was observed that chalcone derivatives showing cytotoxicity against cancer cell lines did not affect the fragility of erythrocytes and hence may be considered as non-toxic to normal cells.

Citral, a monoterpenoid aldehyde interacts synergistically with norfloxacin against methicillin resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus (SA), is a major human pathogen causing wide range of clinical infections, which has been further complicated by drug resistance like methicillin resistant S. aureus (MRSA), vancomycin intermediate S. aureus (VISA)/vancomycin resistant S. aureus (VRSA), etc. The present study was aimed at determining anti-staphylococcal potential of citral against drug resistant clinical isolates alone and in combination with antibiotics. PURPOSE: To assess the potential of citral in combination with norfloxacin in treating drug resistant infections of SA. STUDY DESIGN: In the present study, synergistic interaction of citral and norfloxacin against drug resistant SA strains was evaluated. Further the efficacy and possible mechanism of action of the combination was also evaluated using in vitro and in vivo assays. METHOD: The anti-staphylococcal activity of each of the monoterpene and the antibiotic was determined in terms of MIC and the effective concentration of both compounds in combination was obtained by checkerboard assay. In vivo efficacy and oral acute toxicity was evaluated in Swiss albino mice model. To understand the mechanism of action, time-kill curve, bacteriolysis, leakage, membrane depolarization, salt tolerance and ethidium bromide efflux assays were performed. RESULTS: Citral was found effective against clinical isolates of SA with MIC values ranging from 75 to 150 µg ml-1 exhibiting bacteriostatic activity. Citral interacted synergistically, reducing MIC of norfloxacin up to 32-folds with FICI ≤ 0.50. Citral did not affect cell wall, but could damage cell membrane, inhibit efflux pump and affect the membrane potential. Citral could reduce the staphylococcal load of spleen and liver tissues in a dose-dependent manner which was further reduced when used in combination with norfloxacin. Citral did not exhibit any mortality or morbidity up to 500 mg kg-1 body weight and found to prolong the post-antibiotic effect of norfloxacin. CONCLUSION: Based on these observations, citral could be a lead candidate phytomolecule for further developing it into an anti-staphylococcal agent. The observations of combination study will help in reducing the burden of antibiotics leading to delayed resistance development.

First Report of a 16SrVI Group Phytoplasma Associated with Witches'-Broom Disease on Withania somnifera.

Withania somnifera (L.) Dunal is cultivated in India as an important medicinal cash crop. The whole plant is of great importance in the Indian system of medicine and pharmaceutical industries, but the roots are the main source of active alkaloids. Some of the important alkaloids are tro-pine, pseudotropine, somniferine, colin, withaferin A, withanoides, and a few flavanoides. Typical disease symptoms include phyllody, little leaf, dense clusters of highly proliferating branches with shortened internodes, and resulting witches'-broom. The disease was first observed in and around Lucknow, Uttar Pradesh Province, India during January and February 1992. On the basis of symptoms, transmission electron microscopy (TEM), and antibiotic treatment, the causal organism was identified as a phytoplasma (4). The disease is now spreading to other parts of the country (Gujrat, Haryana, Madhya Pradesh, Punjab, and Rajasthan provinces) with a high disease incidence (70%). In this report, molecular characterization and taxonomic position of the associated phytoplasma is reported. Total genomic DNA was extracted from healthy and infected plants with a modified cetyltrimethylammoniumbromide (CTAB) buffer method. The samples were assayed for the presence of phytoplasma using polymerase chain reaction (PCR) with universal phytoplasma primers P1/P6 (2) for amplification of ribosomal 16S rDNA. PCR product was diluted by 1:200 and used directly as DNA template for nested PCR with primers R16F2n and R16R2 (1). Results showed the presence of an expected 1.5-kb rDNA fragment amplified with the direct PCR and a 1.2-kb product of the nested PCR from infected W. somnifera samples. No PCR product was observed in the healthy counterparts. The PCR assay confirmed the presence of phytoplasma as causal agent. The PCR product was cloned with TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and isolated plasmids were again assessed by restriction enzyme (EcoRI) digestion before sequencing. Purified plasmids were sequenced. Partially sequenced nucleotide sequence analysis of 16SrRNA gene cloned from W. somnifera phytoplasma showed high similarity with several isolates of the 16SrVI group of phytoplasmas. The highest nucleotide matching (99 and 98%) was observed with Centaurea solstitialis virescence phytoplasma (Genbank Accession No. AY270156) and Periwinkle little leaf phytoplasma (PPL-Bd; Genbank Accession No. AF 228053) reported in Italy and Bangladesh, respectively. In restriction fragment length polymorphism (RFLP) analysis, AluI, EcoRI, HhaI, HincII, KpnI, and Sau3AI (Promega, Madison, WI; 5 U per reaction) were used for comparison of restriction pattern of present/reference phytoplasma and with that previously reported (3). The present phytoplasma produced identical restriction profile to those of periwinkle infected by PPL-Bd (periwinkle little leaf phytoplasma, Bangladesh, group 16SrVI). On the basis of PCR studies, absence of virus particles under TEM in infected samples, RFLP analysis and nucleotide sequence matching with previously characterized phytoplasma, this phyto-plasma is classified as a member of Clover proliferation group (16SrVI). To our knowledge, this is the first report of a phytoplasma belonging to 16Sr VI group from W. somnifera. References: (1) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr.35:144, 1996. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) M. Zaim and A. Samad. Plant Sci. 109:225,1995.

Molecular modeling based semi-synthesis and in vitro evaluation of anticancer activity in indolyl chalcones.

A series of indolyl chalcones were synthesized and evaluated in vitro for their anticancer activity against four human cancer cell lines. Compounds 1a, 1b, 1d, 1f-1j, 2c, 2e, 2i showed significant cytotoxicity. Chalcones 1b and 1d were identified as the most potent and selective anticancer agents with IC50 values <1µg/ml and 1.51µg/ml, against WRL-68 cell line, respectively.

Development of a novel plant system as biosensor for detecting environmental hazards and bioactive molecules through distinct responses.

Currently, chemicals are used excessively in medicine, industry, and agriculture throughout the world. Because of the current rise in environmental pollutants and hazardous exposures of human beings, there is an imperative need for monitoring the environment for biosafety. In addition, the importance of natural products with novel activity for drug or agrochemical scopes is being more and more realized. We report on a novel test system that can act as biosensors for detecting useful compounds while simultaneously monitoring or forecasting the biohazard exposure of organisms to chemicals as well as to physical factors in a given environment. Using different compounds and factors with known biological and cytotoxic activities, a detail plant test procedure has been developed that can be used in detecting and analyzing the value and/or danger of any given compound or treatment, including cell division inhibition, cytotoxicity, growth inhibition, and anticancer activities. The method provides a highly efficient single biosensor system that can replace several individual biotesting procedures. This plant assay procedure is a highly sensitive system for monitoring physical stress factors in the environment, including ionizing and nonionizing radiation. The procedure can be followed throughout the year because of the rapid growth rate of the plant used and its regeneration in ambient conditions. Therefore, the described system is highly useful to thoroughly monitor the environment and detection of new chemicals/compounds.

Modulation of inflammatory mediators by coumarinolignoids from Cleome viscosa in female swiss albino mice.

The effect of coumarinolignoid cleomiscosins A, B and C isolated from the plant Cleome viscosa on inflammatory mediators were studied in female swiss albino mice. A mixture of coumarinolignoid A, B, and C at 10, 30 and 100 mg/kg body weight once a day for 14 consecutive days were administered orally to the mice. Pro-inflammatory mediators such as IL-6, TNF-alpha and nitric oxide were estimated from culture supernatant obtained from peritoneal macrophages stimulated by LPS and anti-inflammatory mediator IL-4 was estimated from culture supernatant obtained from spleenocytes stimulated by Con-A. For further confirmation, expressions of inflammatory mediators from serum and mortality rate were studied in LPS-induced toxicity model in mice. The expression of Pro-inflammatory mediators was significantly (P <0.05) decreased in coumarinolignoids treatment group in dose dependent manner, whereas the anti-inflammatory mediator expression was significantly increased in coumarinolignoids at 10 mg/kg treatment. Mortality rate was also significantly reduced in treatment group in LPS-induced toxicity model. The result of this study concluded that the oral administration of coumarinolignoids inhibited the pro-inflammatory mediators and enhances the production of anti-inflammatory mediator in dose dependent manner.

Cytotoxic agents from Terminalia arjuna.

Although a number of chemicals have been isolated from Terminalia arjuna, only a few have been evaluated for their biological significance. As a part of our drug discovery programme for cytotoxic agents from Indian medicinal plants, four novel cytotoxic agents arjunic acid (1), arjungenin (2), arjunetin (3) and arjunoglucoside I (4) were isolated from the bark of T. ARJUNA. Out of the four compounds, arjunic acid (1) was significantly active against the human oral (KB), ovarian (PA 1) and liver (HepG-2 & WRL-68) cancer cell lines. Further, the most active compound arjunic acid was converted into seven semi-synthetic ester derivatives 5 - 11. 2-O-Palmitoyl arjunic acid (6) showed two times more activity, while 2, 3-di-O-acetyl-, 2-O-p-anisoyl-, 2, 3-di-O-benzoyl- and 2, 3-di-O-p-nitrobenzoyl arjunic acid (7 - 10) showed 1.7 - 2.3 times less activity than the cytotoxic drug vinblastine against the liver cancer cell lines HepG-2 and WRL-68 respectively.

Synthesis of 1-(3',4',5'-trimethoxy) phenyl naphtho[2,1b]furan as a novel anticancer agent.

3',4',5'-Trimethoxy benzoyl-naphthalene 2-O-acetic acid (5) underwent base catalysed intramolecular condensation to yield exclusively 1-(3',4',5'-trimethoxy) phenyl naphtho[2,1-b]furan 8. The cyclised product 8 has been characterised by spectroscopy. The product 8 showed significant anticancer activity against human cancer cell lines COLO320DM (colon), CaCO2 (colon) and WRL68 (liver) at 0.7, 0.65 and 0.50 microg/ml concentrations, respectively, in the in vitro MTT assay.

Use of RAPD and AFLP markers to identify inter- and intraspecific hybrids of Mentha.

Three controlled crosses were carried out involving Mentha arvensis and Mentha spicata [M. spicata CIMAP/C30 x M. spicata CIMAP/C33 (cv. Neera); M. arvensis CIMAP/C18 x CIMAP/C17 (cv. Kalka); and M. arvensis CIMAP/C17 x M. spicata CIMAP/C33]. The parents were subjected to random amplified polymorphic DNA (RAPD) analysis with 80 primers, and polymorphic primers were tested for detecting coinherited RAPD profiles among the progeny of these crosses. Of 50 seedlings tested from each intraspecific cross, all demonstrated dominant profiles with the selected RAPD primers except the detected hybrid from respective crosses. Coinherited markers could be detected with the primers OPJ 01, MAP 06, OPT 08, and OPO 20 for M. arvensis; OPJ 05, OPJ 14, OPO 19, and OPT 09 for M. spicata; and OPJ 07, OPJ 10, OPJ 11, OPJ 14, and OPO 02 for the cross M. arvensis x M. spicata. In our amplified fragment length polymorphism (AFLP) analysis, 40 coinherited marker fragments were identified for the cross involving M. arvensis, 32 for the cross involving M. spicata, and 41 for the interspecific cross between M. arvensis and M. spicata. In all crosses, similarity values between the parents were less than those between the parents and the hybrids. Although RAPD markers are generally considered dominant, it is possible to identify a few codominant markers that behave like restriction fragment length polymorphism (RFLP) markers. This molecular marker system may be helpful in rapidly screening out hybrids in crops where cross-pollination is a problem.

Antibacterial activity of Ailanthus excelsa (Roxb).

The antibacterial activity of different fractions of a methanol extract obtained from the dried stem bark of Ailanthus excelsa (Roxb) was studied using different bacterial strains. The ethyl acetate fraction inhibited the growth of all test bacteria. The MIC of the EA fraction was found to be 6 mg/disc.