Suman P. S. Khanuja

Rapid Isolation of DNA from Dry and Fresh Samples of Plants Producing Large Amounts of Secondary Metabolites and Essential Oils

The presence of certain metabolites has been observed to interfere with DNA isolation procedures and downstream reactions such as DNA restriction, amplification and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yields with a single protocol, and thus, even closely related species may require different isolation protocols. Here we describe the essential steps of a rapid DNA isolation protocol that can be used for diverse medicinal and aromatic plants, which produce essential oils and secondary metabolites such as alkaloids, flavanoids, phenols, gummy polysaccharides, terpenes and quinones. The procedure is applicable to dry as well as fresh plant tissues. This protocol, in our experiments, permitted isolation of DNA from tissues of diverse plant species and produced fairly good yields. The isolated DNA proved amenable to PCR amplification and restriction digestion.

Repellency and toxicity of oil from Artemisia annua to certain stored-product beetles.

Abstract The essential oil of Artemisia annua L. was tested for its toxic repellent and development inhibitory activities against 2 economically important stored product insects: Tribolium castaneum (Herbst) and Callosobruchus maculatus (L.). Adult beetles of T. castaneum were repelled significantly by oil of A. annua at 1% concentration (vol:vol) and above in filter paper arena test. Dose–response relationship of A. annua oil revealed a significant negative correlation between larval survival; pupal survival and adult emergence of T. castaneum (i.e., increase in dose caused decrease in survival and adult emergence). Effective concentration (EC50) to reduce F1 progeny by 50% was calculated to be 2.6 and 4.1 μl/ml solvent against both the insect species, C. maculatus and T. castaneum, respectively. The relationship between bioactivity of oil from A. annua and responses of T. castaneum and C. maculatus is discussed. We found that oil from A. annua was largely responsible for both repellent (behavioral) and toxic (physiological) actions on 2 species of insect tested.

Bioactivities of the Leaf Essential Oil of Curcuma Longa (Var. Ch-66) On Three Species of Stored-Product Beetles (Coleoptera)

Abstract Essential oil extracted from the leaves of turmeric, Curcuma longa L., was investigated for contact and fumigant toxicity and its effect on progeny production in three stored-product beetles, Rhyzopertha dominica F. (lesser grain borer), Sitophilus oryzae L. (rice weevil), and Tribolium castaneum Herbst (red flour beetle). Oviposition-deterrent and ovicidal actions of C. longa leaf oil were also evaluated against T. castaneum. The oil was insecticidal in both contact and fumigant toxicity assays. The adults of R. dominica were highly susceptible to contact action of C. longa leaf oil, with LD50 value of 36.71 μg/mg weight of insect, whereas in the fumigant assay, adults of S. oryzae were highly susceptible with LC50 value of 11.36 mg/liter air. Further, in T. castaneum, the C. longa oil reduced oviposition and egg hatching by 72 and 80%, respectively at the concentration of 5.2 mg/cm2. At the concentration of 40.5 mg/g food, the oil totally suppressed progeny production of all the three test insects. Nutritional indices indicate >81% antifeedant action of the oil against R. dominica, S. oryzae and T. castaneum at the highest concentration tested.

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.

Assessment of genetic relationships in Mentha species

A set of 60 random primers was used to analyse 11accessions from six taxa of Mentha developed byCIMAP. These accessions were maintained in the nationalgene bank for medicinal and aromatic plants at CIMAP.A total of 630 bands could be detected as amplifiedproducts upon PCR amplification, out of which 589 werepolymorphic (93.5%). Further analysis of these RAPDprofiles for band similarity indices clearlydifferentiated five of the Mentha arvensis L.accessions from the rest. Among two accessions of Mentha spicata L. CIMAP/C33 could bedistinguished from CIMAP/C32. Mentha × gracilis Sole cv. cardiaca showed a muchhigher similarity with Mentha spicata L. as wellas Mentha arvensis L. which amongst themselvesshowed rather a greater distance indicating that Mentha × gracilis Sole cv. cardiaca might have evolved as a natural hybridbetween M arvensis L. and M. spicataL. In terms of uniqueness of amplified bands fordeveloping RAPD markers, it was observed that at taxalevel 298 bands were unique to one of the six taxa,singly amounting to 47.3% of total amplifiedfragments. Primers MAP 10 and 17 produced polymorphismonly in case of M. spicata L. and Menthaspicata L. cv. viridis while MAP 08 producedpolymorphic bands in all 4 other species than thesetwo. Similarly unique patterns were observeddifferentiating all six species and could be used asRAPD markers for differentiating Mentha species.

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.

Piperitenone Oxide as Toxic, Repellent, and Reproduction Retardant Toward Malarial Vector Anopheles stephensi (Diptera: Anophelinae)

Abstract Anopheles stephensi (Liston) is a well-known vector of malarial parasite in tropical countries. The developing trend of resistance in mosquitoes toward synthetic mosquitocidal agents makes their management extremely difficult. Effectiveness of essential oils with aroma therapeutic values seems to be an emerging tool to combat this vector. Piperitenone oxide isolated from essential oil of a new genotype, Mentha spicata L. variety viridis, has been evaluated for larvicidal, ovicidal, oviposition-deterrent, developmental toxicity, and repellent properties against various stages of A. stephensi. The results indicated the higher efficacy of piperitenone oxide than the crude essential oil of M. spicata variety viridis in all the bioassay experiments. The lethal response of piperitenone oxide and the oil toward fourth instar larvae showed LD50 values of 61.64 and 82.95 μg/ml, respectively. Female adults of A. stephensi exposed to the oil laid ≈42 times less number of eggs at the dose of 60.0 μg/ml as compared with control, whereas exposure of piperitenone oxide at the same dose completely inhibited the oviposition. Furthermore, piperitenone oxide also completely inhibited egg hatching at the dose of 75.0 μg/ml in ovicidal assay. Developmental toxicity studies showed the significant developmental inhibition potential of the compound and oil. Additionally, piperitenone oxide was found to be highly toxic and repellent toward adults of A. stephensi as compared with oil.

Synergistic effect of silymarin and standardized extract of Phyllanthus amarus against CCl4-induced hepatotoxicity in Rattus norvegicus

In search of the effective and standardized hepatoprotective combination therapy, silymarin and standardized extract of Phyllanthus amarus has been evaluated against CCl(4)-induced hepatotoxicity in rats. Eight groups of rats were used. The animals of group A served as normal and were given only vehicle. The animals of group B served as toxin control and were administered with CCl(4) (50% solution of CCl(4) in liquid paraffin, 2 ml/kg b.w., intraperitoneally). The animals of groups C-H received silymarin (100 mg/kg b.w.), Phyllanthus amarus aqueous extract (100 mg/kg b.w.), Phyllanthus amarus ethanolic extract (100 mg/kg b.w.), silymarin (50 mg/kg b.w.)+P. amarus aq. ext. (50 mg/kg b.w.), silymarin (50 mg/kg b.w.)+P. amarus eth. ext. (50 mg/kg b.w.) and marketed formulation (M.F.) 5 ml/kg b.w. for 6 days orally as well as CCl(4) (2 ml/kg b.w.) on 4th day intraperitoneally. The test materials were found effective as hepatoprotective as evidenced by plasma and liver biochemical parameters. The combination of silymarin and Phyllanthus amarus showed synergistic effect for hepatoprotection and silymarin with ethanolic extract of P. amarus showed better activity due to the higher concentration of phyllanthin in ethanolic extract in comparison to aqueous extract of P. amarus as estimated by HPLC.

Morphogenetic variation for artemisinin and volatile oil in Artemisia annua

Seeds of Artemisia annua cv. Jeevanraksha were sown in the nursery in the middle of December in 1997, 1998 and 1999. About 1-month old seedlings were transplanted in the field having sandy loam soil in the subtropical agroclimate of Lucknow, India. The plant tops were sampled fortnightly for leaves during vegetative phase and for leaves and capitula during post-flowering stages for the estimation of artemisinin content. The A. annua plants continued to grow logarithmically in height from the end of rosette phase at about 9 weeks to the pre-flowering stage at about 44 weeks age and attained a height of 3.4 m. The artemisinin content of the leaves was observed to be high from 0.8 to 1.0% in May and 0.8 to 1.3% through late July to late September. Subsequently, plants entered the reproductive phase. While in the vegetative phase, 90% of artemisinin was in the leaves, in the mature plants, about 30% of the artemisinin was in the leaves and 40% was in capitula. In the vegetative stage plants the younger leaves born on the tops of secondary and higher order branches were richer in the artemisinin than the older leaves. The tops of A. annua plants in their vegetative growth phase possessed low levels of essential oil at about 0.2% as compared to 1.2% of essential oil in the full blooming stage plants. The extraction of artemisinin from leaves is more economic than from the mixture of leaves and capitula on account of higher levels of lipids in the extract of the latter. Since A. annua plants grew logarithmically all through vegetative phase from March to late September and artemisinin content in the leaves was high in May and from late July to late September, it is suggested that under the subtropical agroclimates, A. annua crops may be harvested more than once. The ratooning is expected to reduce losses in artemisinin yield resulting from senescence caused dropping of old leaves and favour preponderance of young leaves found richer in artemisinin content.

Antitubercular potential of plants: a brief account of some important molecules.

Mycobacterium tuberculosis is the most lethal pathogen causing tuberculosis in human. After the discovery of antitubercular drugs pyrazinamide, rifampicin, isoniazid, streptomycin, and ethambutol (PRISE), the disease was controlled for a limited period. However, over the course of their usage, the pathogen acquired resistance and evolved into multi‐drug resistant, single‐drug resistant, and extensive drug resistant forms. A good number of plant secondary metabolites are reported to have antitubercular activity comparable to the existing antitubercular drugs or sometimes even better in potency. A well‐defined strategy is required to exploit these phytomolecules as antitubercular drugs. This review gives concise up‐to‐date information regarding the chemistry and pharmacology of plant‐based leads and some insight into their structure–activity relationship.