Ameliorative effect of alkaloid extract of Cyclea peltata(Poir.) Hook.f.& Thoms.roots(ACP) on APAP/CC1_4 induced liver toxicity in Wistar rats and in vitro free radical scavenging property

Objective:To evaluate the hepatoprotective and antioxidant properties of alkaloid extract of Cyclea peltata(C.peltata) against paracetamol/carbon tetra chloride induced liver damage in Wistar rats.Methods:In viro paracetamol/carbon tetrachloride induced liver damage in Wistar rats,in vitro free radical scavenging studies,HPTLC estimation of letrandrine and direct analysis in real timemass spectrometry of alkaloid extract of C.peltata were used for the validation.Results:The results showed that pretreatment with alkaloid extract of C.peltata caused significant reduction of serum glutamate pyruvate transaminase,serum glutamate oxaloacetate transaminase,serum alkaline phosphatase,serum cholesterol,liver malondialdehyde levels.The reduced glutathione,catalase,superoxide dismutase levels in liver were increased with alkaloid extract of C.peltata treatment.These results were almost comparable to silymarin and normal control.Histopathological studies also substantiated the biochemical findings.The in vitro hydroxyl,superoxide and DPPH scavenging study of alkaloid extract of C. peltata showed significant free radical scavenging property.Conclusions:The hepatoprotective property of alkaloid extract of C.peltata against paracetamol/carbon tetrachloride may be due the synergistic action of alkaloids especially tetrandrine,fangchinoline through free radical scavenging and thus preventing oxidative stress.

Intraspecific genetic variability,differentiation and evolutionary relationships revealed through microsatellite loci in seven economically important Calamus species

Population density,species richness and critical population parameters are crucial in determining the levels of gene diversity in dioecious species of the genus Calamus.The extent of intraspecific and intrageneric genetic variability in Calamus from the southern Western Ghats of India was studied using 26 microsatellite markers by sampling 227 individuals belonging to seven economically important species.The heterozygosity of microsatellite loci ranged from zero to 0.78.Average gene diversity within species was 0.13;in all species it was 0.18 and amongst species was 0.06.The Shannon Information Index was the lowest for Calamus metzianus(0.11),whereas it ranged from 0.16 to 0.26 for other species.The expected heterozygosity varied from 0.08 to 0.18.Calamus hookerianus and Calamu travancoricus showed the highest genetic differentiation(44%)revealed through Fst values,whereas the lowest(22%)was observed between Calamus gamblei and Calamu thwaitesii.Population structuring and phylogenetic analysis differentiated the seven species.Due to overexploitation and loss of rare alleles,small populations could lead to fertilization between closely related individuals,resulting in inbreeding and increasing the risk of extinction.This could be important for species such as C.metzianus where allelic polymorphism was 23%,whereas for all other species it was 38%to 46%.Genetic diversity“micro-hotspots”were identified from the protected area network of the southern and central Western Ghats with highest observed heterozygosity.Four microhotspots from the Agasthyamalai Biosphere Reserve and the Pushpagiri Wildlife Sanctuary may be possible for long-term conservation programs.The findings of this study lay a strong foundation for strengthening protected area networks,especially areas with intermediate levels of disturbance.

Improved Clonal Propagation of <i>Alpinia calcarata</i>Rosc., a Commercially Important Medicinal Plant and Evaluation of Chemical Fidelity through Comparison of Volatile Compounds

An efficient and improved clonal propagation of Alpinia calcarata, a commercially important medicinal plant was established on Murashige and Skoog medium. The axillary shoot proliferation was achieved with maximum 5.2 ± 0.7 shoots in 92.8% of rhizome explants in medium with 2.0 mg/L 6-benzylamiopurine (BAP) and 0.2 mg/L indole-3-acetic acid (IAA). Axillary shoot buds (60%) upon subculture for 8 weeks in the same medium produced multiple shoot initials (12.1 ± 0.4) mediated with meristemoids (4.0 ± 0.5) and callus. A gradual reduction in the concentration of BAP or elimination of IAA was required for rapid induction of normal plants devoid of callus from propagules during subsequent subculture. Single clump of 3-4 multiple shoot initials during second subculture on medium with 1.0 mg/L BAP and 0.1 mg/L IAA yielded an average of 21 shoots which was best among different propagules tried. The shoot multiplication rate was further enhanced to 32 shoots when the similar propagules passed to third subculture on medium with 1.0 mg/L BAP alone. Clumps of multiple shoot initials upon subculture on medium with 1.0 mg/L BAP alone exhibited 10 fold multiplication rates. Use of liquid medium in culture bottles with polypropylene caps supported fast growth of the shoots and spontaneous root formation on 50% of the shoots. Shoots transferred to half-strength MS liquid medium with 0.2 mg/L of IAA and IBA was optimum for maximum roots (8.14 ± 1.34) in 100% shoots. The rooted plants were hardened in mist chamber showed 95% survival and well established in the field. The acclimatized plants showed rhizome formation after 4-6 weeks of growth under shade house. Volatile chemicals profile of the leaves, rhizome and root of the in vitro and conventionally propagated plants analyzed by gas chromatography-mass spectrometry were qualitatively and quantitatively similar. The analysis of growth characteristics of 36 month old in vitro and conventionally propagated plants showed a 50% increment of rhizome fresh biomass with prolific root and leaf growth in the former than the latter ones. The protocol described herein will have practical applications for the large scale production of uniform efficient plants for commercial cultivation of A. calcarata.

Antineoplastic effects of deoxyelephantopin,a sesquiterpene lactone from Elephantopusscaber, on lung adenocarcinoma (A549) cells

OBJECTIVE： Deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, showed inhibition of the growth of various tumor cells in vitro. In the present study, we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma （A549） cells. METHODS： The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 50% inhibitory concentration （IC50） value was determined. The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay. Cellular morphology of deoxyelephantopin-treated cells was observed using phase- contrast microscopy. The induction of apoptosis was evaluated using acddine orange and ethidium bromide staining, Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling （TUNEL） assay, DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry. Activation of caspases was detected using fluorogenic substrate specific to caspases 2, 3, 8 and 9 and flow cytometric analysis. The total cellular DNA content and expression of cleaved poly （ADP-ribose） polymerase was also analyzed. RESULTS： Deoxyelephantopin exhibited cytotoxicity to A549 cells （IC50 = 12.287 μg/mL）, however, there was no toxicity towards normal human lymphocytes. Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner. Acridine orange, ethidium bromide and Hoechst 33342 staining showed cell shrinkage, chromosomal condensation and nuclear fragmentation, indicating induction of apoptosis. Deoxyelephantopin increased apoptosis of A549 cells, as evidenced by more TUNEL-positive cells. DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population. Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through both extrinsic and intrinsic pathways. CONCLUSION： These results suggest that deoxyelephantopin has great potential as a new chemotherapeutic agent to be developed further for the treatment of lung cancer.

New records of Graphidaceae(lichenized fungi)from the Western Ghats of Kerala state,India

In the present paper,15 lichenized fungal species belonging to family Graphidaceae are described,of which Ocellularia albomaculata,O.ascidioidea,O.kanneliyensis,O.monosporoides,O.pertusariiformis and O.rhicnopora are new records to Indian lichen biota and Pallidogramme chrysenteron is new to Peninsular India,whereas Asteristion alboolivaceum,Chapsa hiata,Diploschistes rampoddensis,Fissurina rubiginosa,Pseudochapsa phlyctidioides,Thelotrema canarense,T.piluliferum and T.subtile are new to lichen biota of Kerala state.