Screening Of In Vitro Antiviral Activity Of Purified Terpenoid Extracts Of Selected Sea Weeds Against Chikungunya Virus

Currently, the search of novel phytochemicals with unique biological potentialities is a pre-requisite for the designing ideal drugs for the human kind. Sea weeds are bioresources with a broad spectrum of medicinal properties with minimal side effects. Kerala, the Southern state of India reported high incidence of Chikungunya virus (CHIKV) infections in the last several tears. No specific virucidal therapy or effective vaccines are available. This emphasizes the need of searching for phytochemicals as drugs with less cost and more effective. Therefore, an attempt was made in screening purified terpenoid extracts of selected sea weeds as anti-CHIKV potential. In this study the terpenoids composition from the red algae Hypnea musciformis, Kappaphycus alvarezii and Gracillaria dura were identified and analyzed by thin layer chromatography and Gas chromatography- Mass spectrum. The methanolic extract of seaweeds was purified by column chromatography and each fraction was eluted by using petroleum ether and ethyl acetate as solvent combination. The analysis of the purified fraction of H. musciformis and K. alvarezii revealed the presence of 8 terpenoid fractions, and G. dura showed only 4 major components respectively. Vero cell lines were employed in the antiviral assays, infected to CHIKV, and treated with varied doses of purified terpenoid extracts. In the antiviral activity, terpenoid extracts of G. dura showed remarkable and promising EC50 inhibitory effect at 1.25 μg/ml. Further, the terpenoid extracts displayed efficient virucidal activity against CHIKV (inhibit around 90%) with 5 μg/ml dosage. As the last phase, terpenoid extracts added at time intervals of 0, 1, 2, 3 post-infection periods still maintained a significant inhibitory potential against CHIKV viral replication. Thus, the overall study suggests that the terpenoid extracts of G. dura may be effectively used in the prevention and treatment of CHIKV infections. Clinical studies may be warranted for designing a promising new anti-CHIKV drug.

In Vitro Antioxidant Potentiality Of Purified Anthocyanin From Floral Petals Of Wild Balsam Species

Phenolics are the largest group of phytochemicals ubiquitous in plant species with considerable interest economically. Recently, search of novel polyphenols increasingly becomes an area of intensive pharmacological research due to their multiple bioactive features such as antioxidant, antimicrobial, anti-carcinogenic, anti-viral and anti-inflammatory potentialities. Anthocyanins are flavonoid group of polyphenols, a group predominant in flowers, fruits and vegetables. The flavonoids, perhaps the unique single group of phenolics in foods, comprise a group of over 4200 C15 aromatic molecules with multiple structural patterns. The functions of anthocyanins as medicinal have been well-accepted in folk medicine throughout the world. In fact, these molecules are connected to an amazingly broad-based range of health benefits. In this juncture, the aim of this work was to evaluate the antioxidant activities of purified anthocyanin from wild balsam species. Initially, anthocyanin was extracted from floral leaves of wild balsam species and purified by chromatographic techniques. Subsequently, it was subjected to NMR and LC MS analysis. The major fractions identified were hesperidin, dimethoxy antirrhinin and trimethoxy antirrhinin. Further, the anthocyanin extracts were subjected to in vitro protocols like 2,2’-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation, DPPH scavenging assay, ferric reducing antioxidant power (FRAP), beta carotene bleaching assay, metal chelating and H2O2 scavenging power. Interestingly, ABTS, FRAP analyzes yielded significant results as compared to others. The data were comparable with that of synthetic antioxidants like ascorbate and catechin. Meanwhile, beta carotene and H2O2 scavenging assay showed moderate results. DPPH and metal chelating protocols displayed the values 71% and 64% respectively at 25 µg/ml concentration. This study provides model systems for the evaluation of natural antioxidants like anthocyanin. Future in vivo clinical studies are warranted to confirm the obtained data.

Bioactivity And Apoptotic Efficacy Of The Purified Terpenoid Extract From The Moss Brachythecium Buchananii (Hook.) A. Jaeger Against Mg 63 Cells

Bryophytes are primitive non vascular plants. A little is explored regarding the medicinal effects of bryophytes on carcinoma. This study investigated the biological effects of purified terpenoids from Brachythecium buchananii on selected cell lines such as HeLa, MDAMB-231 and MG63 human osteosarcoma cells and also elucidating the regu­latory signaling pathways underlying the effects of terpenoids towards caspase cascade and the antioxidant enzyme system. The cell lines were treated with various concentrations of purified terpenoid extracts interms of evaluating viability (MTT assay). Interestingly, MG63 cell lines showed poor viability as compared to other ongo cells and was subjected to further molecular evaluations. Migration and invasion assay results using wound-healing and transwell assays, respectively reveal the pro-antimetastatic potential of the purified terpenoids from the moss. The flow cytometry study substantiated terpenoid induced apoptosis in MG63 cells. Cell cycle analysis revealed the significant increase in the number of cells arrested at the S growth phase. Terpenoid extract also displayed DNA fragmentation in the cells. Western blot analysis revealed the down regulation of the anti‑apoptotic proteins Bcl‑2, pro‑caspase 3 and over expression of the pro‑apoptotic protein Bax. In addition, the caspase cascade profile of the terpenoid extract substantiated their efficacy in tumour inhibition. Thus, the overall results confirmed the biological features of terpenoid induced apoptosis in the MG63 cells.

Comparison of Bioactive Anthocyanin Components from Begonia Malabarica Lam. and Begonia Rex-Cultorum ‘Baby Rainbow’ L.H.Bailey by GC-MS Analysis.

Anthocyanins, derivatives of 2-phenylbenzopyryliumare are water-soluble natural pigments. Owing to extensive scientific research they have become not only the colouring food products but also therapeutic sources of bioactive compounds and continue to play role in the maintenance of human health. Begonia malabarica Lam. and Begonia rex-cultorum ‘baby rainbow’ L.H. Bailey are common ornamentals with varied leaf colour combinations were analyzed by Gas Chromatography-Mass Spectrometry to determine the bioactive constituents present in the methanolic extracts of the leaves. Powdered leaf samples were subjected to Soxhlet extraction method with methanol and the extract was fractioned by GC-MS. Totally, 7 different compounds each from B. malabarica [phenol, 2,4-bis (1,1-dimethylethyl), hexadecanoic acid, methyl ester, 9,12-octadecadienoic acid (Z,Z), methyl ester, 9,12,15- octadecatrienoic acid, methyl ester, (Z,Z,Z), 1,2-benzenedicarboxylic acid, diisooctyl ester, Spiro[furan-2(5H),2'(1'H)- naphtho[2,1-b]furan]-5-one, 3'a,4',5',5'a,6',7',8',9',9'a,9'b-decahydro-3,3'a,6',6',9'a-pentamethyl and androst-1-en-3- one, 4,4-dimethyl-, (5.alpha.)] and B. rex ‘baby rainbow’ [diethyl Phthalate, 1-(3,6,6-trimethyl-1,6,7,7atetrahydrocyclopenta[ c]pyran-1-yl) ethanone, hexadecanoic acid, methyl ester, 7,10-octadecadienoic acid, methyl ester, 9,12,15-octadecatrienoic acid, methyl ester, (Z,Z,Z), 5,9 methanobenzocycloocten-1(2H)-one,3,4,5,6,7,8,9,10- octahydro-5,10-dihydroxy-3,3,7,7,9-pentamethyl and 1,2-benzenedicarboxylic acid, diisooctyl ester] were identified. All the compounds identified were medicinally proven for the treatment of various human ailments. Further studies are warranted to purify the lead molecules and to evaluate their biological properties including toxicological aspects for the development of novel drugs.

Analysis of Bioactive Compounds by GC-MD from the Young Leaves of Tectona Grandisl.F.

The timber value of Tectona grandis has been established from decades. Teak is an exotic species cultivated along most parts of tropical regions. Anthocyanins are natural colourants which have raised a growing demand due to their extensive range of colours, innocuous and beneficial health effects. Anthocyanins belong to large group of polyphenolics - flavonoids, which are secondary metabolites synthesized by higher plants. Despite the remarkable application of anthocyanins in food, pharmaceutical and cosmetic industries, still it is not properly exploited. In the present study, the bioactive components of Tectona grandis young leaves have been evaluated using Perkin-Elmer Gas chromatography-Mass spectrometry. GC-MS analysis revealed the existence of eleven compounds. 5,9-Methanobenzocycloocten-1(2H)-one,3,4,5,6,7,8,9,10-octahydro-5,10-dihydroxy-3,3,7,7,9- pentamethyl (76.02%) and 1-naphthalenecarboxylic acid,5-[2-(3-furanyl)ethyl]decahydro-1,4a-dimethyl-6- methylene-[1R-(1.alpha.,4a.)] (13.95%). Other compounds present in minor quantities were ledol(0.92%), 3- Buten-2-one,4-(2,6,6-trimethyl-1-cyclohexen-1-yl)orionone(0.49%),9,12,15 Octadecatrienoic acid, methyl ester, (Z,Z,Z)- or Linolenic acid methyl ester (0.82%), Phytol (0.69%), Cedran-diol, 8S,14- (0.60%), Lupeol (0.71%), 3- Methoxymethyl-2,5,5,8a-tetramethyl-6,7,8a-tetrahydro-5H-chromene (3.45%) and Retinol(1.27%). This is the first report of identification of active constituents from the young leaves of Tectona grandis.

Taxonomic Implications with Special Reference to Stomatal Variations in Solanum Species Using Light and Scanning Electron Microscope.

The systematic analysis between 15 different species and one species with two accessions in Solanum has been carried out and need all over revisions and investigations. In continuation to our previous morphometric analysis, currently stomatal variation is analyzed using light microscopy and scanning electron microscopy. In addition to classical anomocytic and anisocytic stomatal types, new types of stomata (anisotricytic, paratetracytic, diacytic and paracytic) are noticed for the first time in the genus Solanum. The scanning electron microscopic study reveals the nature of stomatal pore, nature of peristomatal rims as well as the presence of epicuticular wax deposits and wax flakes which are of taxonomic significance. This study also indicates that foliar stomatal characteristics are valuable taxonomic traits, which can be utilized to address the taxonomic issues within the genus.

Insecticidal Potentiality of Flavonoids from Cell Suspension Culture of Marchantia Linearis Lehm. & Lindenb Against Spodoptera Litura F.

Bryophytes were diverse, primitive non vascular amphibious taxa distributed worldwide and form the second largest category of plants. Bryophytes synthesize an array of phytochemicals to combat against the unhospitable environmental conditions including predation, UV radiation, high temperature and pest and pathogens. The present investigation was undertaken to elucidate flavonoids from in vitro cell cultures of the liverwort Marchantia linearis Lehm. & Lindenb. its fractionation and analysis of insecticidal potentialities. Initially, callus culture was initiated from spores in MS/5 media containing growth regulators BAP and NAA at the concentration of 2 mg/L and 0.5 mg/L. Agitation of the friable callus at lower rpm bring about lower level of cell dispersion, on the contrary at higher rpm might have risk of cell collision that is why rpm was kept at moderate speed i.e., 110 rpm. Continuous sub culturing process substantially improves cell growth and biomass. In the second phase, the flavonoids were isolated from cell suspension cultures of M. linearis and were fractionated by TLC and HPLC PAD chromatogram, which revealed the presence of quercetin, luteolin, apigenin, rutin and kaempferol. In vivo insecticidal analysis revealed significant antifeedant, larvicidal and pupicidal activities at all the concentrations against 5th instar larvae of Spodoptera litura. The extract also exhibited feeding deterrent activity with M. linearis. Similarly, the nutritional parameters were also affected i.e., reduced ECI (Efficiency of conversion of ingested food) and ECD (Efficiency of conversion of digested food) and increased AD (Approximate digestibility) and metabolic cost for the larvae, when compared with the control. The consumption of the basal diet with the incorporation of flavonoids by S. litura larvae was not significantly different compared to the consumption of the control diet by the larvae. Faecal production reduced proportionally with concentrations of the extract.Bryophytes were diverse, primitive non vascular amphibious taxa distributed worldwide and form the second largest category of plants. Bryophytes synthesize an array of phytochemicals to combat against the unhospitable environmental conditions including predation, UV radiation, high temperature and pest and pathogens. The present investigation was undertaken to elucidate flavonoids from in vitro cell cultures of the liverwort Marchantia linearis Lehm. & Lindenb. its fractionation and analysis of insecticidal potentialities. Initially, callus culture was initiated from spores in MS/5 media containing growth regulators BAP and NAA at the concentration of 2 mg/L and 0.5 mg/L. Agitation of the friable callus at lower rpm bring about lower level of cell dispersion, on the contrary at higher rpm might have risk of cell collision that is why rpm was kept at moderate speed i.e., 110 rpm. Continuous sub culturing process substantially improves cell growth and biomass. In the second phase, the flavonoids were isolated from cell suspension cultures of M. linearis and were fractionated by TLC and HPLC PAD chromatogram, which revealed the presence of quercetin, luteolin, apigenin, rutin and kaempferol. In vivo insecticidal analysis revealed significant antifeedant, larvicidal and pupicidal activities at all the concentrations against 5th instar larvae of Spodoptera litura. The extract also exhibited feeding deterrent activity with M. linearis. Similarly, the nutritional parameters were also affected i.e., reduced ECI (Efficiency of conversion of ingested food) and ECD (Efficiency of conversion of digested food) and increased AD (Approximate digestibility) and metabolic cost for the larvae, when compared with the control. The consumption of the basal diet with the incorporation of flavonoids by S. litura larvae was not significantly different compared to the consumption of the control diet by the larvae. Faecal production reduced proportionally with concentrations of the extract.

Evaluation of Biological Efficacy of Trichoderma Species Isolates Against Alternaria Leaf Spot Disease of Sesame.

Alternaria leaf spot disease is a major threat to sesame (Sesamum orientale L.) caused by Alternari asesami. Induced resistance is an alternative to systemic disease resistance response of plants. The present study aims to evaluate Trichoderma species efficacy as biocontrol via induction of resistance against A. sesami in sesame species. During in vitro bio control test, T. harzianum colonize and parallely inhibit the growth of the fungal pathogen. Expression of various defence related enzymes observed in sesame induce resistance against the pathogen infection in the host. T. harzianum coupled with inoculation of A. sesami enhance the remarkable induction of defence enzyme such as peroxidase (POX), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) and also the phenolic content compared with the control. The enzyme activity increased from 48 h of sampling and peaked at 72 h and then decreased after 72 h. In greenhouse and field experiments, soil treatment with a powder formulation of T. harzianum two weeks before planting or at the time of planting reduced significantly the incidence of diseases on both the wild and cultivar Thilarani.The results demonstrate that T. harzianumcan be successfully applied as a biological control against Alternaria leaf spot disease in sesame.

Ultrastructural changes and oxidative stress markers in wild and cultivar Sesamum orientale L. following Alternaria sesami (Kawamura) Mohanty and Behera inoculation.

Alternaria sesami causes leaf spot disease in Sesamum orientale. Conidium germination, inoculation, penetration and colonization of the pathogen on the plant surfaces were studied using scanning electron microscopy. Electron microscopy analysis revealed multiple germ tubes from conidium that spread in all direction across the leaf surfaces. Penetration in the plant surface occured, directly through the epidermis or via stomata with or without the appressoria formation. Hyphal penetration continued through the substomata cavity and some of hyphal branches grew in the intercellular space of mesophyll tissue. Hyphal toxin, caused cell and cell wall damages. Changes in different biochemical parameters in the diseased sesame plants (both in wild and cultivar) were compared to control. Transmission electron microscopy showed structural changes in the chloroplast of diseased plants. Isozyme pattern and assays of different enzymes, namely catalase, acid phosphatase and peroxidase expressed varied level of activities. Meanwhile, esterase, polyphenol oxidase and superoxide dismutase in diseased plants showed remarkable levels compared to control. Due to the infection, chlorophyll content, carbohydrates and total soluble protein decreased whereas free amino acid, proline, phenols and disease-related proteins increased in the host plants. Differential SDS-PAGE band profiling of total soluble proteins were also observed in plants due to the infection.

Effect of methanolic and water extract of Leucobryum bowringii Mitt. on growth, migration and invasion of MCF 7 human breast cancer cells in vitro.

Inhibitory effects of methanol and water extract of L. bowringii. on the adhesion, migration, invasion and matrix metalloproteinase (MMP) activities of MCF 7 human breast cancer cell line are reported. Cells were cultured with 10, 25, 50 µg/mL methanolic or water extract of L. bowringii. Culture medium containing 0.1% DMSO was used as a solvent control. Ultra structural analysis by electron microscopy revealed typical features of apoptosis. A remarkable dose-response parallelism was observed between methanolic extract with growth, migration and invasion of breast cancer cells. Fractionation of methanolic extract by RP-HPLC revealed a pool of phenolic acids. Hoechst 33342 staining assay reveals massive chromatin condensation and subsequent cleavage of structural components of nucleus. The results indicate that methanol extracts inhibit the growth of human breast cancer cells partially through the inhibition of metallo proteinases MMP-2 and MMP-9 activities. Methanolic extract has more anti-metastatic effects in cell based assay than water extract. Clinical application of L. bowringii extract as a bioactive chemopreventive compound may be helpful in limiting breast carcinoma invasion and metastasis.

Wound healing activity of methanolic and aqueous extracts of Plagiochila beddomei Steph. thallus in rat model.

Wound healing occurs as a fundamental response to tissue injury. Polyphenols are considered to be principle constituent to promote wound healing. Plagiochila beddomei was applied clinically by Paliyar tribes of Madurai district to treat intractable wounds. To validate the ethnotherapeutic claims of the plant in skin diseases, the effect of methanolic and aqueous extracts from P. beddomei on wound healing as well as angiogenesis was studied. 7th day and 10th day after excision wounds creation, the percent wound contraction of the extracts group was higher than that of Madecassol® (reference drug) group. On 3rd, 7th and 10th day after wounds creation, the wound healing quality of the extracts group was better than that of Madecassol® group on terms of granulation formation and collagen organization. On 3rd day after wounds creation, the micro vessel density and vascular endothelial growth factor expression of methanolic extracts group was higher than that of Madecassol® group. Phytochemical analysis of the extracts showed the presence of flavonoids, saponins, tannins and phenols. The results show that P. beddomei extract has potent wound healing property probably resulting from the remarkable angiogenic activity.

Antimicrobial activity of protease inhibitor from leaves of Coccinia grandis (L.) Voigt.

Antimicrobial activity of protease inhibitor isolated from Coccinia grandis (L.) Voigt. has been reported. A 14.3 kDa protease inhibitor (PI) was isolated and purified to homogeneity by ammonium sulfate precipitation (20-85% saturation), sephadex G-75, DEAE sepharose column and trypsin-sepharose affinity chromatography from the leaves of C. grandis. The purity was checked by reverse phase high performance liquid chromatography. PI exhibited marked growth inhibitory effects on colon cell lines in a dose-dependent manner. PI was thermostable and showed antimicrobial activity without hemolytic activity. PI strongly inhibited pathogenic microbial strains, including Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris, Eschershia coli, Bacillus subtilis and pathogenic fungus Candida albicans, Mucor indicus, Penicillium notatum, Aspergillus flavus and Cryptococcus neoformans. Examination by bright field microscopy showed inhibition of mycelial growth and sporulation. Morphologically, PI treated fungus showed a significant shrinkage of hyphal tips. Reduced PI completely lost its activity indicating that disulfide bridge is essential for its protease inhibitory and antifungal activity. Results reported in this study suggested that PI may be an excellent candidate for development of novel oral or other anti-infective agents.

Antioxidant modulation in response to heavy metal induced oxidative stress in Cladophora glomerata.

The present investigation was carried out to study the induction of oxidative stress subjected to heavy metal environment. Lipoperoxides showed positive correlation at heavy metal accumulation sites indicating the tissue damage resulting from the reactive oxygen species and resulted in unbalance to cellular redox status. The high activities of ascorbate peroxidase and superoxide dismutase probably counter balance this oxidative stress. Glutathione and soluble phenols decreased, whereas dehydroascorbate content increased in the algae from polluted sites. The results suggested that alga responded to heavy metals effectively by antioxidant compounds and scavenging enzymes.

Influence of host plant on growth and reproduction of Aphis nerii and feeding and prey utilization of its predator Menochilus sexmaculatus.

Influence of different physiological stages (young, mature and senescent) of Calotropis gigantea leaves on growth and reproductive ability of A. nerii and feeding, prey utilization, fecundity and lipid content of its predator M. sexmaculatus were investigated. Increased reproductive period, total life span and reproduction of nymphs per female of A. nerii were observed when reared on mature leaves. This relative preference of A. nerii and maximum utilization of mature leaves as compared to other physiological aged leaves are mainly due to changes in the chemical composition such as protein, carbohydrate, lipid, amino acid, nitrogen and phenolic of C. gigantea. Further, aphids reared on mature leaves influenced its predator's (M. sexmaculatus) growth, prey utilization and reproductive performances. Fecundity and longevity were high, while developmental time of predator was shorter on mature leaves fed aphid. Maximum prey utilization and increased efficiency of ingested and digested food of predator was observed on mature leaves reared aphid. The results are interpreted and discussed in relation to plant aphid and predator interaction (tritrophic).