PRELIMINARY SCREENING OF PHYTOCHEMICALS AND ANTIPROLIFERATIVE AND ANTI-INFLAMMATORY PROPERTIES OF THESPESIA POPULNEA (L.) SOLAND LEAF EXTRACTS

Objective: Plant-derived bioactive molecules are providing infinite opportunities for new drug development as they possess a wide range of actions against diseases with lesser side effects. The present study is made to analyze the qualitative phytochemicals and to evaluate in vitro antiproliferative and anti-inflammatory potentials of leaf extracts of Thespesia populnea. Methods: T. populnea leaf extracts were prepared sequentially according to the increasing polarity of the solvents, i.e., petroleum ether, chloroform, ethyl acetate, and methanol. Qualitative phytochemical analysis was performed to identify the chemical constituents of the extracts, and antiproliferative properties were evaluated against different cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay. Bovine serum albumin anti-denaturation assay was done to identify the anti-inflammatory activity.Results: Phytochemical analysis of the extracts revealed the presence of alkaloids, terpenoids, glycosides, tannins, flavonoids, and phenolics. The chloroform extract (CHFE) of T. populnea has a dose-dependent antiproliferative effect against acute T-cell leukemia (Jurkat E6-1), prostate Grade IV adenocarcinoma (PC-3), mouse fibroblast (L-929), and monkey kidney normal (Vero) cells. Their inhibitory concentration 50% (IC50) values were found to be 35.73±0.94 μg/ml for Jurkat E6-1, 60.79±1.84 μg/ml for PC-3, 60.88±1.45 μg/ml for L-929, and 83.482±2.05 μg/ml for Vero, respectively. CHFE also displayed the anti-inflammatory potential.Conclusion: The chloroform leaf extract of T. populnea possesses potent antiproliferative and anti-inflammatory activity. These properties present in leaf extract may be explained by the presence of biologically active constituents

Genetic Diversity Assessment of Rarely Cultivated Traditional Indica Rice (Oryza sativa L.) Varieties

Random amplified polymorphic DNA fingerprinting was performed to assess the genetic diversity among rarely cultivated traditional indica rice (Oryza sativa L.) varieties collected from a tribal hamlet of Kerala State, India. A total of 664 DNA bands amplified by 15 primers exhibited 72.9% polymorphism (an average of 32.3 polymorphic bands per primer). The varieties Jeerakasala and Kalladiyaran exhibited the highest percent (50.19%) polymorphism, while Thondi and Adukkan showed the lowest (9.85%). Adukkan (78 bands) and Jeerakasala (56 bands) yielded the highest and the lowest number of amplicons, respectively. Unweighted Pair Group Method with Arithmetic mean analysis using the Dice similarity coefficient showed the highest value of similarity coefficient between the varieties Adukkan and Thondi, both shared higher level of similarity (0.81), followed by Kanali and Thondi (0.88). Of the three subclusters, the varieties of Adukkan, Thondi, Kanali, Mannuveliyan, Thonnuranthondi, and Chennellu grouped together with a similarity of 0.77. The second group represented by Navara, Gandhakasala, and Jeerakasala with a similarity coefficient of 0.76 formed a cohesive group. The variety Kalladiyaran formed an isolated position that joined the second cluster. The Principal Coordinate Analysis also showed separation of Kalladiyaran from the other varieties

Induction of TLR-2 and TLR-5 Expression by Helicobacter pylori Switches cagPAI-Dependent Signalling Leading to the Secretion of IL-8 and TNF-α

Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI) encoding a type-IV secretion system (T4SS) for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs) and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5) during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-α cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-α cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IκB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-κB. Finally, NF-κB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression of TLR-2 and TLR-5 can qualitatively shift cagPAI-dependent to cagPAI-independent pro-inflammatory signalling pathways with possible impact on the outcome of H. pylori-associated diseases

Rapid <i>in vitro </i>propagation of the threatened endemic orchid, <i>Ipsea malabarica </i>(Reichb.f.) J D Hook through protocorm-like bodies

829-834Rapid propagation of I. malabarica (Reichb. f.) J D Hook, an endemic and endangered orchid of the Western Ghats of Kerala. India through conversion of axillary buds to protocorm-like bodies (PLBs), and subsequent plant regeneration was achieved. Growth regulators and sugar displayed significant influence in the induction of PLBs. In vitro derived shoots from field grown rhizomes of Ipsea cultured on Murashige and Skoog (MS) medium with 13.3μM N6-benzyladenine (BA) containing 2% commercial grade sugar turned the axillary buds to PLBs within 25 days, and developed a mean of 33.1 PLBs within 50 days. Kinetin (KIN) did not induce PLBs, but facilitated axillary bud proliferation. Transfer of PLBs on medium having same concentration of BA and sugar facilitated rapid multiplication, and developed a mean of 47.5 PLBs. No decline of PLB proliferation was observed up to 10th subculture. Half strength MS medium with 6.97μM KIN facilitated conversion of 98% PLBs to plantlets. On this media, a mean of 5.8 roots were also developed per shoot. Shoots developed bulbs during culture were grown to rhizomes. Increase of sugar to 6 or 8% hastened the development of bulbs/rhizomes. Reintroduction of PLB-derived plantlets in the natural habitat i.e. at Vellarimala (at 1300 m height) of the Western Ghats of Kerala was attempted as a means to assist in situ conservation. This is the first report of conversion of axillary buds to PLBs. The protocol enables to surmount the threat of extinction of this endemic and endangered orchid

<i>In vitro </i>propagation of <i>Dendrobium </i>hybrids using flower stalk node explants

280-285Large-scale in vitro propagation protocol for Dendrobium hybrids Sonia 17 and 28, two highly prized commercial cut flower cultivars through shoot multiplication using flower stalk node explants and protocorm-like bodies (PLBs) formation was accomplished. Both hybrids did not exhibit significant differences in initiation, multiplication, rooting, and field establishment. Flower stalk nodes cultured on half strength Murashige and Skoog (MS) medium supplemented with 6.97 μM kinetin (Kn), or 15% coconut water (CW) or 13.3 μM of N6-benzyladenine (BA) evoked bud break. Kn showed better growth of the initiated bud. Excision and culture of the initiated shoots on medium having same amount of Kn developed more than 5 shoots per shoot directly from the base. Subsequent culture enhanced the rate of shoot induction. Transfer of isolated shoots onto 44.4μM of BA enriched medium displayed induction of more than 6 PLBs from the base within 60 days. PLBs underwent rapid multiplication upon transferral to medium having the same concentration of BA (44.4 μM). Subsequent culture increased the proliferation of PLBs. No decline was observed in the proliferation of shoots as well as PLBs up to 15th subculture. PLBs transferred onto half strength MS medium with 6.97 μM of Kn underwent conversion of more than 90% PLBs to shoots. The shoots were rooted at the best on half strength MS medium with 2 g 1-1 activated charcoal. Survival rate of the plantlets of the two hybrid cultivars after acclimatization was more than 80%

<i>In vitro </i>propagation of three commercial cut flower cultivars of <i><span style="font-size:14.0pt;line-height:115%;font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";color:#414141;mso-ansi-language:EN-IN; mso-fareast-language:EN-IN;mso-bidi-language:HI" lang="EN-IN">Anthurium andraeanum </span></i><span style="font-size:14.0pt;line-height:115%;font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";color:#2C2C2C;mso-ansi-language:EN-IN; mso-fareast-language:EN-IN;mso-bidi-language:HI" lang="EN-IN">Hort.</span>

154-159In vitro propagation of Anthurium andraeanum Hort. cut flower cultivars viz. Lima White, Tropical White and Tropical Red through organogenesis using mature plant derived leaf explants was established on Murashige and Skoog (MS) medium fortiried with different growth regulators. Cultivar, stage and different regions of the source leaf, and type of growth regulators significantly influenced callus induction. Explants from folded brown leaves were superior in induction of<span style="font-size:14.0pt;font-family:HiddenHorzOCR;mso-hansi-font-family: " times="" new="" roman";mso-bidi-font-family:hiddenhorzocr;color:black"="" lang="EN-IN"> callus. Half strength MS medium fortified with 0.88 μM of benzyladenine (BA), 0.9μM of 2,4-dichlorophenoxyacetic acid (2,4- D) and 0.46μM of kinetin (Kn) at pH 5.5 was most effective for callus induction. Transfer of callus to medium with 0.54 μM of NAA in place of 2,4-D induced higher number of shoots. Subsequent cultures displayed enhanced rate of shoot initiation and multiplication. Transfer of shoots onto half strength MS medium supplemented with 0.54 μM of NAA favoured rooting of shoots. Cultivar Tropical White was superior in callus, shoot and root induction compared to Lima White and Tropical Red. Plantlets after acclimation in greenhouse were transferred to net-house, that exhibited ninety seven percent surviva1. Plants flowered normally between 12 and15 months and were morphologically similar to that of the mother plants.</span

Tetracycline and Glutathione Inhibit Matrix Metalloproteinase Activity: An In Vitro Study Using Culture Supernatants of L929 and Dalton Lymphoma Cell Lines

Tetracycline and glutathione inhibited the protease activities of matrix metalloproteinase-2 and matrix metalloproteinase-9 expressed by mouse fibrosarcoma cells (L929) and Dalton lymphoma cells, respectively. The inhibitory activity of the tetracycline may be due to its ability to chelate metal ions such as calcium and zinc. Gelatin-zymography technique was used to demonstrate the inhibitory activity of both tetracycline and glutathione. The intensity of the bands corresponding to metalloproteinase activity in zymography gel was reduced in the presence of 50–100 μg/mL of tetracycline. The presence of 10–100 μg/mL of tetracycline in the medium increased the adherence of L929 cancer cells. These results clearly indicate the antimetastatic property of tetracycline. Reduced glutathione, a compound which is produced endogenously by the cells to maintain the redox status, was shown to inhibit the matrix metalloproteinase activity (in vitro). Therefore, it is assumed that decreased glutathione levels in synovial fluids or plasma might increase the activity of MMP. Reduced glutathione at 100 μg/mL inhibited the metalloproteinase activity in gelatin-zymographic gel. As both tetracycline and glutathione exhibited an inhibitory effect on matrix metalloproteinase activity, it was of great interest to check their clinical effects on various MMP associated pathological conditions such as cancer metastasis and arthritis. Here we report that tetracycline and reduced glutathione inhibited the activity of MMP2 completely and activity of MMP9 partly