DELONIX REGIA BOJ. EX. HOOK RAFFIN; FAMILY: FABACEAE, BARK METHANOL EXTRACT PHYTOCHEMICAL PROFILE AND POTENTIAL THERAPEUTIC EVALUATION

Objectives: The aim of this study was to evaluate the antioxidant, anti-inflammatory, and anti-cancer potencies of the Delonix regia bark, a first of its kind. Methods: The bark was extracted sequentially in Soxhlet apparatus with hexane, chloroform, and methanol in the increasing order of polarity. These extracts were subjected to find its antioxidant activity and total phenol content. Antibacterial activity against human pathogenic bacteria was tested. The anti-inflammatory properties were elucidated by its capacity to inhibit 15-lipoxygenase (LOX) and human cyclooxygenase (COX)-2. Cell cytotoxic capacity was evaluated against MCF-7 cells breast cancer cell lines. Results: Liquid chromatography (LC)-Mass Spectroscopy (MS) fingerprint of the methanol extract identified a total of 14 polyphenols, of which five were structurally characterized based on their mass-charge ratio [M-H]− peak, UV-vis absorption in comparison to published data. Antibacterial activity by disk diffusion inhibited human pathogenic bacteria. Bacterial biofilm inhibition capacity of extract (750 mg) imaged by confocal laser scanning microscopy revealed loss of microcolonies. Extract when tested for 15-LOX inhibition exhibited IC50 values of 94.5 ± 1.23 mg.mL−1 by enzyme kinetics studies using spectrophotometric techniques. Similarly, it could inhibit COX-2 enzyme at relatively lower concentrations (32.18 ± 1.91 mg.mL−1). Further, it quenched free radicals produced by Fentons’ reagent studied by DNS-nicking assay indicating its strong antioxidant property with the capacity to protect DNA. In vitro cytotoxicity was evaluated by 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphynyl tetrazolium bromide assay and apoptosis induced in MCF-7 cells was assessed morphologically. Conclusion: Our data suggest that D. regia bark methanol extract exerts its therapeutic activity for further pharmaceutical evaluations. Further studies are necessary to determine the mechanisms of these pharmacological properties

Caspase Activators: Phytochemicals with Apoptotic Properties Targeting Cancer, a Health Care Strategy to Combat this Disease

Context: Caspases, a family of cysteine-aspartic proteases have a pivotal role in apoptotic pathways. Their down-regulation is reported to induce inappropriate cell survival and enhanced carcinogenic potential. Screening of phytochemicals with a capacity to activate caspases enhancing apoptotic capacity has been proven to be effective anticancer agents. Objectives: This review consolidates data on phtochemicals traditionally used to treat cancerous conditions. The scientific validation of caspase-activated apoptosis for this tradition& application has been compiled. Methods: Internet assisted scientific literature was collected from Google, Google Scholar, Research Gate and NCI, restricted to publications from 1997 to 2019. Search terms `caspases and cancer', `assay of caspases', `traditionally used medicinal plants', `Kani tribes', `plant extracts activating caspase', `cytotoxicity assay', `docking phytochemicals to caspase', `technological advancement for anticancer therapy', `clinical studies of plant extracts and phytochemicals' and `herbal drugs approved by FDA' was included. Results: The compilation revealed significance of multiple experiment& strategies, traditional research laboratory practices and advanced in silico molecular docking techniques in anticancer therapy. Technological advancement such as MALDI-TOF assisted phytochemical mediated protein target identification and designing promoter for caspases activation and synthesizing functionalized nano carriers for clinic& studies has been included for identification of hit molecule and lead optimization. Eugenol and berberine were identified as phytochemicals with potential drug characteristics by both in silico and in vivo studies. Conclusion: The phytochemicals from important Kani tribal medicinal plants via in silico docking and in vivo studies identified could be explored at clinical trials

Characterization of a hydroxyproline-rich glycoprotein in pearl millet and its differential expression in response to the downy mildew pathogen Sclerospora graminicola

A monoclonal antibody, JIM 20, derived against an extensin type of hydroxyproline-rich glycoprotein (HRGP) from pea, showed high affinity for HRGP in pearl millet [Pennisetum glaucum (L.) R. Br.]. Electrophoretic separation of Tris-SDS extracted proteins from suspension cells of pearl millet revealed a range of PM-HRGP polypeptides having a glycan epitope, which reacted with JIM 20. A high molecular mass band, probably an HRGP aggregate or polymer, and a few low molecular mass polypeptides were recognized by JIM 20 during Western blot analysis. Treatment of pearl millet suspension cells with hydrogen peroxide in the presence of an endogenous peroxidase resulted in insolubilization of HRGP polypeptides with molecular weights between 45 and 33 kDa. To investigate the gene coding for an extensin type of HRGP, a fosmid-based genomic library of pearl millet having a fourfold genome coverage was constructed. A partial sequence of 378 bp of an HRGP gene was obtained by PCR amplification of pearl millet DNA with a primer pair designed from the conserved regions of monocotyledon extensin type of HRGPs. Screening the genomic library using the homologous probe developed from the 378-bp PCR product resulted in the isolation of five fosmid clones. Restriction mapping of these fosmids resulted in an 11.8-kb region around an HRGP gene in pearl millet. The newly characterized gene, PM-HRGP, had all the characteristic features of a monocotyledon extensin type of HRGP. An intron at the 3' untranslated region of the gene was identified by cDNA cloning. Differential expression of the PM-HRGP gene was observed during compatible and incompatible interactions of pearl millet with the downy mildew pathogen Sclerospora graminicola (Sacc) Schroet. Induced expression of the gene was observed only in case of an incompatible interaction.</p

Purification and characterization of proline/hydroxyproline-rich glycoprotein from pearl millet coleoptiles infected with downy mildew pathogen Sclerospora graminicola

Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall structural components, which are also involved in response to pathogen attack. In pearl millet, deposition and cross-linking of HRGPs in plant cell walls was shown to contribute to the formation of resistance barriers against the phytopathogenic oomycete Sclerospora graminicola. In the present study, the purification and characterization of HRGPs that accumulated in coleoptiles of pearl millet seedlings in response to S. graminicola inoculation has been carried out. Periodic acid Schiff’s staining revealed that the purified protein was a glycoprotein. The protein to carbohydrate ratio was determined to be 95.5:4.5 (w/w). Proline amounted for 20mol of the total amino acids as indicated by amino acid composition analysis. The isolated protein had a pI of 9.8 and was shown to be composed of subunits of 27, 17, and 14kDa. Cross reactivity with the monoclonal antibody MAC 265 and the presence of the signature amino acid sequence, PVYK, strongly suggested to classify the purified glycoprotein as a member of the P/HRGPs class. In the presence of horseradish peroxidase and H2O2 the purified glycoprotein served as a substrate for oxidative cross-linking processes

Role of hydroxyproline-rich glycoproteins in resistance of pearl millet against downy mildew pathogen Sclerospora graminicola

Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall components involved in plant defense response to pathogen attack. In the present study, a resistant pearl millet (Pennisetum glaucum) cultivar, IP18292, was compared with a susceptible cultivar, 7042S, to investigate the contribution of HRGPs in the successful defense against the phytopathogenic oomycete S. graminicola. Northern hybridization using MeHRGP cDNA, a heterologous probe from cassava, indicated steady accumulation of HRGP transcripts, from 2 h.p.i. onwards with a maximum at 6 h.p.i., in the resistant cultivar. This is followed by HRGPs accumulation at about 8 h.p.i. as revealed by Western-blot analysis. Immunocytochemical localization by tissue printing and confocal immunofluorescence microscopy indicated cell walls of parenchymatic cells and the vascular tissue of coleoptile as sites of HRGP deposition. In vitro studies in the presence of horseradish peroxidase and H2O2 showed cross-linking of pearl millet HRGPs, which occurred parallel to isodityrosine accumulation. Inducible high isodityrosine content was also observed in vivo in the resistant cultivar. Here, H2O2 was found to accumulate as twin burst at 1 and 6 h.p.i., whereas in the susceptible cultivar only an early single peak was detectable. Moreover, the amount of hydroxyproline in HRGPs was about twice as high in the resistant as in the susceptible cultivar. These results suggest that cell wall strengthening in S. graminicola-infected resistant pearl millet is brought about by a combination of polypeptide cross-linking of isodityrosine as well as by the high content of hydroxyproline in HRGPs, and H2O2, in contrast to the susceptible plant

Serodiagnosis of pearl millet resistance to downy mildew by quantitating cell wall P/HRGP using polyclonal antiserum Pab-P/HRGP

Proline/hydroxyproline-rich glycoprotein (P/HRGP) level in pearl millet genotypes resistant to downy mildew increase after inoculation with the oomycete pathogen Sclerospora graminicola. Using purified P/HRGPs from pearl millet cell walls, polyclonal antibodies (Pab-P/HRGP) were raised in rabbit. Based on this antiserum, an enzyme immunoassay was developed that displays a linearity detection range from 0.01 to 10 μg P/HRGP. Western blot analysis, confirming the induction of three marker P/HRGPs in the infected resistant genotype, and immunocytochemical studies on P/HRGP localization either in epidermal peelings or in suspension-cultured cells demonstrated the specificity of the antiserum. Besides its characterization, Pab-P/HRGP was employed to screen various genotypes of pearl millet for fast, sensitive and specific detection of induced P/HRGPs upon infections. The results presented are discussed with presumed importance to downy mildew disease and the use of this new antiserum in pearl millet screening for disease resistance

Hydroxyproline-rich glycoproteins accumulate in pearl millet after seed treatment with elicitors of defense responses against Sclerospora graminicola

The accumulation of hydroxyproline-rich glycoproteins (HRGPs) was investigated after induction of resistance in pearl millet against downy mildew caused by Sclerospora graminicola. Treatment of susceptible pearl millet seeds with various biotic and abiotic elicitors resulted in increased HRGP content in the cell walls of coleoptiles at 9 h after inoculation. Similar results with increased accumulation at 4–6 h after inoculation were obtained in suspension cells of pearl millet. Maximum HRGP accumulation was observed in seedlings raised from susceptible seeds treated with chitosan and Pseudomonas fluorescens. Western blot analysis with MAC 265 (a rat monoclonal antibody raised against pea HRGP) identified three proteins of 27, 17 and 14 kDa in resistant cultivars. The absence of the 14 kDa HRGP was observed in susceptible cultivars as reported earlier. The induced accumulation of the 14 kDa HRGP upon elicitor treatments was observed in the present study. Peroxidase and hydrogen peroxide, essential components for HRGP cross-linking, were also increased in samples treated with these elicitors. A tissue specific increase in HRGP at the regions around vascular bundles was observed upon chitosan treatment. The results presented will have a presumed importance in identifying the susceptible pearl millet varieties and improving those using elicitors of defense for field applications.

Characterization of a hydroxyproline-rich glycoprotein in pearl millet and its differential expression in response to the downy mildew pathogen Sclerospora graminicola

A monoclonal antibody, JIM 20, derived against an extensin type of hydroxyproline-rich glycoprotein (HRGP) from pea, showed high affinity for HRGP in pearl millet [Pennisetum glaucum (L.) R. Br.]. Electrophoretic separation of Tris–SDS extracted proteins from suspension cells of pearl millet revealed a range of PM-HRGP polypeptides having a glycan epitope, which reacted with JIM 20. A high molecular mass band, probably an HRGP aggregate or polymer, and a few low molecular mass polypeptides were recognized by JIM 20 during Western blot analysis. Treatment of pearl millet suspension cells with hydrogen peroxide in the presence of an endogenous peroxidase resulted in insolubilization of HRGP polypeptides with molecular weights between 45 and 33 kDa. To investigate the gene coding for an extensin type of HRGP, a fosmid-based genomic library of pearl millet having a fourfold genome coverage was constructed. A partial sequence of 378 bp of an HRGP gene was obtained by PCR amplification of pearl millet DNA with a primer pair designed from the conserved regions of monocotyledon extensin type of HRGPs. Screening the genomic library using the homologous probe developed from the 378-bp PCR product resulted in the isolation of five fosmid clones. Restriction mapping of these fosmids resulted in an 11.8-kb region around an HRGP gene in pearl millet. The newly characterized gene, PM-HRGP, had all the characteristic features of a monocotyledon extensin type of HRGP. An intron at the 30 untranslated region of the gene was identified by cDNA cloning. Differential expression of the PM-HRGP gene was observed during compatible and incompatible interactions of pearl millet with the downy mildew pathogen Sclerospora graminicola (Sacc) Schroet. Induced expression of the gene was observed only in case of an incompatible interaction.