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

Therapeutic targets of medicinal plants of Western Ghats, India

Drugs derived from natural resources represent a significant segment of the pharmaceutical market. Traditional systems of medicines like Ayurveda, Traditional Chinese Medicine (TCM) or the European phytotherapy assumed synergistic approach wherein all ingredients of the plants could bring about maximum therapeutic efficacy. Due to the escalated cost of development of pre-clinical synthetic drugs, there has been a Worldwide demand for phytopharmaceuticals. The modern therapy applies more and more combinatorial approach in treatment of diseases like cancer, cardiovascular or rheumatic diseases. High-throughput methods are available to investigate complex mixtures of whole plant extracts and establish synergistic effects for multi-therapeutic target treatments. Demands are increasing to establish open access data banks in a multidisciplinary effort for medicinal plant research with the aim to develop a common standard molecular/therapeutic "bar codes" for characterization of medicinal plants. In the present chapter an attempt has been made to compile information on medicinal plant extracts with respect to their pharmacological attributes to promote wider acceptance and use of these plant-based drugs in main stream of medicine. The mode of action of phytopharmaceuticals as assessed by up/down-regulation of therapeutic target molecules with respect to anti-cancer, anti-inflammatory, anti-hypertensive, anti-viral, anti-obesity, antidiabetic and pro-gynecological properties complying with multi-target therapy will be described. This approach further helps scientific validation of plant extracts/bioactive molecules that are already being used in traditional medicine

β-Amino butyric acid-induced resistance in pearl millet to downy mildew is associated with accumulation of defence-related proteins

Earlier studies had indicated that β-amino butyric acid (BABA) treatment of pearl millet seeds influenced seedling vigor and protected seedlings from downy mildew disease caused by the oomycetous biotrophic fungus Sclerospora graminicola. Application of 50mMBABAreduced disease severity and offered protection against S. graminicola of ∼74%. The protection induced was durable and operative during the vegetative and reproductive growth periods of the crop. In the present study an attempt was made to understand the biochemical basis for this protection. A close association was found between BABA-induced protection and increased accumulation of defence-related proteins like phenylalanine ammonia lyase, peroxidase, β-1,3-glucanase and cell wall hydroxyproline-rich glycoproteins (HRGP). Isoelectric focusing of β-1,3-glucanases indicated the presence of several isoforms, of which isoforms of pI 9.6 and 8.8 were markedly increased in BABA-treated seedlings upon S. graminicola infection. Increased accumulation of hydroxyproline-rich glycoproteins was also observed at 9 h after inoculation in these samples. Western blots with MAC 265, a monoclonal antibody against pea HRGP identified a 17-kDa HRGP molecule that significantly increased in concentration in BABA-treated pearl millet seedlings as a response to S. graminicola infection. The postinfectional protection offered by BABA involved induction of defence responses which were comparable to the highly resistant cultivar

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

Cloning, expression and purification of resistance gene analogue RGPM 301 from pearl millet in Escherichia coli

Plants combat their pathogens with an array of defense responses. One of the key mechanisms involves products of resistance (R) genes which are responsible for recognition of effector molecules from pathogens and subsequent triggering of defense responses. Resistance gene analogues (RGAs) containing the specific conserved domains of R-genes are isolated from various plants using degenerate oligonucleotide primer based PCR approach. In an earlier study, RGPM 301 an RGA from pearl millet shown to be involved in resistance mechanism against downy mildew disease was isolated and characterized. In the present study, RGPM 301 containing an open reading frame (ORF) of 992 amino acids was cloned into pRSET A expression vector and expressed in Escherichia coli as a Hig-tag fusion protein. The recombinant RGA RGPM 301 was purified to near homogeneity using the Nickel-CL agarose column. Its molecular mass was found to be 120 kDa when separated on the SDS-PAGE which was confirmed by western blotting analysis using the anti-His antibody. The purified protein was subjected to in-gel trypsin digestion followed by mass spectrometric analysis for the confirmation of its identity. These findings facilitate further studies on the exact role of this RGA in the pearl millet downy mildew host pathogen system

Cytotoxic effect of p-coumaric acid on neuroblastoma, n2a cell via generation of reactive oxygen species leading to dysfunction of mitochondria inducing apoptosis and autophagy

p-Coumaric acid (p-CA), an ubiquitous plant phenolic acid, has been proven to render protection against pathological conditions. In the present study, p-CA was evaluated for its capacity to induce cytotoxic effect to neuroblastoma N2a cells and we report here the possible mechanism of its action. p-CA at a concentration of 150 mu mol/L, upon exposure for 72 h, stimulated 81.23 % of cells to apoptosis, as evidenced by flow cytometer studies mediated through elevated levels of ROS (7.5-fold over control). Excess ROS production activated structural injury to mitochondrial membrane, observed as dissipation of its membrane potential and followed by the release of cytochrome c (8.73-fold). Enhanced generation of intracellular ROS correlated well with the decreased levels (similar to 60 %) of intracellular GSH. Sensitizing neuroblastoma cells for induction of apoptosis by p-CA identified p53-mediated upregulated accumulation of caspase-8 messenger RNA (2.8-fold). Our data report on autophagy, representing an additional mechanism of p-CA to induce growth arrest, detected by immunoblotting and fluorescence, correlated with accumulation of elevated levels (1.2-fold) of the LC3-II protein and acridine orange-stained autophagosomes, both autophagy markers. The present study indicates p-CA was effective in production of ROS-dependent mitochondrial damage-induced cytotoxicity in N2a cells