DNA barcoding and NMR spectroscopy-based assessment of species adulteration in the raw herbal trade of Saraca asoca (Roxb.) Willd, an important medicinal plant.

Saraca asoca (Roxb.) Willd, commonly known as "Asoka" or "Ashoka," is one of the most important medicinal plants used in raw herbal trade in India. The bark extracts of the tree are used in the treatment of leucorrhea and other uterine disorders besides also having anti-inflammatory, anti-bacterial, anti-pyretic, anti-helminthic, and analgesic activity. The indiscriminate and rampant extraction of the wood to meet the ever-increasing market demand has led to a sharp decline in naturally occurring populations of the species in the country. Consequently, the species has recently been classified as "vulnerable" by the International Union for Conservation of Nature (IUCN). Increasing deforestation and increasing demand for this medicinal plant have resulted in a limited supply and suspected widespread adulteration of the species in the raw herbal trade market. Adulteration is a serious concern due to: (i) reduction in the efficacy of this traditional medicine, (ii) considerable health risk to consumers, and (iii) fraudulent product substitution that impacts the economy for the Natural Health Product (NHP) Industry and consumers. In this paper, we provide the first attempt to assess the extent of adulteration in the raw herbal trade of S. asoca using DNA barcoding validated by NMR spectroscopic techniques. Analyzing market samples drawn from 25 shops, mostly from peninsular India, we show that more than 80 % of the samples were spurious, representing plant material from at least 7 different families. This is the first comprehensive and large-scale study to demonstrate the widespread adulteration of market samples of S. asoca in India. These results pose grave implications for the use of raw herbal drugs, such as that of S. asoca, on consumer health and safety. Based on these findings, we argue for a strong and robust regulatory framework to be put in place, which would ensure the quality of raw herbal trade products and reassure consumer confidence in indigenous medicinal systems. Graphical Abstract DNA barcoding and NMR spectroscopy-based assessment of adulteration in Saraca asoca.

Identification of Biomarkers for Resistance to Fusarium oxysporum f. sp. cubense Infection and in Silico Studies in Musa paradisiaca Cultivar Puttabale through Proteomic Approach.

Panama wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the major disease constraints of banana production. Previously, we reported the disease resistance Musa paradisiaca cv. puttabale clones developed from Ethylmethanesulfonate and Foc culture filtrate against Foc inoculation. Here, the same resistant clones and susceptible clones were used for the study of protein accumulation against Foc inoculation by two-dimensional gel electrophoresis (2-DE), their expression pattern and an in silico approach. The present investigation revealed mass-spectrometry identified 16 proteins that were over accumulated and 5 proteins that were under accumulated as compared to the control. The polyphosphoinositide binding protein ssh2p (PBPssh2p) and Indoleacetic acid-induced-like (IAA) protein showed significant up-regulation and down-regulation. The docking of the pathogenesis-related protein (PR) with the fungal protein endopolygalacturonase (PG) exemplify the three ionic interactions and seven hydrophobic residues that tends to good interaction at the active site of PG with free energy of assembly dissociation (1.5 kcal/mol). The protein-ligand docking of the Peptide methionine sulfoxide reductase chloroplastic-like protein (PMSRc) with the ligand ß-1,3 glucan showed minimum binding energy (-6.48 kcal/mol) and docking energy (-8.2 kcal/mol) with an interaction of nine amino-acid residues. These explorations accelerate the research in designing the host pathogen interaction studies for the better management of diseases.

Wound healing activity of Delonix elata stem bark extract and its isolated constituent quercetin-3-rhamnopyranosyl-(1-6) glucopyranoside in rats.

Delonix elata L. is a Ceasalpinaceae species and is traditionally used in India for treatment of skin diseases, liver diseases and rheumatic problems. However, systematic evaluation of its wound healing activity is lacking. Thus, in the present study, we aimed to assess the wound healing activity of D. elata stem bark extract (DSE) and its isolated constituent quercetin-3-rhamnopyranosyl-(1-6) glucopyranoside (QRPG) in rats. The formulations effects on wound healing were assessed by the wound contraction rate, epithelialization period, tensile strength, content of the hydroxyproline, hexosamine and uronic acid in granulation tissue, histopathological studies and Col 1 α (I) expression level in wound tissue by reverse transcription polymerase chain reaction (RT-PCR) study. The topical application of DSE ointment caused faster epithelialization, significant wound contraction (100%), and better tensile strength (710.5±10.5 g/cm2), while QRPG showed wound epithelialization with 98.2% contraction, better than that of the control group (78.18%). The biochemical analysis of granulation tissue revealed that DSE and QRPG significantly increased hydroxyproline, hexosamine and uronic acid content. A significant increase in the expression of Col 1 α (I) was observed in the wound tissue of DSE and QRPG treated rats. DSE and QRPG were shown to enhance wound healing by increasing collagen synthesis through up-regulation of Col 1 α (I), thus validating ethnomedicinal uses.

Wound healing activity of Delonix elata stem bark extract and its isolated constituent quercetin-3-rhamnopyranosyl-(1-6) glucopyranoside in rats☆ / 药物分析学报

Delonix elata L. is a Ceasalpinaceae species and is traditionally used in India for treatment of skin diseases, liver diseases and rheumatic problems. However, systematic evaluation of its wound healing activity is lacking. Thus, in the present study, we aimed to assess the wound healing activity of D. elata stem bark extract (DSE) and its isolated constituent quercetin-3-rhamnopyranosyl-(1-6) glucopyranoside (QRPG) in rats. The formulations effects on wound healing were assessed by the wound contraction rate, epithelialization period, tensile strength, content of the hydroxyproline, hexosamine and uronic acid in granulation tissue, histopathological studies and Col 1α(I) expression level in wound tissue by reverse transcription polymerase chain reaction (RT-PCR) study. The topical application of DSE ointment caused faster epithelialization, significant wound contraction (100%), and better tensile strength (710.5 ± 10.5 g/cm2), while QRPG showed wound epithelialization with 98.2%contraction, better than that of the control group (78.18%). The biochemical analysis of granulation tissue revealed that DSE and QRPG significantly increased hydroxyproline, hexosamine and uronic acid content. A significant increase in the expression of Col 1α(I) was observed in the wound tissue of DSE and QRPG treated rats. DSE and QRPG were shown to enhance wound healing by increasing collagen synthesis through up-regulation of Col 1α(I), thus validating ethnomedicinal uses.

Antioxidant and prophylactic effects of Delonix elata L., stem bark extracts, and flavonoid isolated quercetin against carbon tetrachloride-induced hepatotoxicity in rats.

Delonix elata L. (Ceasalpinaceae), is widely used by the traditional medical practitioners of Karnataka, India, to cure jaundice, and bronchial and rheumatic problems. The objective of this study was to screen the in vitro antioxidant and hepatoprotective activity of the stem bark extracts against CCl4-induced liver damage in rats. Among different stem bark extracts tested, the ethanol extract (DSE) has shown significant in vitro antioxidant property in radicals scavenging, metal chelating, and lipid peroxidation inhibition assays. HPLC analysis of the DSE revealed the presence of known antioxidant molecules, namely, gallic acid, ellagic acid, coumaric acid, quercetin, and rutin. Bioassay-guided fractionation of DSE has resulted in the isolation and characterization of quercetin. DSE and quercetin have shown significant prophylactic effects by restoring the liver function markers (AST, ALT, ALP, serum bilirubin, and total protein) and antioxidant enzymes (SOD, CAT, GPx, and GST). These results were proved to be hepatoprotective at par with silymarin and well supported by the histological observations of liver sections with distinct hepatic cells, and mild degree of fatty change and necrosis. The results indicated that the DSE and quercetin were significant for prophylactic activity against CCl4-induced liver damage in rats. This activity could be attributed to the antioxidant constituents in the DSE and hence justified the ethnomedicinal claims.

A new signal sequence for recombinant protein secretion in Pichia pastoris.

Pichia pastoris is one of the most widely used expression systems for the secretory expression of recombinant proteins. The secretory expression in P. pastoris usually makes use of the prepro MATα sequence from Saccharomyces cerevisiae, which has a dibasic amino acid cleavage site at the end of the signal sequence. This is efficiently processed by Kex2 protease, resulting in the secretion of high levels of proteins to the medium. However, the proteins that are having the internal accessible dibasic amino acids such as KR and RR in the coding region cannot be expressed using this signal sequence, as the protein will be fragmented. We have identified a new signal sequence of 18 amino acids from a P. pastoris protein that can secrete proteins to the medium efficiently. The PMT1-gene-inactivated P. pastoris strain secretes a ~30 kDa protein into the extracellular medium. We have identified this protein by determining its N-terminal amino acid sequence. The protein secreted has four DDDK concatameric internal repeats. This protein was not secreted in the wild-type P. pastoris under normal culture conditions. We show that the 18-amino-acid signal peptide at the N-terminal of this protein is useful for secretion of heterologous proteins in Pichia.

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris.

Protein mannosyltransferases (PMTs) catalyze the O-mannosylation of serine and threonine residues of proteins in the endoplasmic reticulum. The five PMT genes coding for protein mannosyltransferases, designated as PMT1, 2, 4, 5 and 6, were identified from Pichia pastoris genome based on the homology to PMT genes in Saccharomyces cerevisiae genome, which has seven PMT genes. The homologues of S. cerevisiae PMT 3 &7 genes are absent in P. pastoris genome. Approximately 5% of the recombinant insulin precursor expressed in P. pastoris is O-mannosylated. In this study, we attempted to prevent O-mannosylation of insulin precursor in vivo, through inactivation of the Pichia PMT genes. Since multiple PMTs are found to be expressed, it was important to understand which of these are involved in O-mannosylation of the insulin precursor. The genes encoding PMT1, 4, 5 and 6 were knocked out by insertional inactivation method. Inactivation of PMT genes 4, 5 and 6 showed ∼16-28% reductions in the O-mannosylation of insulin precursor. The PMT1 gene disrupted Pichia clone showed ∼60% decrease in O-mannosylated insulin precursor, establishing its role as an important enzyme for insulin precursor O-mannosylation.