Production of coloured callus in Orthosiphon stamineus Benth and antioxidant properties of the extracted pigments

Purpose The purpose of the present study is to understand the role of auxin and cytokinin in stimulating the production of pigmented callus in Orthosiphon stamineus and to gain correlation between the callus colours with their antioxidant capacity and bioactive constituents. Design/methodology/approach In this study, plant tissue culture was used to induce production of callus of various colours from leaf explants of O. stamineus, via manipulation of plant hormones (0-2.0 mg L−1 indole-3-acetic acid [IAA] and Kinetin [Kin]). The coloured callus was subjected to solvent extraction and used for quantification of its carotenoid, chlorophyll, anthocyanin and phenolic contents. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of the extracts was also evaluated, before and after four weeks of storage at −20°C. Findings The highest mean (per cent) explants that produced roots (93.33 ± 0.05 per cent) were observed when the cultures were supplemented with 2.0 mg L−1 IAA. The colour of the callus changed with time, from green to cream to brown after two and four months of culture, respectively. Optimum production of green callus was achieved with addition of 2.0 mg L−1 Kin plus 1.0-2.0 mg L−1 IAA to the media, while cream callus in 0.5 mg L−1 Kin plus 2.0 mg L−1 IAA and brown callus in 0.5 mg L−1 Kin plus 1.5 mg L−1 IAA. Green callus was found to contain the highest amount of chlorophylls, carotenoid and anthocyanin, while cream callus contained the highest amount of phenolic compounds. The amount of pigments and secondary metabolites in the callus extracts decreased after four weeks of storage, except anthocyanin. The antioxidant potential of the extracts also increased after storage. Research limitations/implications The major compounds identified in the methanolic extracts of O. stamineus-coloured callus are chlorophylls, carotenoids, flavonoids and phenolic acids. Future research work should include improvements in the extraction and identification methods which may lead to detection of other compounds that could attribute to the antioxidant capacity, to complement the findings of the current study. Practical implications This analysis provides valuable information on the application of IAA and Kinetin (Kin) to manipulate the content of major pigments with medicinal benefits in O. stamineus by using the plant tissue culture system. Originality/value A comparative study on antioxidant capacity and bioactive constituents of pigmented callus from O. stamineus leaves is original. To the best of the authors’ knowledge, this is the first attempt of comparative evaluation on antioxidant potential of O. stamineus-coloured callus produced using IAA and Kin

Analysis of bioactive pigments in coloured callus of Azadirachta indica for possible use as functional natural colourants

Purpose – The purpose of this study is to evaluate the content of bioactive pigments in coloured callus of Azadirachta indica and to understand the correlation between the callus colours with their bioactive constituents, antioxidant properties and cytotoxicity. These assessments will yield valuable insight into the use of in vitro-derived pigments for possible use as functional natural colourants. Design/methodology/approach – In this study, the authors have successfully developed a protocol to produce leaf-derived callus of various colours with enhanced content of bioactive pigments in A. indica through plant tissue culture. Comparative analysis of the pigments content (chlorophyll, carotenoid, phenolics and anthocyanins) in the coloured callus was conducted, followed by evaluation of its bioactive properties. The antioxidant properties against 2,2-diphenyl-1-picrylhydrazyl and 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radicals, ferric reducing antioxidant power and cytotox activities of the coloured callus extracts were also reported. Findings – Callus of various colours were successfully produced in A. indica through plant tissue culture, and their valuable pigment content and bioactivity were evaluated. The green callus contained the highest amount of anthocyanin, followed by brown and cream callus. The total anthocyanin contents in both the green and brown callus was more than two-fold higher than that in cream callus. Contrasting observation was obtained for total phenolic content (TPC), where the TPC of cream callus was significantly higher than that in brown callus. Nevertheless, the green callus also exhibited the highest TPC. Green callus also contained the highest amount of total chlorophyll and carotenoid, as well as exhibited the highest antioxidant potential, and was found to be the only extract with active cytotox activity against SKOV-3 cells. Correlation analysis revealed that the excellent bioactivity exhibited by the coloured extracts was strongly correlated with the bioactive pigments present in the callus. Research limitations/implications – The major bioactive compounds identified in the methanolic extracts of A. indica coloured callus are anthocyanins, phenolics, chlorophylls and carotenoids. Future research work should include improvements in the extraction and identification methods, which may lead to detection and determination of other compounds that could attribute to its bioactivity, to complement the findings of the current study. Practical implications – This analysis provides valuable information on the application of plant tissue culture as an alternative source for sustainable production of major pigments with medicinal benefits in A. indica for possible use as functional natural colourants. Originality/value – A comparative study on bioactive pigment production in coloured callus from A. indica leaves and its antioxidant potential and cytotoxicity is original. To the best of the authors’ knowledge, this is the first report detailing a comparative evaluation on the production of coloured callus in A. indica and its relative biochemical composition and bioactive properties

Molecular docking studies of selected medicinal drugs as dengue virus-2 protease inhibitors

Dengue is a potentially deadly disease with no effective drug. An in silico molecular docking was performed using Autodock 4.2.6 to investigate the molecular interactions between protease inhibitors, comprising antibiotic derivatives namely doxycycline (3), rolitetracycline (5) and a non-steroidal anti-inflammatory drug (NSAID), meclofenamic acid (4), against the NS2B-NS3 protease from dengue virus-2 (DENV-2). The non-competitive inhibitor (3) showed lower binding energy (-5.15 kcal/mol) than the predicted competitive inhibitors 4 and 5 (-3.64 and -3.21 kcal/mol, respectively). Structural analyses showed compound 3 that bound to a specific allosteric site, interacted with Lys74, a significant amino acid residue bonded to one of the catalytic triad, Asp75. Compounds 4 and 5 showed direct binding with two of the catalytic triad, His51 and Ser135, hence, predicted to be competitive inhibitors

A proteomic based assessment on changes in myofirbrillar proteins of goat longissimus muscle as affected by heat treatments

The present study examined the effect of different heat treatments; (1) chilled, (2) boiled at 100°C for 30 min, and (3) autoclaved at 121°C at 15 psi for 20 min, on the expression of goat skeletal muscle proteins using two-dimensional gel electrophoresis. The molecular weight (MW) and isoelectric point (pI) of heat stable proteins were characterised followed by identification of the proteins by MALDI-TOF/TOF mass spectrometry. There were 153 protein spots obtained in the boiled samples, while only 46 protein spots were observed in the autoclaved samples. Thirteen spots that exhibited high intensity of protein were chosen from the autoclaved sample for MALDI-TOF/TOF mass spectrometry analysis. The putative heat stable proteins identified were myosin light chain (MLC), actin, tropomyosin (TPM), troponin T (TnT), myoglobin, and creatine kinase. The Proc-GLM analysis revealed that the heat treatments have resulted in significant differences in spot intensities of actin, troponin T (TnT), myoglobin, and creatine kinase with no significant changes noted in other proteins

Differences in thermostable actin profile of goat meat as observed in two-dimensional gel electrophoresis (2DE)

Different heat treatments, (1) chilled, 4°C (2) boiled at 100°C for 30 min and (3) autoclaved at 121°C at 15 psi for 20 min were employed on goat meat to mimic domestic and industrial cooking. The effects on intensity of actin proteins was observed using two-dimensional gel electrophoresis where significant differences (p>0.05) were observed in the spot intensity between chilled and boiled samples, similarly in chilled and autoclaved samples. However, no significant difference was observed between boiled and autoclaved samples. The slight changes observed in the cooking of meat confirmed that actin protein is susceptible to denaturation cause by heat. MALDI-TOF/TOF analysis revealed the peptide-mass fingerprint between positions 21 - 374 that not affected by heat treatment. Peptides from this position merit the candidature of actin as putative thermostable marker for detecting goat meat (chevon) in food product

Induction of Apoptosis and Cell Cycle Blockade by Helichrysetin in A549 Human Lung Adenocarcinoma Cells

Researchers are looking into the potential development of natural compounds for anticancer therapy. Previous studies have postulated the cytotoxic effect of helichrysetin towards different cancer cell lines. In this study, we investigated the cytotoxic effect of helichrysetin, a naturally occurring chalcone on four selected cancer cell lines, A549, MCF-7, Ca Ski, and HT-29, and further elucidated its biochemical and molecular mechanisms in human lung adenocarcinoma, A549. Helichrysetin showed the highest cytotoxic activity against Ca Ski followed by A549. Changes in the nuclear morphology of A549 cells such as chromatin condensation and nuclear fragmentation were observed in cells treated with helichrysetin. Further evidence of apoptosis includes the externalization of phosphatidylserine and the collapse of mitochondrial membrane potential which are both early signs of apoptosis. These signs of apoptosis are related to cell cycle blockade at the S checkpoint which suggests that the alteration of the cell cycle contributes to the induction of apoptosis in A549. These results suggest that helichrysetin has great potentials for development as an anticancer agent

Strep-tag ii mutant maltose-binding protein for reagentless fluorescence sensing

Maltose-binding protein (MBP) is a periplasmic binding protein found in Gram negative bacteria. MBP is involved in maltose transport and bacterial chemotaxis; it binds to maltose and maltodextrins comprising α(1-4)-glucosidically linked linear glucose polymers and α(1-4)-glucosidically linked cyclodextrins. Upon ligand binding, MBP changes its conformation from an open to a closed form. This molecular recognition-transducing a ligand-binding event into a physical one-renders MBP an ideal candidate for biosensor development. Here, we describe the construction of a Strep-tag II mutant MBP for reagentless fluorescence sensing. malE, which encodes MBP, was amplified. A cysteine residue was introduced by site-directed mutagenesis to ensure a single label attachment at a specific site with a thiol-specific fluorescent probe. An environmentally sensitive fluorophore (IANBD amide) was covalently attached to the introduced thiol group and analysed by fluorescence sensing. The tagged mutant MBP (D95C) was purified (molecular size, ∼42 kDa). The fluorescence measurements of the IANBD-labelled Strep-tag II-D95C in the solution phase showed an appreciable change in fluorescence intensity (dissociation constant, 7.6±1.75 μM). Our mutant MBP retains maltose-binding activity and is suitable for reagentless fluorescence sensin

LC–QTOF-MS identification of porcine-specific peptide in heat treated pork identifies candidate markers for meat species determination

The purpose of this study was to identify porcine-specific peptide markers from thermally processed meat that could differentiate pork from beef, chevon and chicken meat. In the initial stage, markers from tryptic digested protein of chilled, boiled and autoclaved pork were identified using LC–QTOF-MS. An MRM method was then established for verification. A thorough investigation of LC–QTOF-MS data showed that only seven porcine-specific peptides were consistently detected. Among these peptides, two were derived from lactate dehydrogenase, one from creatine kinase, and four from serum albumin protein. However, MRM could only detect four peptides (EVTEFAK, LVVITAGAR, FVIER and TVLGNFAAFVQK) that were consistently present in pork samples. In conclusion, meat species determination through a tandem mass spectrometry platform shows high potential in providing scientifically valid and reliable results even at peptide level. Besides, the specificity and selectivity offered by the proteomics approach also provide a robust platform for Halal authentication

Impact of chill storage on antioxidant status, lipid and protein oxidation, color, drip loss and fatty acids of semimembranosus muscle in goats

The study examined the effect of refrigerated storage on antioxidant activities, lipid and protein oxidation, fatty acids (FAs), drip loss and color of semimembranosus (SM) muscle from goats. Samples of SM were obtained from carcasses of 15 Boer bucks (7 months old; body weight, 32.18 ± 0.81 kg) subjected to an 8 d storage at 4°C. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities were stable while carotenoid, tocopherol, water holding capacity and redness declined (P 0.05) from 14.00 to 13.08 density/mm2. The concentrations of n-3 and n-6 FA decreased while the saturated FA increased over storage. Correlations (P < 0.05) were found between antioxidant vitamins and quality indicators of chevon