Hydroalcoholic root extracts of Houttuynia cordata (Thunb.) standardized by UPLC-Q-TOF-MS/MS promotes apoptosis in human hepatocarcinoma cell HepG2 via GSK-3ß/ß-catenin/PDL-1 axis.

Houttuynia cordata (Thunb.), an important medicinal plant of Northeast India, Korea, and China, is used to treat various ailments and for anticancer research. Knowing its traditional practices, we are interested in the mode-of-action of HCT on HepG2 to co-relate the traditional practice with modern drug therapeutics. UPLC-Q-ToF-Ms analysis of HCT reveals identification of 14 metabolites. Network pharmacology analysis of the 14 compounds showed interaction with 232 different targets with their potential involvement in hepatocellular carcinoma. Whole extracts impart cytotoxicity on variety of cell lines including HepG2. There was a significant morphological alteration in treated HepG2 cells due to impairment of cytoskeletal components like ß and γ- tubulin. Arrest at G1-S checkpoint was clearly indicated downregulation of Cyclin D1. The root extracts actuated apoptosis in HepG2 as evident from altered mitochondrial membrane potential, Annexin V- FITC, BrdU-PI, AO/EtBr assays, and modulations of apoptotic protein expression but without ROS generation. Whole extracts caused abrogation of epithelial to mesenchymal transition with repression of Snail, N-Cadherin, Vimentin, MMP-9, and upregulation of Pan-Cadherin. Pathway analysis found GSK-3ß in Wnt/ß-Catenin signaling cascade to be involved through Hepatocellular carcinoma (hsa05225) pathway. The GSK-3ß/ß-Catenin/PDL-1 signaling was found to be inhibited with the downregulation of pathway components. This was further confirmed by application of EGF, an inducer of the GSK-3ß/ß-Catenin pathway that neutralized the effect of Houttuynia cordata (Thunb.) root extract on the said pathway. Network pharmacology analysis also confirms the synergy network with botanical-bioactive-target-disease which showed Kaempferol to have the highest degree of association with the said pathway.

Socio-economic sustainability with circular economy - An alternative approach.

As the global population and living standards rise, it pushes the demand for basic amenities like food, health, and energy resources. Additionally, manufacturing automation has led to mass production and consumption, triggering waste production. The existing linear economy approach has led to increasing waste production and resource depletion, posing significant environmental and public health threats. To overcome these impediments, an alternative model called the circular economy concept has gained popularity in the global industry community. This closed-loop, restorative, waste-free concept has the potential to protect the environment and improve economics by reducing energy and resource consumption. Thus, major impetus should be given to strengthening the backbone of the economy where tools such as green technologies, decarbonization strategies, bio refinery processes, material flow analysis, life cycle assessment, ecological footprints (water, carbon, and material), substance flow analysis, circularity index, eco-designing, bioresource management, new business models, and policy play an essential role in the areas of socio-economic sustainability, ecological facts, and industrial aspects to enhance socio-economic growth in a sustainable manner. Sectoral awareness, collaborations, and partnerships among the Government, stakeholders, policymakers, and competent authorities are also essential to enabling circularity within the eco-systems.

6-Gingerol contents of several ginger varieties of Northeast India and correlation of their antioxidant activity in respect to phenolics and flavonoids contents.

INTRODUCTION: Ginger constitutes the rhizome part of the plant Zingiber officinale from the Zingiberaceae family. A large number of ginger varieties with high sensorial and functional quality are found in Northeast India. Hence, phytopharmacological screening of different ginger varieties is essential that will serve as a guideline in applied research to develop high-end products and improve economical margins. OBJECTIVE: To determine the variation in total phenolics content (TPC), total flavonoids content (TFC), and antioxidant activities and correlate that with 6-gingerol contents of different ginger varieties collected from Northeast India using Pearson's correlation analysis. MATERIALS AND METHODS: The TPC and TFC values were determined using standard methods. Antioxidant activities were measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical scavenging assays, while reversed-phase high-performance liquid chromatography (RP-HPLC) analysis was utilised for quantitative determination of 6-gingerol content. RESULTS: The result revealed that ginger variety 6 (GV6) contains the highest 6-gingerol content and TPC value showing maximum antioxidant activity, followed by GV5, GV4, GV9, GV3, GV2, GV8, GV1, and GV7. The findings also suggested that the antioxidant activity has much better correlations with TPC as compared with TFC values. Pearson's correlation analysis showed a significant correlation between 6-gingerol contents and TPC values. CONCLUSION: This work underlines the importance of ginger varieties from Northeast India as a source of natural antioxidants with health benefits.

Evaluation of Mollugo oppositifolia Linn. as cholinesterase and ß-secretase enzymes inhibitor.

Mollugo oppositifolia Linn. is traditionally used in neurological complications. The study aimed to investigate in-vitro neuroprotective effect of the plant extracts through testing against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and ß-secretase linked to Alzheimer's disease (AD). To understand the safety aspects, the extracts were tested for CYP450 isozymes and human hepatocellular carcinoma cell (HepG2) inhibitory potential. The heavy metal contents were estimated using atomic absorption spectroscopy (AAS). Further, the antioxidant capacities as well as total phenolic content and total flavonoid content (TFC) were measured spectrophotometrically. UPLC-QTOF-MS/MS analysis was employed to identify phytometabolites present in the extract. The interactions of the ligands with the target proteins (AChE, BChE, and BACE-1) were studied using AutoDockTools 1.5.6. The results showed that M. oppositifolia extract has more selectivity towards BChE (IC50 = 278.23 ± 1.89 µg/ml) as compared to AChE (IC50 = 322.87 ± 2.05 µg/ml). The IC50 value against ß-secretase was 173.93 µg/ml. The extract showed a CC50 value of 965.45 ± 3.07 µg/ml against HepG2 cells and the AAS analysis showed traces of lead 0.02 ± 0.001 which was found to be within the WHO prescribed limits. Moreover, the IC50 values against CYP3A4 (477.03 ± 2.01 µg/ml) and CYP2D6 (249.65 ± 2.46 µg/ml) isozymes justify the safety aspects of the extract. The in silico molecular docking analysis of the target enzymes showed that the compound menthoside was found to be the most stable and showed a good docking score among all the identified metabolites. Keeping in mind the multi-targeted drug approach, the present findings suggested that M. oppositifolia extract have anti-Alzheimer's potential.

Production of bioactive hydrolysate using protease, ß-glucosidase and α-amylase of Bacillus spp. isolated from kinema.

The aim of this study was the production of soybean bioactive hydrolysate using Bacillus spp. isolated from kinema. Totally 251 bacteria isolated from kinema samples, collected at different time period were screened for protease, ß-glucosidase and α-amylase activities and further identified by ARDRA based grouping followed by analysis of 16S rRNA gene sequence similarity. The results showed that Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis were the major Bacillus species. Twelve fermentative strains belonging to these groups and having high protease, α-amylase and ß-glucosidase activity were used for solid state fermentation. The best strains for soybean fermentation that result in production of protein hydrolysates rich in polyphenols that have higher bioactivity were B. subtilis KN12C, B. amyloliquefaciens KN2G and B. licheniformis KN13C. Potential isolates can be applied for the production of soybean hydrolysates and can also find application in production of value added products from by-products of soybean processing industries.

Subtractive transcriptome analysis of leaf and rhizome reveals differentially expressed transcripts in Panax sokpayensis.

In the present study, suppression subtractive hybridization (SSH) strategy was used to identify rare and differentially expressed transcripts in leaf and rhizome tissues of Panax sokpayensis. Out of 1102 randomly picked clones, 513 and 374 high quality expressed sequenced tags (ESTs) were generated from leaf and rhizome subtractive libraries, respectively. Out of them, 64.92 % ESTs from leaf and 69.26 % ESTs from rhizome SSH libraries were assembled into different functional categories, while others were of unknown function. In particular, ESTs encoding galactinol synthase 2, ribosomal RNA processing Brix domain protein, and cell division cycle protein 20.1, which are involved in plant growth and development, were most abundant in the leaf SSH library. Other ESTs encoding protein KIAA0664 homologue, ubiquitin-activating enzyme e11, and major latex protein, which are involved in plant immunity and defense response, were most abundant in the rhizome SSH library. Subtractive ESTs also showed similarity with genes involved in ginsenoside biosynthetic pathway, namely farnesyl pyrophosphate synthase, squalene synthase, and dammarenediol synthase. Expression profiles of selected ESTs validated the quality of libraries and confirmed their differential expression in the leaf, stem, and rhizome tissues. In silico comparative analyses revealed that around 13.75 % of unigenes from the leaf SSH library were not represented in the available leaf transcriptome of Panax ginseng. Similarly, around 18.12, 23.75, 25, and 6.25 % of unigenes from the rhizome SSH library were not represented in available root/rhizome transcriptomes of P. ginseng, Panax notoginseng, Panax quinquefolius, and Panax vietnamensis, respectively, indicating a major fraction of novel ESTs. Therefore, these subtractive transcriptomes provide valuable resources for gene discovery in P. sokpayensis and would complement the available transcriptomes from other Panax species.

ß-aminobutyric acid mediated drought stress alleviation in maize (Zea mays L.).

The present study highlights the role of ß-aminobutyric acid (BABA) in alleviating drought stress effects in maize (Zea mays L.). Chemical priming was imposed by pretreating 1-week-old plants with 600 µM BABA prior to applying drought stress. Specific activities of key antioxidant enzymes and metabolites (ascorbate and glutathione) levels of ascorbate-glutathione cycle were studied to unravel the priming-induced modulation of plant defense system. Furthermore, changes in endogenous ABA and JA concentrations as well as mRNA expressions of key genes involved in their respective biosynthesis pathways were monitored in BABA-primed (BABA+) and non-primed (BABA-) leaves of drought-challenged plants to better understand the mechanistic insights into the BABA-induced hormonal regulation of plant response to water-deficit stress. Accelerated stomatal closure, high relative water content, and less membrane damage were observed in BABA-primed leaves under water-deficit condition. Elevated APX and SOD activity in non-primed leaves found to be insufficient to scavenge all H2O2 and O2 (·-) resulting in oxidative burst as evident after histochemical staining with NBT and DAB. A higher proline accumulation in non-primed leaves also does not give much protection against drought stress. Increased GR activity supported with the enhanced mRNA and protein expressions might help the BABA-primed plants to maintain a high GSH pool essential for sustaining balanced redox status to counter drought-induced oxidative stress damages. Hormonal analysis suggests that in maize, BABA-potentiated drought tolerance is primarily mediated through JA-dependent pathway by the activation of antioxidant defense systems while ABA biosynthesis pathway also plays an important role in fine-tuning of drought stress response.

An RNA isolation system for plant tissues rich in secondary metabolites.

BACKGROUND: Secondary metabolites are reported to interfere with the isolation of RNA particularly with the recipes that use guanidinium-based salt. Such interference was observed in isolation of RNA with medicinal plants rheum (Rheum australe) and arnebia (Arnebia euchroma). A rapid and less cumbersome system for isolation of RNA was essential to facilitate any study related to gene expression. FINDINGS: An RNA isolation system free of guanidinium salt was developed that successfully isolated RNA from rheum and arnebia. The method took about 45 min and was successfully evaluated on twenty one tissues with varied secondary metabolites. The A260/280 ratio ranged between 1.8 - 2.0 with distinct 28 S and 18 S rRNA bands visible on a formaldehyde-agarose gel. CONCLUSIONS: The present manuscript describes a rapid protocol for isolation of RNA, which works well with all the tissues examined so far. The remarkable feature was the success in isolation of RNA with those tissues, wherein the most commonly used methods failed. Isolated RNA was amenable to downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), differential display (DD), suppression subtractive hybridization (SSH) library construction, and northern hybridization.

Expression of 3-hydroxy-3-methylglutaryl-CoA reductase, p-hydroxybenzoate-m-geranyltransferase and genes of phenylpropanoid pathway exhibits positive correlation with shikonins content in arnebia [Arnebia euchroma (Royle) Johnston].

BACKGROUND: Geranyl pyrophosphate (GPP) and p-hydroxybenzoate (PHB) are the basic precursors involved in shikonins biosynthesis. GPP is derived from mevalonate (MVA) and/or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway(s), depending upon the metabolite and the plant system under consideration. PHB, however, is synthesized by only phenylpropanoid (PP) pathway. GPP and PHB are central moieties to yield shikonins through the synthesis of m-geranyl-p-hydroxybenzoate (GHB). Enzyme p-hydroxybenzoate-m-geranyltransferase (PGT) catalyses the coupling of GPP and PHB to yield GHB. The present research was carried out in shikonins yielding plant arnebia [Arnebia euchroma (Royle) Johnston], wherein no molecular work has been reported so far. The objective of the work was to identify the preferred GPP synthesizing pathway for shikonins biosynthesis, and to determine the regulatory genes involved in the biosynthesis of GPP, PHB and GHB. RESULTS: A cell suspension culture-based, low and high shikonins production systems were developed to facilitate pathway identification and finding the regulatory gene. Studies with mevinolin and fosmidomycin, inhibitors of MVA and MEP pathway, respectively suggested MVA as a preferred route of GPP supply for shikonins biosynthesis in arnebia. Accordingly, genes of MVA pathway (eight genes), PP pathway (three genes), and GHB biosynthesis were cloned. Expression studies showed down-regulation of all the genes in response to mevinolin treatment, whereas gene expression was not influenced by fosmidomycin. Expression of all the twelve genes vis-à-vis shikonins content in low and high shikonins production system, over a period of twelve days at frequent intervals, identified critical genes of shikonins biosynthesis in arnebia. CONCLUSION: A positive correlation between shikonins content and expression of 3-hydroxy-3-methylglutaryl-CoA reductase (AeHMGR) and AePGT suggested critical role played by these genes in shikonins biosynthesis. Higher expression of genes of PP pathway was a general feature for higher shikonins biosynthesis.