Investigations of Limeum Indicum Plant for Diabetes Mellitus and Alzheimer's Disease Dual Therapy: Phytochemical, GC-MS Chemical Profiling, Enzyme Inhibition, Molecular Docking and In-Vivo Studies.

Limeum indicum has been widely utilized in traditional medicine but no experimental work has been done on this herb. The primary objective of this study was to conduct a phytochemical analysis and assess the multifunctional capabilities of aforementioned plant in dual therapy for Alzheimer's disease (AD) and Type 2 diabetes (T2D). The phytochemical screening of ethanol, methanol extract, and their derived fractions of Limeum indicum was conducted using GC-MS, HPLC, UV-analysis and FTIR. The antioxidant capacity was evaluated by DPPH method. The inhibitory potential of the extracts/fractions against α-, ß-glucosidase acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and monoaminine oxidases (MAO-A & B) was evaluated. Results revealed that acetonitrile fraction has highest inhibitory potential against α-glucosidase (IC50=68.47±0.05 µg/mL), methanol extract against ß-glucosidase (IC50=91.12±0.07 µg/mL), ethyl acetate fraction against AChE (IC50=59.0±0.02 µg/mL), ethanol extract against BChE (28.41±0.01 µg/mL), n-hexane fraction against MAO-A (IC50=150.5±0.31 µg/mL) and methanol extract for MAO-B (IC50=75.95±0.13 µg/mL). The docking analysis of extracts\fractions suggested the best binding scores within the active pocket of the respective enzymes. During the in-vivo investigation, ethanol extract produced hypoglycemic effect (134.52±2.79 and 119.38±1.40 mg/dl) after 21 days treatment at dose level of 250 and 500 mg/Kg. Histopathological findings further supported the in-vivo studies.

Short-term responses of soil enzyme activities and stoichiometry to litter input in Castanopsis carlesii and Cunninghamia lanceolata plantations.

Litter input triggers the secretion of soil extracellular enzymes and facilitates the release of carbon (C), nitrogen (N), and phosphorus (P) from decomposing litter. However, how soil extracellular enzyme activities were controlled by litter input with various substrates is not fully understood. We examined the activities and stoichiometry of five enzymes including ß-1,4-glucosidase, ß-D-cellobiosidase, ß-1,4-N-acetyl-glucosaminidase, leucine aminopeptidase and acidic phosphatase (AP) with and without litter input in 10-year-old Castanopsis carlesii and Cunninghamia lanceolata plantations monthly during April to August, in October, and in December 2021 by using an in situ microcosm experiment. The results showed that: 1) There was no significant effect of short-term litter input on soil enzyme activity, stoichiometry, and vector properties in C. carlesii plantation. In contrast, short-term litter input significantly increased the AP activity by 1.7% in May and decreased the enzymatic C/N ratio by 3.8% in August, and decreased enzymatic C/P and N/P ratios by 11.7% and 10.3%, respectively, in October in C. lanceolata plantation. Meanwhile, litter input increased the soil enzymatic vector angle to 53.8° in October in C. lanceolata plantations, suggesting a significant P limitation for soil microorganisms. 2) Results from partial least squares regression analyses showed that soil dissolved organic matter and microbial biomass C and N were the primary factors in explaining the responses of soil enzymatic activity to short-term litter input in both plantations. Overall, input of low-quality (high C/N) litter stimulates the secretion of soil extracellular enzymes and accelerates litter decomposition. There is a P limitation for soil microorganisms in the study area.

Discovery of plant chemical defence mediated by a two-component system involving ß-glucosidase in Panax species.

Plants usually produce defence metabolites in non-active forms to minimize the risk of harm to themselves and spatiotemporally activate these defence metabolites upon pathogen attack. This so-called two-component system plays a decisive role in the chemical defence of various plants. Here, we discovered that Panax notoginseng, a valuable medicinal plant, has evolved a two-component chemical defence system composed of a chloroplast-localized ß-glucosidase, denominated PnGH1, and its substrates 20(S)-protopanaxadiol ginsenosides. The ß-glucosidase and its substrates are spatially separated in cells under physiological conditions, and ginsenoside hydrolysis is therefore activated only upon chloroplast disruption, which is caused by the induced exoenzymes of pathogenic fungi upon exposure to plant leaves. This activation of PnGH1-mediated hydrolysis results in the production of a series of less-polar ginsenosides by selective hydrolysis of an outer glucose at the C-3 site, with a broader spectrum and more potent antifungal activity in vitro and in vivo than the precursor molecules. Furthermore, such ß-glucosidase-mediated hydrolysis upon fungal infection was also found in the congeneric species P. quinquefolium and P. ginseng. Our findings reveal a two-component chemical defence system in Panax species and offer insights for developing botanical pesticides for disease management in Panax species.

Discovery of enzymes to biotransform ginsenoside Rd into ginsenosides F2 and CK using metagenomics and genomic mining.

Ginsenosides are the main active components of ginseng, including many types and different contents. Among them, minor ginsenosides have better biological functions and pharmacological activities than those of the major ginsenosides. However, minor ginsenosides cannot be obtained in large quantities, but by means of enzymatic transformation technology, some major ginsenosides can be de-glycosylated at a specific position to generate minor ginsenosides. In this study, we report two glycosidase genes associated with the conversion of ginsenoside Rd to ginsenosides F2 or CK. SWMU-CK-1 was identified among the total genes extracted from the feces of plum deer by local Blast screening for putative ginsenoside conversion function, which could cause the conversion of ginsenoside Rd → F2 → CK. The other gene was found in the Bifidobacterium breve 689b SGAir 0764 chromosome genome, which might have the same function as the ß-glucosidase gene testified by the gene matching, named SWMU-F2-2, and can achieve the Rd → F2 transformation. This study reports two genes that enable achieving the biotransformation of rare ginsenosides, while it provides a new insight and a promising approach to explore new genes and develop new functions of existing genes.

A Novel Beta-Glucosidase Gene for Plant Type Was Identified by Genome-Wide Association Study and Gene Co-Expression Analysis in Widespread Bermudagrass.

Bermudagrass (Cynodon spp.) is one of the most widely distributed warm-season grasses globally. The growth habits and plant type of bermudagrass are strongly associated with the applied purpose of the landscape, livestock, and eco-remediation. Therefore, persistent efforts are made to investigate the genetic basis of plant type and growth habits of bermudagrass. Here, we dissect the genetic diversity of 91 wild bermudagrass resources by genome-wide association studies (GWAS) combined with weighted gene co-expression analysis (WGCNA). This work is based on the RNA-seq data and the genome of African bermudagrass (Cynodon transvaalensis Burtt Davy). Sixteen reliable single-nucleotide polymorphisms (SNPs) in transcribed regions were identified to be associated with the plant height and IAA content in diverse bermudagrass by GWAS. The integration of the results from WGCNA indicates that beta-glucosidase 31 (CdBGLU31) is a candidate gene underlying a G/A SNP signal. Furthermore, both qRT-PCR and correlation coefficient analyses indicate that CdBGLU31 might play a comprehensive role in plant height and IAA biosynthesis and signal. In addition, we observe lower plant height in Arabidopsis bglu11 mutants (homologs of CdBGLU31). It uncovers the breeding selection history of different plant types from diverse bermudagrass and provides new insights into the molecular function of CdBGLU31 both in plant types and in IAA biosynthetic pathways.

Lowering effect of combined sweet potato and onion intake on plasma quercetin concentration and underlying mechanism involving intestinal ß-glucosidase activity.

A combined intake of cooked sweet potato and fried onion in humans was found to suppress the increase of plasma quercetin metabolite concentration. Experiments using rat ß-glucosidase indicated that excess carbohydrate digestion products, especially glucose-containing saccharides, interfere with the deglycosylation of quercetin glucosides during intestinal epithelial uptake. Combined meals of sweet potato and onion may lower the bioavailability of onion quercetin glucosides.

New prenylated flavonoid glycosides derived from Epimedium wushanense by ß-glucosidase hydrolysis and their testosterone production-promoting effects.

Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A-D (1-4) and two new prenyl flavonoid derivatives wushepimedoside E-F (5-6), and one know analog epimedkoreside B (7) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1-7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3ß-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.

Production, concentration and partial characterization of an enzymatic extract produced by an Aspergillus niger mutant in solid state fermentation.

An enzymatic extract from Aspergillus niger 3T5B8 was produced by Solid State Fermentation (SSF) in aerated columns, using wheat bran as substrate. A combination of extracts produced using three different process conditions varying temperature, pH and aeration formed the final extract (Mixture). The Mixture was concentrated by an ultrafiltration process that partially purified and provided an efficient recovery of the enzymatic activities of xylanase (88.89%), polygalacturonase (89.3%), ß-glucosidase (93.15%), protease (98.68%) and carboxymethylcellulase (CMCase) (98.93%). SDS-PAGE analysis showed 15 visible protein bands in the crude and concentrated Mixture with molecular weights ranging from 15.1 to 104.6 kDa. Thin layer chromatography confirmed the effective action of ß-glucosidase and xylanase hydrolysis activities over cellobiose and xylan, respectively. A central composite design (CCD) with two variables and four replicates at the center points was used to determine the optimal temperature and pH for CMCase and ß-glucosidase. The optimal temperature was 78.9 °C and pH 3.8 for CMCase and 52.8 °C and pH 4.8 for ß-glucosidase, respectively.

Rice ß-glucosidase Os12BGlu38 is required for synthesis of intine cell wall and pollen fertility.

Glycoside hydrolase family1 ß-glucosidases play a variety of roles in plants, but their in planta functions are largely unknown in rice (Oryza sativa). In this study, the biological function of Os12BGlu38, a rice ß-glucosidase, expressed in bicellular to mature pollen, was examined. Genotype analysis of progeny of the self-fertilized heterozygous Os12BGlu38 T-DNA mutant, os12bglu38-1, found no homozygotes and a 1:1 ratio of wild type to heterozygotes. Reciprocal cross analysis demonstrated that Os12BGlu38 deficiency cannot be inherited through the male gamete. In cytological analysis, the mature mutant pollen appeared shrunken and empty. Histochemical staining and TEM showed that mutant pollen lacked intine cell wall, which was rescued by introduction of wild-type Os12BGlu38 genomic DNA. Metabolite profiling analysis revealed that cutin monomers and waxes, the components of the pollen exine layer, were increased in anthers carrying pollen of os12bglu38-1 compared with wild type and complemented lines. Os12BGlu38 fused with green fluorescent protein was localized to the plasma membrane in rice and tobacco. Recombinant Os12BGlu38 exhibited ß-glucosidase activity on the universal substrate p-nitrophenyl ß-d-glucoside and some oligosaccharides and glycosides. These findings provide evidence that function of a plasma membrane-associated ß-glucosidase is necessary for proper intine development.

Cloning and Production of Thermostable Enzymes for the Hydrolysis of Steryl Glucosides in Biodiesel.

Vegetable oil-derived biodiesels have a major quality problem due to the presence of precipitates formed by steryl glucosides, which clog filters and injectors of diesel engines. An efficient, scalable, and cost-effective method to hydrolyze steryl glucosides using thermostable enzymes has been developed. Here, methods to discover, express in recombinant microorganisms and manufacture enzymes with SGase activity, as well as methods to treat biodiesel with such enzymes, and to measure the content of steryl glucosides in biodiesel samples are presented.

Microbial Application to Improve Olive Mill Wastewater Phenolic Extracts.

Olive mill wastewater (OMW) contains valuable and interesting bioactive compounds, among which is hydroxytyrosol, which is characterized by a remarkable antioxidant activity. Due to the health claims related to olive polyphenols, the aim of this study was to obtain an extract from OMW with an increased level of hydroxytyrosol by means of microbial enzymatic activity. For this purpose, four commercial adsorbent resins were selected and tested. The beta-glucosidase and esterase activity of strains of Wickerhamomyces anomalus, Lactiplantibacillus plantarum, and Saccharomyces cerevisiae were also investigated and compared to those of a commercial enzyme and an Aspergillus niger strain. The W. anomalus strain showed the best enzymatic performances. The SP207 resin showed the best efficiency in selective recovery of hydroxytyrosol, tyrosol, oleuropein, and total phenols. The bioconversion test of the OMW extract was assessed by using both culture broths and pellets of the tested strains. The results demonstrated that the pellets of W. anomalus and L. plantarum were the most effective in hydroxytyrosol increasing in phenolic extract. The interesting results suggest the possibility to study new formulations of OMW phenolic extracts with multifunctional microorganisms.

Enzymatic hydrolysis and auto-isomerization during ß-glucosidase treatment improve the aroma of instant white tea infusion.

The aroma changes in instant white tea resulting from ß-glucosidase treatment was investigated by quantitative descriptive analysis (QDA), gas chromatography-mass spectrometry (GC-MS), odour activity value analysis (OAV), aroma reconstruction and omission tests. The grassy, floral and sweet notes increased significantly (P < 0.05), and the roasted note decreased significantly (P < 0.05) upon ß-glucosidase treatment. Quantitative analysis showed that the concentrations of benzaldehyde, benzeneacetaldehyde, (Z)-3-hexen-1-ol, linalool, phenylethyl alcohol, cis-linalool oxide, trans-linalool oxide, hexanol, hotrienol and (E)-2-hexen-1-ol increased significantly (P < 0.05) after treatment; however, (Z)-3-hexen-1-ol isomerized to (E)-2-hexen-1-ol. OAV analysis, aroma reconstruction and the omission test showed that the grassy, floral and sweet notes increased as the (Z)-3-hexen-1-ol, cis/trans-linalool oxide and benzeneacetaldehyde increased, whereas the roasted note declined under the same conditions. The enzymatic hydrolysis of glycosidic precursors and the auto-isomerization of volatile compounds provide new information for understanding how ß-glucosidase treatment improves the aroma of tea products.

Polyphenol oxidase dominates the conversions of flavonol glycosides in tea leaves.

Flavonol glycosides are associated with astringency and bitterness of teas. To clarify the dominant enzymatic reaction of flavonol glycosides in tea leaves, the catalytic effects of polyphenol oxidase (PPO), peroxidase (POD) and ß-glucosidase were studied, with the maintaining rates of total flavonol glycosides (TFG) being 73.0%, 99.8% and 94.3%. PPO was selected for further investigations, including the effects of pH value (3.5 ~ 6.5), temperature (25 °C ~ 55 °C) and dosage (39 ~ 72 U/mL PPO and 36 U/mL PPO, 3 ~ 36 U/mL POD). The oxidation of flavonol glycosides were intensified at pH 6.5, with 51.8% and 15.4% of TFG maintained after PPO and PPO + POD treatments, suggesting an enhancement from POD. The sensitivity ranking to PPO was: myricetin glycosides > quercetin glycosides > kaempferol glycosides. The inhibitor treatment testified the leading role of PPO in catalyzing flavonol glycosides in tea leaves. Sugar moiety enhanced the docking affinity of flavonol glycosides for PPO. PPO shows the potential of modifying flavonol glycoside composition.

Light Limitation Impacts Growth but Not Constitutive or Jasmonate Induced Defenses Relevant to Emerald Ash Borer (Agrilus planipennis) in White Fringetree (Chionanthus virginicus) or Black Ash (Fraxinus nigra).

White fringetree is a host for the invasive emerald ash borer (EAB) but is of lower quality than the related and highly susceptible black ash. Field observations suggest that host trees grown in full sun are more resistant to EAB than those in shade, however the impact of light limitation on chemical defenses has not been assessed. We quantified constitutive and jasmonate-induced phloem defenses and growth patterns of white fringetree and black ash under differential light conditions and related them to EAB larval performance. White fringetree had significantly lower constitutive and induced activities of peroxidase, polyphenol oxidase, ß-glucosidase, chitinase and lignin content, but significantly higher gallic acid equivalent soluble phenolic, soluble sugar, and oleuropein concentrations compared to black ash. Multivariate analyses based on tissue chemical attributes displayed clear separation of species and induced defense responses. Further, EAB performed significantly worse on white fringetree than black ash, consistent with previous studies. Light limitation did not impact measured defenses or EAB larval performance, but it did decrease current year growth and increase photosynthetic efficiency. Overall our results suggest that phenolic profiles, metabolite abundance, and growth traits are important in mediating white fringetree resistance to EAB, and that short-term light limitation does not influence phloem chemistry or larval success.

Biotransformation of Protopanaxadiol-Type Ginsenosides in Korean Ginseng Extract into Food-Available Compound K by an Extracellular Enzyme from Aspergillus niger.

Compound K (C-K) is one of the most pharmaceutically effective ginsenosides, but it is absent in natural ginseng. However, C-K can be obtained through the hydrolysis of protopanaxadiol-type ginsenosides (PPDGs) in natural ginseng. The aim of this study was to obtain the high concentration of food-available C-K using PPDGs in Korean ginseng extract by an extracellular enzyme from Aspergillus niger KACC 46495. A. niger was cultivated in the culture medium containing the inducer carboxymethyl cellulose (CMC) for 6 days. The extracellular enzyme extracted from A. niger was prepared from the culture broth by filtration, ammonium sulfate, and dialysis. The extracellular enzyme was used for C-K production using PPDGs. The glycoside-hydrolyzing pathways for converting PPDGs into C-K by the extracellular enzyme were Rb1 → Rd → F2 → C-K, Rb2 → Rd or compound O → F2 or compound Y → C-K, and Rc → Rd or compound Mc1 → F2 or compound Mc → C-K. The extracellular enzyme from A. niger at 8.0 mg/ml, which was obtained by the induction of CMC during the cultivation, converted 6.0 mg/ml (5.6 mM) PPDGs in Korean ginseng extract into 2.8 mg/ml (4.5 mM) food-available C-K in 9 h, with a productivity of 313 mg/l/h and a molar conversion of 80%. To the best of our knowledge, the productivity and concentration of C-K of the extracellular enzyme are the highest among those by crude enzymes from wild-type microorganisms.

Biotransformation of Major Ginsenoside Rb1 to Rd by Dekkera anomala YAE-1 from Mongolian Fermented Milk (Airag).

Dekkera anomala YAE-1 strain separated from "airag" (Mongolian fermented mare's milk) produces ß-glucosidase, which can convert ginsenoside Rb1 from Panax ginseng. Ginseng-derived bioactive components such as ginsenoside Rb1 have various immunological and anticancer activities. Airag was collected from five different mare milk farms located near Ulaanbaatar, Mongolia. YAE-1 strains were isolated from airag to examine the hydrolytic activities of ß-glucosidase on Korean Panax ginseng using an API ZYM kit. Supernatants of selected cultures having ß-glucosidase activity were examined for hydrolysis of the major ginsenoside Rb1 at 40°C, pH 5.0. The YAE-1 strain was found to be nearly identical at 99.9% homology with Dekkera anomala DB-7B, and was thus named Dekkera anomala YAE-1. This strain exerted higher ß-glucosidase activity than other enzymes. Reaction mixtures from Dekkera anomala YAE-1 showed great capacity for converting ginsenoside Rb1 to ginsenoside Rd. The ß-glucosidase produced by Dekkera anomala YAE-1 was able to hydrolyze ginsenoside Rb1 and convert it to Rd during fermentation of the ginseng. The amount of ginsenoside Rd was highly increased from 0 to 1.404 mg/ml in fermented 20% ginseng root at 7 days.

Establishment and optimization of a hemp (Cannabis sativa L.) agroinfiltration system for gene expression and silencing studies.

Industrial hemp (Cannabis sativa L.) is a high-yielding annual crop primarily grown for fiber, seeds, and oil. Due to the phytochemical composition of hemp, there has been an increased interest in the market for nutraceuticals and dietary supplements for human health. Recent omics analysis has led to the elucidation of hemp candidate genes involved in the syntheses of specialized metabolites. However, a detailed study of these genes has not been undertaken due to the lack of a stable transformation system. We report for the first time an agroinfiltration system in hemp utilizing vacuum infiltration, which is an alternative method to stable transformation. A combination of 0.015% Silwett L-77, 5 mM ascorbic acid, and thirty second sonication followed by a 10-minute vacuum treatment resulted in the highest ß-glucuronidase expression in the leaf, male and female flowers, stem, and root tissues. The phytoene desaturase gene was silenced with a transient hairpin RNA expression, resulting in an albino phenotype in the leaves and the male and female flowers. This agroinfiltration system would be useful for overexpression and silencing studies of target genes to regulate the yield of specialized metabolites in hemp.

Aroma enhancement of instant green tea infusion using ß-glucosidase and ß-xylosidase.

ß-Glucosidase and ß-xylosidase were investigated for their ability to improve the aroma of instant green tea. The aroma and corresponding contributors were analyzed by sensory evaluation, gas chromatography-mass spectrometry, and odor activity value. Their specific contributions to aroma attributes were further examined by aroma reconstruction and omission experiments. The ß-glucosidase treatment significantly enhanced floral and grassy notes, on account of the increases of geraniol, nonanal, and cis-3-hexen-1-ol, and weakened the caramel note, attributable to the increases of nonanal, cis-3-hexen-1-ol, geraniol, methyl salicylate, and decanal. The co-treatment with ß-glucosidase and ß-xylosidase further enhanced the grassy note, with further increase in nonanal and cis-3-hexen-1-ol, and further weakened the caramel note, with additional increase in nonanal, cis-3-hexen-1-ol, methyl salicylate, and decanal. The synergistic action of ß-glucosidase and ß-xylosidase provides new clues to the production of instant green tea infusions with high aroma quality.

Tyrosinase-mediated dopamine polymerization modified magnetic alginate beads for dual-enzymes encapsulation: Preparation, performance and application.

In this study, a simple and efficient method to obtain entrapment of mixtures of double enzymes is developed. As a proof of principle, double enzymes (tyrosinase (TYR) and ß-glucosidase (ß-Glu)) were co-immobilized in magnetic alginate-polydopamine (PDA) beads using in situ TYR-mediated dopamine polymerization and internal setting strategy-mediated magnetic alginate-PDA gelation. The leakage of enzymes from the magnetic alginate beads was significantly reduced by exploiting the double network cross-linking of alginate and PDA, which was induced by the d-(+)-Gluconic acid δ-lactone (GDL) and TYR, respectively. The physicochemical properties of the prepared magnetic alginate beads were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) analysis. After that, the enzymatic reaction conditions and the performance of the entrapped TYR and ß-Glu, such as enzyme kinetics and inhibition kinetics, were investigated. The Michaelis-Menten constants (Km) of the entrapped TYR and ß-Glu were determined as 2.72 and 3.45 mM, respectively. The half-maximal inhibitory concentrations (IC50) of kojic acid and castanospermine for the entrapped TYR and ß-Glu were determined as 13.04 and 56.23 µM, respectively. Finally, the entrapped double enzymes magnetic alginate beads were successfully applied to evaluate the inhibitory potency of six kinds of tea polyphenols extracts. Black tea and white tea showed high inhibition activity against TYR were (36.14 ± 1.43)% and (36.76 ± 2.35)%, respectively, while the black tea and dark tea showed high inhibition activity against ß-Glu were (37.89 ± 6.70)% and (21.28 ± 4.68)%, respectively.

Multistarter fermentation of glutinous rice with Fu brick tea: Effects on microbial, chemical, and volatile compositions.

A higher fermentation efficiency was achieved, using multistarter fermentation of glutinous rice supplemented with Fu brick tea (FGR-FBT), than when using traditional fermentation. The effects of multistarter fermentation on the microbial, chemical, and volatile compositions were determined. When FBT was incorporated during glutinous rice fermentation, increased population of yeasts and fungi, as well as enhanced α-amylase, proteinase and ß-glucosidase activities, were observed. Specific fungi were isolated and identified as Aspergillus spp., which are known to secrete extracellular enzymes that modify the chemical properties, including ethanol levels, pH, total acids, and total soluble solids. The aroma profile of fermented glutinous rice was studied in the absence and presence of FBT, using HS-SPME-GC-MS and the electronic-nose. This analysis indicated that 35 characteristic volatile compounds were only found in FGR-FBT. The results show that FBT can be added during the fermentation of food products to enhance microbial biotransformation and modify flavour metabolism.