The Inhibitory Effects of Plant Derivate Polyphenols on the Main Protease of SARS Coronavirus 2 and Their Structure-Activity Relationship.

The main protease (Mpro) is a major protease having an important role in viral replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus that caused the pandemic of 2020. Here, active Mpro was obtained as a 34.5 kDa protein by overexpression in E. coli BL21 (DE3). The optimal pH and temperature of Mpro were 7.5 and 37 °C, respectively. Mpro displayed a Km value of 16 µM with Dabcyl-KTSAVLQ↓SGFRKME-Edans. Black garlic extract and 49 polyphenols were studied for their inhibitory effects on purified Mpro. The IC50 values were 137 µg/mL for black garlic extract and 9-197 µM for 15 polyphenols. The mixtures of tannic acid with puerarin, daidzein, and/or myricetin enhanced the inhibitory effects on Mpro. The structure-activity relationship of these polyphenols revealed that the hydroxyl group in C3', C4', C5' in the B-ring, C3 in the C-ring, C7 in A-ring, the double bond between C2 and C3 in the C-ring, and glycosylation at C8 in the A-ring contributed to inhibitory effects of flavonoids on Mpro.

Fermented Wild Ginseng by Rhizopus oligosporus Improved l-Carnitine and Ginsenoside Contents.

We conducted this study to investigate the beneficial effects of Rhizopus oligosporus fermentation of wild ginseng on ginsenosides, l-carnitine contents and its biological activity. The Rhizopus oligosporus fermentation of wild ginseng was carried out at 30 °C for between 1 and 14 days. Fourteen ginsenosides and l-carnitine were analyzed in the fermented wild ginseng by the ultra high pressure liquid chromatography-mass spectrometry (UPLC-MS) system. Our results showed that the total amount of ginsenosides in ginseng increased from 3,274 to 5,573 mg/kg after 14 days of fermentation. Among the 14 ginsenosides tested, the amounts of 13 ginsenosides (Rg1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg2, Rg3, Rh1, compound K, F1 and F2) increased, whereas ginsenoside Rb1 decreased, during the fermentation. Furthermore, l-carnitine (630 mg/kg) was newly synthesized in fermented ginseng extract after 14 days. In addition, both total phenol contents and DPPH radical scavenging activities showed an increase in the fermented ginseng with respect to non-fermented ginseng. These results show that the fermentation process reduced the cytotoxicity of wild ginseng against RAW264.7 cells. Both wild and fermented wild ginseng showed anti-inflammatory activity via inhibition of nitric oxide synthesis in RAW264.7 murine macrophage cells.

Anti-cariogenic Characteristics of Rubusoside.

Streptococcus mutans plays an important role in the development of dental caries in humans by synthesizing adhesive insoluble glucans from sucrose by mutansucrase activity. To explore the anti-cariogenic characteristics of rubusoside (Ru), a natural sweetener component in Rubus suavissimus S. Lee (Rosaceae), we investigated the inhibitory effect of Ru against the activity of mutansucrase and the growth of Streptococcus mutans. Ru (50 mM) showed 97% inhibitory activity against 0.1 U/mL mutansucrase of S. mutans with 500 mM sucrose. It showed competitive inhibition with a K i value of 1.1 ± 0.2 mM and IC50 of 2.3 mM. Its inhibition activity was due to hydrophobic and hydrogen bonding interactions based on molecular docking analysis. Ru inhibited the growth of S. mutans as a bacteriostatic agent, with MIC and MBC values of 6 mM and 8 mM, respectively. In addition, Ru showed synergistic anti-bacterial activity when it was combined with curcumin. Therefore, Ru is a natural anti-cariogenic agent with anti-mutansucrase activity and antimicrobial activity against S. mutans. ELECTRONIC SUPPLEMENTARY MATERIAL ESM: The online version of this article (doi: 10.1007/s12257-018-0408-0) contains supplementary material, which is available to authorized users.

Decrease of insoluble glucan formation in Streptococcus mutans by co-cultivation with Enterococcus faecium T7 and glucanase addition.

OBJECTIVES: To develop preventive canine oral health bio-materials consisting of probiotics and glucanase to reduce insoluble glucan and volatile sulfur compound formation. RESULTS: Co-cultivation of Enterococcus faecium T7 with Streptococcus mutans at inoculation ratio of 3:1 (v/v) resulted in 25% reduction in the growth of Streptococcus mutans. Amounts of soluble and insoluble glucans produced by S. mutans were decreased to 70 and 55%, respectively. Insoluble glucan was decreased from 0.6 µg/ml in S. mutans culture to 0.03 µg/ml in S. mutans co-cultivated with E. faecium T7 in the presence of Lipomyces starkeyi glucanase. Volatile sulfur compound, a main component of halitosis produced by Fusobacteria nucleatum, was decreased by co-cultivating F. nucleatum with E. faecium. CONCLUSION: E. faecium and glucanase can be combined as potentially active ingredients of oral care products for pets by reducing plaque-forming bacteria growth and their by-products that cause cavity and periodontal disease.

Production of steviol from steviol glucosides using ß-glucosidase from a commercial pectinase, Sumizyme PX.

OBJECTIVE: To purify and characterize a specific enzyme from a commercial pectinase for the production of steviol from stevioside (Ste) without adding organic solvent and to improve steviol production. RESULTS: Commercial Sumizyme PX converted Ste to steviol with a yield of 98%. ß-Glucosidase from Sumizyme PX (ßglyPX) was purified in three steps with 12.5-fold purification and 51% yield. The specific activity of the purified ßglyPX was 141 U/mg. The molecular weight of ßglyPX was ~ 116 kDa on SDS-PAGE. Its optimum activity was at pH 3.5 and 65 °C. It was stable for 12 h up to 55 °C and for 24 h at pH 2-9.5. K m values of ßglyPX for pNPGal, oNPGlc, lactose, and Ste were 2.4, 0.7, 18, and 7.8 mM, respectively. The optimum conditions for steviol production were 55 °C, 900 U/ml, 80 mg Ste/ml, 12 h. CONCLUSION: ßglyPX contains great potential for industrial steviol production from Ste.

Inhibitory effect of flavonoids against NS2B-NS3 protease of ZIKA virus and their structure activity relationship.

OBJECTIVES: To determine the inhibitory activities of flavonoids against NS2B-NS3 protease of ZIKA virus (ZIKV NS2B-NS3pro) expressed in Escherichia coli BL21 (DE3) and their structure activity relationship. RESULTS: ZIKV NS2B-NS3pro was expressed in E. coli BL21(DE3) as a 35 kDa protein. It had a K m of 26 µM with the fluorogenic peptide Dabcyl-KTSAVLQSGFRKME-Edan. The purified ZIKV NS2B-NS3pro was used for inhibition and kinetic assays to determine the activities of 22 polyphenol compounds. These polyphenol compounds at 100 µM inhibited the activity of ZIKV NS2B-NS3pro by 6.2-88%. Seven polyphenol compounds had IC50 ranging from 22 ± 0.2 to 112 ± 5.5 µM. Myricetin showed a mixed type inhibitory pattern against ZIKV NS2B-NS3pro protease. Its IC50 value was 22 ± 0.2 µM with a K i value of 8.9 ± 1.9 µM. CONCLUSION: The chemical structure of a polyphenol compound and its inhibitory activity against ZIKV NS2B-NS3pro can be explored to develop highly selective inhibitors against ZIKV NS2B-NS3pro.

The effect of fermented buckwheat on producing l-carnitine- and γ-aminobutyric acid (GABA)-enriched designer eggs.

BACKGROUND: The potential of fermented buckwheat as a feed additive was studied to increase l-carnitine and γ-aminobutyric acid (GABA) in designer eggs. Buckwheat contains high levels of lysine, methionine and glutamate, which are precursors for the synthesis of l-carnitine and GABA. Rhizopus oligosporus was used for the fermentation of buckwheat to produce l-carnitine and GABA that exert positive effects such as enhanced metabolism, antioxidant activities, immunity and blood pressure control. RESULTS: A novel analytical method for simultaneously detecting l-carnitine and GABA was developed using liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS. The fermented buckwheat extract contained 4 and 34 times more l-carnitine and GABA respectively compared with normal buckwheat. Compared with the control, the fermented buckwheat extract-fed group showed enriched l-carnitine (13.6%) and GABA (8.4%) in the yolk, though only l-carnitine was significantly different (P < 0.05). Egg production (9.4%), albumen weight (2.1%) and shell weight (5.8%) were significantly increased (P < 0.05). There was no significant difference in yolk weight, and total cholesterol (1.9%) and triglyceride (4.9%) in the yolk were lowered (P < 0.05). CONCLUSION: Fermented buckwheat as a feed additive has the potential to produce l-carnitine- and GABA-enriched designer eggs with enhanced nutrition and homeostasis. These designer eggs pose significant potential to be utilized in superfood production and supplement industries. © 2016 Society of Chemical Industry.

Production of a low calorie mandarin juice by enzymatic conversion of constituent sugars to oligosaccharides and prevention of insoluble glucan formation.

Over 99% of sucrose in mandarin juice (57.1 g/l in original juice to 428.4 g/l in concentrated juice) was enzymatically converted to glucooligosaccharides using 3 U dextransucrase/ml prepared from Leuconostoc mesenteroides at 28 °C. The oligosaccharide synthesis yields were 51 and 47% for the original and the concentrated mandarin juice, respectively. The degree of polymerization of oligosaccharides in the enzyme-modified juice was 2-7. Calories in the original and modified mandarin juice were 433 and 301 kcal/l (30.5% reduction). Compared with the original juice, the enzyme-modified juice showed 82% decrease of insoluble glucan formation by mutansucrase from Streptococcus mutans. A sensory evaluation of the juices revealed that the original and modified mandarin juices had sweetness values of 4.5 and 4.9 and the same values for overall acceptability.

Inhibition effect of flavonoid compounds against neuraminidase expressed in Pichia pastoris.

Neuraminidase (NA) is one of the two glycoproteins on the surface of influenza virus, which cleaves terminal sialic acid residues and facilitates the release of virions from infected cells. The recombinant NA from H5N1 influenza virus strain A/Vietnam/1203/04 was expressed in Pichia pastoris X33 as a 45 kDa protein that displayed a K m of 9.96 ± 1.26 µM with fluorogenic substrate, 2'-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid. Partially purified NA was used for the inhibition and kinetic assays with eight flavonoid compounds and gallic acid. Among them, gallocatechin gallate (GCG) showed the best inhibition against NA with the IC50 of 8.98 ± 0.46 µM and showed a competitive inhibition pattern with K i value of 8.34 ± 0.25 µM. In molecular docking experiments, GCG displayed a binding energy of -13.71 kcal/mol to the active site of NA and the galloyl moiety was required for NA inhibition activity.

In vitro evaluation of novel inhibitors against the NS2B-NS3 protease of dengue fever virus type 4.

The discovery of potent therapeutic compounds against dengue virus is urgently needed. The NS2B-NS3 protease (NS2B-NS3pro) of dengue fever virus carries out all enzymatic activities needed for polyprotein processing and is considered to be amenable to antiviral inhibition by analogy. Virtual screening of 300,000 compounds using Autodock 3 on the GVSS platform was conducted to identify novel inhibitors against the NS2B-NS3pro. Thirty-six compounds were selected for in vitro assay against NS2B-NS3pro expressed in Pichia pastoris. Seven novel compounds were identified as inhibitors with IC50 values of 3.9 ± 0.6-86.7 ± 3.6 µM. Three strong NS2B-NS3pro inhibitors were further confirmed as competitive inhibitors with Ki values of 4.0 ± 0.4, 4.9 ± 0.3, and 3.4 ± 0.1 µM, respectively. Hydrophobic and hydrogen bond interactions between amino acid residues in the NS3pro active site with inhibition compounds were also identified.

Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris.

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42 kDa protein that displayed a K ( m ) of 15 ± 2 µM with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds. The IC(50) of six flavonoid compounds were 47-381 µM. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CL(pro) with IC(50) values of 73, 73 and 47 µM, respectively. GCG showed a competitive inhibition pattern with K ( i ) value of 25 ± 1.7 µM. In molecular docking experiments, GCG displayed a binding energy of -14 kcal mol(-1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.

Inhibitory effects of epigallocatechin gallate and its glucoside on the human intestinal maltase inhibition.

Human intestinal maltase (HMA) is an α-glucosidase responsible for the hydrolysis of α-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA has become an important target in the treatment of type-2 diabetes. In this study, epigallocatechin gallate (EGCG) and EGCG glucoside (EGCG-G1) were identified as inhibitors of HMA by an in vitro assay with IC50 of 20 ± 1.0 and 31.5 ± 1.0 µM, respectively. A Lineweaver-Burk plot confirmed that EGCG and EGCG-G1 were competitive inhibitors of maltose substrate against HMA and inhibition kinetic constants (K i ) calculated from a Dixon plot were 5.93 ± 0.26 and 7.88 ± 0.57 µM, respectively. Both EGCG and EGCG-G1 bound to the active site of HMA with numerous hydrophobic and hydrogen bond interactions.

Phytochemical properties and functional characteristics of wild turmeric (Curcuma aromatica) fermented with Rhizopus oligosporus.

This study investigated the effect of solid-state fermentation of wild turmeric (Curcuma aromatica) with Rhizopus oligosporus, a common fungus found in fermented soy tempeh, on phytochemical and biological properties. Ultra-performance liquid chromatography-tandem mass spectrometry showed that fermented wild turmeric has higher concentrations of curcumin, demethoxycurcumin, bisdemethoxycurcumin, phenolic compounds and total flavonoid-curcuminoid after fermentation for 1-, 3-, and 5-day relative to non-fermented turmeric. The l-carnitine content reached 242 µg g-1 extract after fermentation for 7-day. Wild turmeric had 1.47- and 2.25-fold increases in ORAC and FRAP, respectively, after 3-day fermentation. The inhibitory effects of fermented wild turmeric on lipid accumulation from 3T3-L1 cells, nitric oxide production from lipopolysaccharide-stimulated RAW264.7 murine macrophages, and melanin formation by B16F10 mouse melanoma cells with α-MSH increased 1.08-, 1.44-, and 1.52-fold, respectively, after 3-day fermentation. Based on these results, fermented wild turmeric product can be used as a functional ingredient in the cosmeceutical and nutraceutical industries.

The bifidogenic effects and dental plaque deformation of non-digestible isomaltooligosaccharides synthesized by dextransucrase and alternansucrase.

Non-digestible isomaltooligosaccharides (NDIMOS) are functional food and beverage ingredients that contributed to human health benefits through metabolism of gastrointestinal microorganism. In this study, NDIMOS were synthesized by combine dextransucrase from Leuconostoc mesenteroides B512F/KM and alternansucrase from L. mesenteroides NRRL 1355CF10/KM using sucrose as substrate and maltose as acceptor. Their digestibility was confirmed by using digestive enzymes including α-amylase and amyloglucosidase. NDIMOS inhibited insoluble glucan formation through mutansucrase from Streptococcus mutans. The bifidogenic effect of NDIMOS was investigated by growth of four strains of Bifidobacterium in MRS broth containing NDIMOS, compared with MRS broth contain glucose and negative control. Additionally, Bifidobacterium bifidum or Bifidobacterium adolescentis inhibited the growth of Salmonella enterica serovar typhimurium when they were co-cultivation in MRS broth containing NDIMOS. These results suggested that NDIMOS is potential functional ingredient for food, beverage, and pharmaceutical application.

Virtual screening identification of novel severe acute respiratory syndrome 3C-like protease inhibitors and in vitro confirmation.

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Structure based virtual screening of 308307 chemical compounds was performed using the computation tool Autodock 3.0.5 on a WISDOM Production Environment. The top 1468 ranked compounds with free binding energy ranging from -14.0 to -17.09 kcal mol(-1) were selected to check the hydrogen bond interaction with amino acid residues in the active site of 3CL(pro). Fifty-three compounds from 35 main groups were tested in an in vitro assay for inhibition of 3CL(pro) expressed by Escherichia coli. Seven of the 53 compounds were selected; their IC(50) ranged from 38.57±2.41 to 101.38±3.27 µM. Two strong 3CL(pro) inhibitors were further identified as competitive inhibitors of 3CL(pro) with K(i) values of 9.11±1.6 and 9.93±0.44 µM. Hydrophobic and hydrogen bond interactions of compound with amino acid residues in the active site of 3CL(pro) were also identified.

Discovery of novel inhibitors for human intestinal maltase: virtual screening in a WISDOM environment and in vitro evaluation.

Human intestinal maltase (HMA) is an α-glucosidase that hydrolyses α-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA is an important target to discover of new drugs for the treatment of type 2 diabetes. In this study, 308,307 compounds were virtually screened with HMA using Autodock 3.0.5 in a WISDOM production environment to discover novel inhibitors. The 42 top-scoring free binding energy compounds, representing 17 groups containing potential hydrogen bonding with key residues in the active site pocket of HMA, were tested in vitro for their inhibitory activities against recombinant HMA expressed from Pichia pastoris. Compounds 17 and 18 were competitive inhibitors exclusively for HMA without any in vitro inhibition for human pancreatic α-amylase. The K(i) values were 20 µM for both compound 17 and 18.

Brewing of glucuronic acid-enriched apple cider with enhanced antioxidant activities through the co-fermentation of yeast (Saccharomyces cerevisiae and Pichia kudriavzevii) and bacteria (Lactobacillus plantarum).

Co-fermentation using yeast (Saccharomyces cerevisiae and Pichia kudriavzevii) and the bacteria (Lactobacillus plantarum) as starters isolated from spontaneous sourdough was conducted for the brewing of glucuronic acid (GlcA)-enriched apple cider. The concentration of GlcA in the apple cider co-fermented for 14 d with commercial S. cerevisiae and L. plantarum was 37.7 ± 1.7 mg/mL while a concentration of 62.8 ± 3.1 mg/mL was recorded for fermentation with P. kudriavzevii and L. plantarum, which was higher than the corresponding single yeast fermentation. The co-fermented apple cider revealed higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of 171.67 ± 0.79 µg trolox equivalents (TE)/mL using P. kudriavzevii and L. plantarum, compared to the control (143.89 ± 7.07 µg TE/mL) just using S. cerevisiae. Thus, the co-fermentation of S. cerevisiae and L. plantarum and P. kudriavzevii and L. plantarum provided a new strategy for the development of GlcA-enriched apple cider with enhanced antioxidant capacity.

Characteristics of sourdough bread fermented with Pediococcus pentosaceus and Saccharomyces cerevisiae and its bio-preservative effect against Aspergillus flavus.

Six lactic acid bacteria (LAB) and four yeast strains were isolated from Pyeongchang spontaneous sourdough. In combination with the segregated Saccharomycopsis fibuligera and Saccharomyces cerevisiae, Pediococcus pentosaceus was employed for sourdough bread starters because of its antifungal action against Aspergillus flavus. The sourdough bread fermented with P. pentosaceus and S. cerevisiae displayed 56.4% ± 5.5% antifungal movement counter to A. flavus expansion at 96 h. The concentration of lactic and acetic acids in the sourdough bread was 4.5- and 1.6-folds above the control bread, respectively, contributing to the balanced sensory properties with a fermentation quotient (FQ) of 2.08-2.86. SPME- GC/MS newly distinguished twenty-two volatile compounds including six aldehydes, five alcohols, one phenol, three ketones, one acid, and six esters. The results suggest the P. pentosaceus and S. cerevisiae combination as promising sourdough starters for making enhanced quality bread free of preservatives.

Biflavonoids from Torreya nucifera displaying SARS-CoV 3CL(pro) inhibition.

As part of our search for botanical sources of SARS-CoV 3CL(pro) inhibitors, we selected Torreya nucifera, which is traditionally used as a medicinal plant in Asia. The ethanol extract of T. nucifera leaves exhibited good SARS-CoV 3CL(pro) inhibitory activity (62% at 100µg/mL). Following bioactivity-guided fractionation, eight diterpenoids (1-8) and four biflavonoids (9-12) were isolated and evaluated for SARS-CoV 3CL(pro) inhibition using fluorescence resonance energy transfer analysis. Of these compounds, the biflavone amentoflavone (9) (IC(50)=8.3µM) showed most potent 3CL(pro) inhibitory effect. Three additional authentic flavones (apigenin, luteolin and quercetin) were tested to establish the basic structure-activity relationship of biflavones. Apigenin, luteolin, and quercetin inhibited 3CL(pro) activity with IC(50) values of 280.8, 20.2, and 23.8µM, respectively. Values of binding energy obtained in a molecular docking study supported the results of enzymatic assays. More potent activity appeared to be associated with the presence of an apigenin moiety at position C-3' of flavones, as biflavone had an effect on 3CL(pro) inhibitory activity.

Composition and biochemical properties of ale beer enriched with lignans from Schisandra chinensis Baillon (omija) fruits.

To develop a beverage with high antioxidant capacity and desirable sensory characteristics, Schisandra chinensis (omija) fruits were added to ale type beer at different time points of the brewing process. The phenolic compounds contents in beer were found to be dependent at the moment of the addition of omija fruit. Addition of omija fruits at the initiation of boiling imparted highest oxidative stability to beer and resulted in highest total phenolic and flavonoid contents in ale beer (606.82 mg GAE/L and 406.75 mg QE/L, respectively). The amounts of schisandrin, gomisin A and gomisin B in beer were 12.10 mg/mL, 3.12 mg/mL and 0.86 mg/mL, respectively. Taken together, it is hypothesized that the addition of omija fruits to traditional brewing process can improve the development of value-added beer products.