Inhibitory effects of senkyuchachosan on SARS-CoV-2 papain-like protease activity in vitro.

Papain-like protease (PLpro) enzyme plays a vital role in viral replication as it breaks down polyproteins and disrupts the host's immune response. There are few reports on Kampo formulas that focus on PLpro activity. In this study, we evaluated the inhibitory effects of senkyuchachosan, a traditional Japanese medicine, on PLpro of SARS-CoV-2, the virus responsible for causing COVID-19. We purified the PLpro enzyme and conducted in vitro enzymatic assays using specific substrates. Among the nine crude drugs present in senkyuchachosan, four (Cyperi Rhizoma, Schizonepetae Spica, Menthae Herba, and Camelliae sinensis Folium [CsF]) strongly inhibited PLpro activity. CsF, derived from Camellia sinensis (green tea), contains polyphenols, including catechins and tannins. To confirm that the PLpro inhibitory effects of senkyuchachosan predominantly stem from tannins, the tannins were removed from the decoction using polyvinylpolypyrrolidone (PVPP). The inhibitory effect of senkyuchachosan on PLpro activity was reduced by the removal of PVPP. In addition, the tannin fraction obtained from the CsF extracts showed significant PLpro inhibitory effects. These findings lay the groundwork for the potential development of therapeutic agents that target SARS-CoV-2 infection by intervening in proteolytic cleavage of the virus.

Isolation of Pro-Osteogenic Compounds from Euptelea polyandra That Reciprocally Regulate Osteoblast and Osteoclast Differentiation.

Plants contain a large number of small-molecule compounds that are useful for targeting human health and in drug discovery. Healthy bone metabolism depends on the balance between bone-forming osteoblast activity and bone-resorbing osteoclast activity. In an ongoing study searching for 22 plant extracts effective against osteoporosis, we found that the crude extract of Euptelea polyandra Sieb. et Zucc (E. polyandra) had osteogenic bioactivity. In this study, we isolated two compounds, isoquercitrin (1) and astragalin (2), responsible for osteogenic bioactivity in osteoblastic MC3T3-E1 cells from the leaf of E. polyandra using column chromatography and the spectroscopic technique. This is the first report to isolate astragalin from E. polyandra. Compounds (1) and (2) promoted osteoblast differentiation by increasing alkaline phosphatase (ALP) activity and alizarin red S stain-positive calcium deposition, while simultaneously suppressing tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation in RAW264.7 cells at non-cytotoxic concentrations. Isoquercitrin (1) and astragalin (2) increased the expression of osteoblastic differentiation genes, Osterix, ALP, and Osteoprotegerin in the MC3T3-E1 cells, while suppressing osteoclast differentiation genes, TRAP, Cathepsin K, and MMP 9 in the RAW264.7 cells. These compounds may be ideal targets for the treatment of osteoporosis due to their dual function of promoting bone formation and inhibiting bone resorption.

Inhibition of furin-like enzymatic activities and SARS-CoV-2 infection by osthole and phenolic compounds with aryl side chains.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread as a pandemic and caused damage to people's lives and countries' economies. The spike (S) protein of SARS-CoV-2 contains a cleavage motif, Arg-X-X-Arg, for furin and furin-like enzymes at the boundary of the S1/S2 subunits. Given that cleavage plays a crucial role in S protein activation and viral entry, the cleavage motif was selected as the target. Our previous fluorogenic substrate study showed that osthole, a coumarin compound, inhibits furin-like enzyme activity. In this study, we examined the potential activities of 15 compounds with a structure-activity relationship with osthole, and evaluated their protective ability against SARS-CoV-2 infection. Of the 15 compounds tested, compounds C1 and C2 exhibited the inhibitory effects of osthole against furin-like enzymatic activity; however, little or no inhibitory effects against furin activity were observed. We further examined the inhibition of SARS-CoV-2 activity by compounds C1 and C2 using a Vero E6 cell line that expresses the transmembrane protease serine 2 (TMPRSS2). Compounds C1, C2, and osthole effectively inhibited SARS-CoV-2 infection. Therefore, osthole and its derivatives can potentially be used as therapeutic agents against SARS-CoV-2.

Characterization of Carbohydrates, Amino Acids, Viscosity, and Antioxidant Capacity in Rice Wines Made in Saitama, Japan, with Different Sake Rice.

We investigated the physicochemical properties of Japanese rice wines, including their functional properties and carbohydrate and amino acid content in solution and solid state. Three samples were tested. The glucose, allose, and raffinose contents in samples (A, B, C) in g/100 g were (3.47, 3.45, 7.05), (1.60, 1.63, 1.61), and (2.14, 2.75, 1.49), respectively. The total amino acid in µmol/mL was (3.1, 3.5, 4.4). Glutamic acid, alanine, and arginine varied in content across the samples. The viscosity (10 °C) and activation energy (ΔE) calculated using the Andrade equation were (2.81 ± 0.03, 2.74 ± 0.06, 2.69 ± 0.03) mPa-s and (22.3 ± 1.1, 22.0 ± 0.2, 21.3 ± 0.5) kJ/mol, respectively. Principal component analysis using FT-IR spectra confirmed the separation of the samples into principal components 2 and 3. The IC50 values from the DPPH radical scavenging test were (2364.7 ± 185.3, 3041.9 ± 355.1, 3842.7 ± 228.1) µg/mL. Thus, the three rice wines had different carbohydrate and amino acid contents, viscosities, and antioxidant capacities.

Anti-Helicobacter pylori activity of swertianolin, isolated from swertia herb.

Helicobacter pylori is a Gram-negative, spiral-shaped, motile bacterium present in human stomachs that causes gastric ulcers. A preliminary screening revealed that a methanolic extract of swertia herb demonstrated anti-H. pylori activity. Swertia herb (Swertia japonica Makino, Gentianaceae) is a well-known Japanese traditional medicine to treat gastrointestinal diseases. In this study, we explored the active compounds in methanolic extract of swertia herb. The dried extract was dissolved in water and partitioned with n-hexane, ethyl acetate, and n-butanol, successively. The part soluble in ethyl acetate showed effective anti-H. pylori activity, and two compounds, swertianolin (1) and isoorientin (2), were isolated. The IC50 values of 1, 2, and amoxicillin (AMPC) which is used as positive control were 6.1, 177.0, and 0.044 µM, respectively. The minimum bactericidal concentration (MBC) values of 1 and AMPC were 91.7 and 0.21 µM, respectively. The MBC of 2 could not be determined (> 892.9 µM). Furthermore, synergy was observed when compound 1 was used in combination with AMCP. Therefore, 1 could be considered as one of the active compounds of swertia herb. To our knowledge, the anti-H. pylori activities of methanolic extract of swertia herb and its isolated compound have never been reported.

Effect of Maillard reaction on the quality of clarified butter, ghee.

In Ayurveda, a traditional Indian medicine system, clarified butter is called ghee and is used for food and medicinal purposes. Since butter is subjected to heat to prepare ghee, the heating process affects the ghee quality, such as oxidation, flavor, nutritional value, and biological activity. Therefore, this study focused on the Maillard reaction progress and free-radical scavenging activity with temperature and time during ghee preparation. First, ghee was prepared at low to high temperatures, and its quality (milk fat content, retinol, α-tocopherol, peroxide value, Maillard reaction progress, and free radical scavenging activity) was evaluated. Maillard reaction progress was enhanced at medium and high temperatures (120-160 â„ƒ), and the free radical-scavenging activity of ghee corresponded to the Maillard reaction progress. Since ghee is often reheated during use, we further evaluated the effect of the reheating process. The reheating process did not alter the Maillard reaction progress or the free radical scavenging activity. Our findings serve as good quality control measures for ghee preparation.

1H-NMR-Based Metabolomics for the Classification of the Roots of Paeonia lactiflora, a Constituent of Kampo Medicines.

The root of Paeonia lactiflora (PAEONIAE RADIX) is a constituent of the traditional Japanese medicines (Kampo) and is known to have various effects. Peony roots cultivated in Japan and China are available in the Japanese market for medicinal use. In this study, the chemical diversity of ten available peony roots in the market that differed in their cultivation area was investigated using 1H-NMR metabolomics techniques. Principal component analysis and hierarchical cluster analysis of the 1H-NMR spectra of the peony roots methanolic extracts revealed a clear difference between the metabolic profiles of Japanese and Chinese peony roots. By preparative procedures using chromatography based on 1H-NMR spectra measurements, oxypaeoniflorin and (+)-catechin were found to be specific compounds for Japanese peony root. All peony roots used in this study were listed in the Japanese Pharmacopoeia. Therefore, the differences in the constituents of these peony roots might be attributed to growing conditions than differences in species. Cultivation conditions also influence the quality of natural medicines.

Lignin, an active component in the corn silk water extract, inhibits glycation.

The formation of advanced glycation end products is associated with aging and diabetic complications such as neuropathy, retinopathy, and nephropathy. Thus, the suppression of AGEs formation could prevent and/or treat their related disorders. Corn silk is used as a traditional medicine for the prevention of diabetic complications and treatment of edema in Japan and China. Previous studies revealed the anti-glycation activity of flavonoids in the methanolic extract of corn silk. The anti-glycation activity of the corn silk water extract was higher than that of the methanolic extract; however, the active components of the water extract remained unidentified. The purpose of this study is to make clear the components showing anti-glycation activity in the corn silk water extract and elucidated their structural characteristics. The evaluation of anti-glycation activity was carried out by enzyme-linked immunosorbent assay to detect glycated bovine serum albumin. Remarkable anti-glycation activity was observed in the > 3 kDa fraction. Reversed-phase HPLC analysis of this fraction showed broad peaks characteristic of high-molecular-weight polyphenols. Decomposition reactions did not provide evidence of condensed or acid-hydrolyzable tannins. Therefore, polyphenols contained in the corn silk water extract were considered to be lignin-carbohydrate complex. The 1H- and 13C-nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectroscopy spectra of the > 3 kDa fraction were in agreement with the values reported for lignin. Consequently, we concluded that lignin-carbohydrate complex is one of the active components against glycation in the corn silk water extract.

Effect of Underwater Exercise on Salivary Metabolites of Older Persons With Disability.

BACKGROUND/AIM: Underwater exercise is aimed at preventing aging, maintaining, and improving motor function, and improving physical function. However, its rehabilitation effects have not been well evaluated. In order to gain insight into the molecular basis of its rehabilitation effects, possible changes in the salivary metabolites of four older persons with disability (mean age: 72.5 years) during underwater exercise were investigated. MATERIALS AND METHODS: Halitosis was measured by Breathtron; salivary bacterial number by bacterial counter; amino acids by amino acid analyzer; 8-oxoguanine by ELISA; and intracellular metabolites by capillary electrophoresis, time-of-flight mass spectrometry, liquid chromatography, and triode quadrupole mass spectrometry. RESULTS: Underwater exercise induced apparent declines in two major salivary amino acids (glycine and proline) and bacterial numbers in the cheek mucosa and salivary, without apparent changes in the halitosis and urine 8-oxoguanine concentration. Older subjects showed higher concentrations of most of 166 metabolites compared to young volunteers (mean age: 38.8 years old). Fifteen compounds were significantly reduced with the progression of underwater exercise. CONCLUSION: Improvement of upright balance function with underwater exercise is correlated with several salivary components.

Degradative Effect of Nattokinase on Spike Protein of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged as a pandemic and has inflicted enormous damage on the lives of the people and economy of many countries worldwide. However, therapeutic agents against SARS-CoV-2 remain unclear. SARS-CoV-2 has a spike protein (S protein), and cleavage of the S protein is essential for viral entry. Nattokinase is produced by Bacillus subtilis var. natto and is beneficial to human health. In this study, we examined the effect of nattokinase on the S protein of SARS-CoV-2. When cell lysates transfected with S protein were incubated with nattokinase, the S protein was degraded in a dose- and time-dependent manner. Immunofluorescence analysis showed that S protein on the cell surface was degraded when nattokinase was added to the culture medium. Thus, our findings suggest that nattokinase exhibits potential for the inhibition of SARS-CoV-2 infection via S protein degradation.

Effect of Apple Consumption on Postprandial Blood Glucose Levels in Normal Glucose Tolerance People versus Those with Impaired Glucose Tolerance.

The present study investigated the effect of apple consumption on postprandial blood glucose and insulin levels in subjects with normal versus impaired glucose tolerance. The study participants were ten healthy subjects with no glucose intolerance (normal subjects) (mean, 24.4 ± 4.8 years) and nine subjects with impaired glucose tolerance (mean, 45.2 ± 11.1 years, including 2 on insulin therapy). The test meal included white rice (148 g) and a Fuji apple (150 g). The normal subjects were randomly divided into two groups: the apple-first group, wherein the subjects consumed white rice 5 min after consuming the apple, and the rice-first group, wherein the subjects consumed an apple 5 min after consuming the white rice. Blood samples were then taken from both groups for 3 h. In addition, the subjects with impaired glucose tolerance received the same treatment as the normal subjects, with the difference being glucose level monitoring according to the order in which the apples were consumed. In the normal subjects, the Cmax of Δblood glucose and Δinsulin levels were 54.0 ± 5.0 mg/dL and 61.9 ± 7.2 µU/dL versus 46.2 ± 5.9 mg/dL and 49.8 ± 8.5 µU/dL in the rice-first and apple-first groups, respectively. The incremental area under the curve (iAUC) of insulin tended to decrease in the apple-first group. In the impaired glucose tolerance subjects, the Cmax of Δblood glucose was 75.2 ± 7.2 mg/dL in the apple-first group compared to 90.0 ± 10.0 mg/dL in the rice-first group, which was a significant difference (p < 0.05). The iAUC of blood glucose was lower in the apple-first group. Eating an apple before a meal may be a simple and effective strategy for managing the glycaemic response in individuals with impaired glucose tolerance.

Fruit body formation and intra-species DNA polymorphism in Japanese Wolfiporia cocos strains.

Poria, the dried sclerotium of Wolfiporia cocos, is a medicinal mushroom that is widely used in traditional Japanese medicine. The fruit body of W. cocos is rarely found in the natural environment in Japan, therefore an optimized technique for fruit body formation is essential for producing new strains through crossbreeding and for biological research. Here, we developed a cultivation technique for fruit body formation of W. cocos using three strains collected from different areas of Japan. When mycelia were cultured on sawdust-based medium after liquid medium culture, all strains successfully formed fruit bodies as a brown honeycomb-like structure. Furthermore, we analyzed single nucleotide polymorphisms of the three strains using the STE3-like pheromone receptor protein gene, STE3.2, and found a genetic marker for discriminating one strain from the others. The results are expected to promote extensive studies on crossbreeding and domestic production of W. cocos.

Comparison of various commercially available cinnamon barks using NMR metabolomics and the quantification of coumarin by quantitative NMR methods.

Cinnamon bark is an important spice worldwide. In this study, the chemical diversity of various commercially available cinnamon barks that differed in their production areas and utility applications (culinary spice or medicines) were investigated by the use of 1H NMR metabolomics. Our results indicated that principle component analysis (PCA) and hierarchical cluster analysis (HCA) of the 1H NMR spectra of the cinnamon bark methanolic extracts including the deduction of their species by nucleotide sequence analysis enabled differentiation of the cinnamon barks according to their species, production areas and utility applications. The constituents of Vietnam cinnamon were found to differ significantly from the other samples investigated based on PCA score plots and HCA constellation dendrograms. Coumarin was found to be a key compound for the discrimination of Vietnamese cinnamon by multivariate analysis of the 1H NMR spectral data and direct comparison of the 1H NMR spectra. In addition, coumarin was quantified using quantitative NMR methods. As a result, coumarin was contained in Vietnamese cinnamon at a higher level compared to other cinnamons. This study indicated that 1H NMR metabolomics could deduce spices, utility, and producing area of commercially available cinnamon barks. Furthermore, combining quantitative 1H NMR methods with 1H NMR metabolomics enable quantification of coumarin in cinnamon bark on a single measurement.

Inhibitory effect of honokiol on furin-like activity and SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a pandemic and has caused damage to the lives of the people and economy of countries. However, the therapeutic reagents against SARS-CoV-2 remain unclear. The spike (S) protein of SARS-CoV-2 contains a cleavage motif at the S1/S2 boundary, known to be cleaved by furin. As cleavage is essential for S protein activation and viral entry, furin was selected as the target compound. In this study, we examined the inhibitory effects of two lignans (honokiol and magnolol) on furin-like enzymatic activity using a fluorogenic substrate with whole-cell lysates. Of two compounds tested, honokiol partially inhibited furin-like enzymatic activity. We further examined the anti-SARS-CoV-2 activity of honokiol using VeroE6 cell line, which is stably expressing a transmembrane protease serine 2 (TMPRSS2). It was shown that honokiol exhibited remarkable inhibition of SARS-CoV-2 infection. Therefore, honokiol and crude drugs which contain honokiol such as Magnolia species have a potential therapeutic reagents for SARS-CoV-2.

Screening for inhibitory effects of crude drugs on furin-like enzymatic activities.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a cleavage motif R-X-X-R for furin-like enzymes at the boundary of the S1/S2 subunits. The cleavage of the site by cellular proteases is essential for S protein activation and virus entry. We screened the inhibitory effects of crude drugs on in vitro furin-like enzymatic activities using a fluorogenic substrate with whole-cell lysates. Of the 124 crude drugs listed in the Japanese Pharmacopeia, aqueous ethanolic extract of Cnidii Monnieris Fructus, which is the dried fruit of Cnidium monnieri Cussion, significantly inhibited the furin-like enzymatic activities. We further fractionated the plant extract and isolated the two active compounds with the inhibitory activity, namely, imperatorin and osthole, whose IC50 values were 1.45 mM and 9.45 µM, respectively. Our results indicated that Cnidii Monnieris Fructus might exert inhibitory effects on furin-like enzymatic activities, and that imperatorin and osthole of the crude drug could be potential inhibitors of the motif cleavage.

Cyanobacterial branching enzymes bind to α-glucan via surface binding sites.

Besides their catalysis, specific interactions between starch/glycogen processing enzymes and their substrates have been reported. Multiple branching enzyme (BE) isoforms, BE1, BE2, and BE3, have been found in a limited number of cyanobacterial species that are characterized by amylopectin accumulation. Seven surface binding sites (SBSs) located away from the active site have been identified in crystal structures of cyanobacterial BE1 from Crocosphaera subtropica (Cyanothece sp.) ATCC 51142 (51142BE1). In the present study, binding affinity toward amylopectin, amylose, and glycogen was investigated for wild-type 51142BE1 and its mutants (residues at SBSs important for sugar-binding were replaced by alanine). These enzymes showed retarded mobility during electrophoresis in non-denaturing polyacrylamide gels in the presence of polysaccharides. This was caused by interactions between the enzymes and the polysaccharides, enabling calculation of the dissociation constants (Kd values) of the enzymes toward the polysaccharides. Mutational analysis indicated that particular domains of the protein (domains A and C) were involved in the polysaccharide binding. Kd values toward the polysaccharides were also measured for 10 BE isoforms (five BE1, three BE2, and two BE3) from 5 cyanobacterial strains. All BEs displayed much lower Kd values (higher affinity) toward amylopectin and amylose than toward glycogen, as described for plant BEs. In addition, one BE2 displayed exceptionally high Kd values (low affinity), while two BE3 exhibited multiple Kd values to all polysaccharides. These results could be ascribed to sequence variations in the SBSs, irrespective of the catalytic specificity.

Use of 13C-NMR Chemical Shifts; Application of Principal Component Analysis for Categorizing Structurally Similar Methoxyflavones and Correlation Analysis between Chemical Shifts and Cytotoxicity.

The 13C-NMR spectral data for the 15-carbon flavonoid skeleton in eleven methoxyflavones isolated from Kaempferia parviflora (Zingiberaceae) were processed by principal component analysis (PCA). Based on the PCA score plots, the methoxyflavones were categorized into three groups according to their structural features. The cytotoxicities of the methoxyflavones toward 3T3-L1 murine preadipocyte cells were evaluated by 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and found to differ according to structure. The relationship between the 13C-NMR chemical shifts of the methoxyflavones and their cytotoxicities was investigated using Pearson's correlation analysis. The 13C-NMR signal at C-10, a quaternary carbon, was correlated with cytotoxicity. Based on these results, a structural design which lowers the 13C-NMR chemical shift at C-10 would be important for the development of cytotoxic compounds. Although quantitative structure-activity and structure-property relationships are well established paradigms for predicting trends among a series of compounds, quantitative property-activity relationships have been relatively unstudied. This approach offers a new strategy for directing structure-activity relationship research.

Cannabidiol Content and In Vitro Biological Activities of Commercial Cannabidiol Oils and Hemp Seed Oils.

Background: Hemp (Cannabis sativa L.) seed contains high contents of various nutrients, including fatty acids and proteins. Cannabidiol (CBD) is a non-psychoactive compound that can be extracted from C. sativa and used for treating epilepsy and pain. Industrial hemp products, including CBD and hemp seed oils, have become increasingly popular. Some products are marketed without a clear distinction between CBD and hemp seed oils. Herein, the CBD content and biological activities of commercial CBD and hemp seed oils were examined. Methods: CBD content was measured by high-performance liquid chromatography. For in vitro antioxidant activity determination, 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging assays were performed. Results: The CBD concentrations in the two CBD oil samples were 18.9 ± 0.5 and 9.2 ± 0.4 mg/mL. Of the seven hemp seed oil samples, six samples contained CBD in concentrations ranging from 2.0 ± 0.1 to 20.5 ± 0.5 µg/mL, but it was not detected in one sample. Antioxidant activity was observed in both CBD oil samples. Conclusions: The results indicate that (1) CBD content varied by hemp seed oil sample and that (2) antioxidant activity could be a useful landmark for discriminating CBD oils from hemp seed oils.

Physical Properties of an Ionic Liquid Composed of Two Water-Soluble Vitamins and Enhanced Skin Permeation of Both Vitamins.

: A highly viscous substance was prepared by evaporating an ethanol solution containing two hydrophilic vitamins; vitamin C, and vitamin B6. The viscous substance and physical mixture of the two vitamins were tested using a differential scanning calorimeter and an X-ray diffractometer. The highly viscous substance was found to be a liquid crystal (LC) made of these two hydrophilic vitamins. Determination by proton nuclear magnetic resonance measurement suggested that intramolecular hydrogen bonding in vitamin B6 was eliminated by the LC formation. This LC compound showed high solubility in 1,3-butanediol (almost 87%). Much higher skin permeation of both vitamin C and B6 was also observed from the LC compound than that from the physical mixture. The present LC compound containing vitamin C and vitamin B6 may be useful for pharmaceutical and cosmeceutical applications.

Improving quality control of yucca extracts used as food additives by screening antimicrobial activity using NMR metabolomics.

Yucca schidigera is mainly distributed in southwestern US and the northern desert of Mexico. Its extract is widely used as a food additive for its antimicrobial activity. However, this antimicrobial activity is subject to significant variability across production lots. Yucca extracts are natural products and their composition is affected by their cultivation area and weather. Manufacturer deal with natural products such as food additives pay particularly close attention to quality control. In the present study, NMR metabolomics methods were used to screen the antimicrobial activity of yucca extracts. Yucca extracts were subjected to principal component analysis (PCA) and categorized on a score plot of their 1H NMR spectral data according to their antimicrobial activity. Furthermore, hierarchical cluster analysis (HCA) was also used to classify yucca extracts based on their antimicrobial activity. Classification using PCA and HCA was dependent upon saponin content, particularly that of schidigera-saponin A1 and D1, which was further confirmed by HPLC analysis of the yucca extracts. We demonstrated that NMR-based metabolomics is a potentially useful tool to use in combination with conventional quality control methods for yucca extracts used as food additives. We envisage this method as tool for initially screening the extracts prior to carrying out the officially recommended quality control tests.