Inhibition of Protease and Egg Hatching of Haemonchus contortus by Soybean Seed Exudates.

Gastrointestinal nematode infection of small ruminants causes losses in livestock production. Plant compounds show promises as alternatives to commercial anthelmintics that have been exerting selective pressures that lead to the development of drug-resistant parasites. Soybean (Glycine max) is an economical value crop, with a higher protein content compared to other legumes. The objective of this study was to evaluate whether the protease inhibitors exuded from the G. max mature seeds have anthelmintic activity against Haemonchus contortus. To obtain the soybean exudates (SEX), mature seeds were immersed in 100 mM sodium acetate buffer, pH 5.0, at 10 C, for 24 hr. Then the naturally released substances present in SEX were collected and exhaustively dialyzed (cutoff 12 kDa) against distilled water. The dialyzed seed exudates (SEXD) were heated at 100 C for 10 min and centrifuged (12,000 g, at 4 C for 15 min). The supernatant obtained was recovered and designated as the heat-treated exudate fraction (SEXDH). The protein content, protease inhibitor activity, and the effect of each fraction on H. contortus egg hatch rate were evaluated. The inhibition extent of SEX, SEXD, and SEXDH on H. contortus egg proteases was 31.1, 42.9, and 63.8%, respectively. Moreover, SEX, SEXD, and SEXDH inhibited the egg hatching with EC50 of 0.175, 0.175, and 0.241 mg ml-1, respectively. Among the commercial protease inhibitors tested, only EDTA and E-64 inhibited the H. contortus hatch rate (79.0 and 28.9%, respectively). We present evidence demonstrating that soybean exudate proteins can effectively inhibit H. contortus egg hatching. This bioactivity is displayed by thermostable proteins and provides evidence that protease inhibitors are a potential candidate for anthelmintic use.

An Updated Review on Betacoronavirus Viral Entry Inhibitors: Learning from Past Discoveries to Advance COVID-19 Drug Discovery.

Even after one year of its first outbreak reported in China, the coronavirus disease 2019 (COVID-19) pandemic is still sweeping the World, causing serious infections and claiming more fatalities. COVID-19 is caused by the novel coronavirus SARS-CoV-2, which belongs to the genus Betacoronavirus (ß-CoVs), which is of greatest clinical importance since it contains many other viruses that cause respiratory disease in humans, including OC43, HKU1, SARS-CoV, and MERS. The spike (S) glycoprotein of ß-CoVs is a key virulence factor in determining disease pathogenesis and host tropism, and it also mediates virus binding to the host's receptors to allow viral entry into host cells, i.e., the first step in virus lifecycle. Viral entry inhibitors are considered promising putative drugs for COVID-19. Herein, we mined the biomedical literature for viral entry inhibitors of other coronaviruses, with special emphasis on ß-CoVs entry inhibitors. We also outlined the structural features of SARS-CoV-2 S protein and how it differs from other ß-CoVs to better understand the structural determinants of S protein binding to its human receptor (ACE2). This review highlighted several promising viral entry inhibitors as potential treatments for COVID-19.

Potential Leads from Liquorice Against SARS-CoV-2 Main Protease using Molecular Docking Simulation Studies.

AIM AND OBJECTIVE: At present, the world is facing a global pandemic threat of SARSCoV- 2 or COVID-19 and to date, there are no clinically approved vaccines or antiviral drugs available for the treatment of coronavirus infections. Studies conducted in China recommended the use of liquorice (Glycyrrhiza species), an integral medicinal herb of traditional Chinese medicine, in the deactivation of COVID-19. Therefore, the present investigation was undertaken to identify the leads from the liquorice plant against COVID-19 using molecular docking simulation studies. MATERIALS AND METHODS: A set of reported bioactive compounds of liquorice were investigated for COVID-19 main protease (Mpro) inhibitory potential. The study was conducted on Autodock vina software using COVID-19 Mpro as a target protein having PDB ID: 6LU7. RESULTS: Out of the total 20 docked compounds, only six compounds showed the best affinity towards the protein target, which included glycyrrhizic acid, isoliquiritin apioside, glyasperin A, liquiritin, 1-methoxyphaseollidin and hedysarimcoumestan B. From the overall observation, glycyrrhizic acid followed by isoliquiritin apioside demonstrated the best affinity towards Mpro representing the binding energy of -8.6 and -7.9 Kcal/mol, respectively. Nevertheless, the other four compounds were also quite comparable with the later one. CONCLUSION: From the present investigation, we conclude that the compounds having oxane ring and chromenone ring substituted with hydroxyl 3-methylbut-2-enyl group could be the best alternative for the development of new leads from liquorice plant against COVID-19.

GB-2 inhibits ACE2 and TMPRSS2 expression: In vivo and in vitro studies.

After the first case of Coronavirus disease 2019 (COVID-19) was reported in Wuhan, COVID-19 has rapidly spread to almost all parts of world. Angiotensin converting enzyme 2 (ACE2) receptor can bind to spike protein of SARS-CoV-2. Then, the spike protein of SARS-CoV-2 can be cleaved and activated by transmembrane protease, serine 2 (TMPRSS2) of the host cells for SARS-CoV-2 infection. Therefore, ACE2 and TMPRSS2 are potential antiviral targets for treatment of prevention of SARS-CoV-2 infection. In this study, we discovered that 10-250 µg/mL of GB-2, from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, can inhibit ACE2 mRNA expression and ACE2 and TMPRSS2 protein expression in HepG2 and 293 T cells without cytotoxicity. GB-2 treatment could decrease ACE2 and TMPRSS2 expression level of lung tissue and kidney tissue without adverse effects, including nephrotoxicity and hepatotoxicity, in animal model. In the compositions of GB-2, we discovered that 50 µg/mL of theaflavin could inhibit protein expression of ACE2 and TMPRSS2. Theaflavin could inhibit the mRNA expression of ACE2. In conclusion, our results suggest that GB-2 and theaflavin could act as potential compounds for ACE2 and TMPRSS2 inhibitors in the further clinical study.

In Silico Identification of Potential Natural Product Inhibitors of Human Proteases Key to SARS-CoV-2 Infection.

Presently, there are no approved drugs or vaccines to treat COVID-19, which has spread to over 200 countries and at the time of writing was responsible for over 650,000 deaths worldwide. Recent studies have shown that two human proteases, TMPRSS2 and cathepsin L, play a key role in host cell entry of SARS-CoV-2. Importantly, inhibitors of these proteases were shown to block SARS-CoV-2 infection. Here, we perform virtual screening of 14,011 phytochemicals produced by Indian medicinal plants to identify natural product inhibitors of TMPRSS2 and cathepsin L. AutoDock Vina was used to perform molecular docking of phytochemicals against TMPRSS2 and cathepsin L. Potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of TMPRSS2 and cathepsin L. Further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. This led to the identification of 96 inhibitors of TMPRSS2 and 9 inhibitors of cathepsin L among phytochemicals of Indian medicinal plants. Further, we have performed molecular dynamics (MD) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of TMPRSS2 namely, qingdainone, edgeworoside C and adlumidine, and of cathepsin L namely, ararobinol, (+)-oxoturkiyenine and 3α,17α-cinchophylline. Interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. Further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here.

Polyprenylated acylphloroglucinols as deubiquitinating protease USP7 inhibitors from Hypericum hookerianum.

Hookerianones A - E (1-5), five new polyprenylated acylphloroglucinols (PPAPs), along with six known ones, were isolated from the aerial parts of Hypericum hookerianum. Their structures were elucidated by analyses of MS, NMR, chiroptical properties, biogenetic pathway, and/or single crystal X-ray diffraction. A ubiquitin-rhodamine 110 assay showed that furohyperforin and hypercalin C, two representative PPAPs in this plant, inhibited more than 90% USP7 at the concentration of 10 µM.

Syzygium samarangense Volatile Oil Inhibited Bacteria Growth and Extracellular Protease of Salmonella typhimurium.

BACKGROUND AND OBJECTIVE: Medicinal plants are fast becoming essential pharmaceuticals for disease and infection management. The vast antimicrobial properties of these plants reside in the inhibitory properties of their endogenous secondary metabolites. Therefore, this study aimed to assess if the volatile oil of Syzygium samarangense inhibits enteric bacteria growth and its effect against the caseinolytic activity of the extracellular protease of Salmonella typhimurium. MATERIALS AND METHODS: The volatile oil was extracted by hydrodistillation, while the antimicrobial assay was assessed with the microdilution method. The extracellular protease was partially purified by salting out, followed by size-exclusion chromatography. The mode of inhibition of this enzyme was deduced from the enzyme-substrate kinetics using a line-weaver burke plot. RESULTS: The antimicrobial properties of the oil were reported against ten enteric bacteria. Proteus vulgaris has the highest IC50 value of 0.75±0.004% v/v, while S. typhimurium, the most sensitive bacterium, showed the lowest IC50 value of 0.17±0.005% v/v. The extracellular protease of S. typhimurium was partially purified to achieve 3.73 purification fold and 314.2 µmol min-1 mg-1 protein. The optimal caseinolytic activity of this enzyme was found at pH 7.5 and 40 °C. The protease showed significantly higher activity in the presence of Zn2+ (9.3±0.33 U min-1) as compared to the control (7.0±0.58 U min-1) (p<0.05), however, K+, Ca2+, Co2+, Ba2+, Pb2+ and Hg2+ considerably reduced the enzyme activity. The activity of this enzyme was competitively inhibited by the volatile oil as an inhibitor. CONCLUSION: The volatile oil of S. samarangense inhibited a wide range of enteric bacteria and, therefore proposed as a potential antimicrobial agent. Inhibiting the extracellular protease of S. typhimurium may be one of its modes of action against these pathogens.

Proteomic Comparison of Three Extraction Methods Reveals the Abundance of Protease Inhibitors in the Seeds of Grass Pea, a Unique Orphan Legume.

Grass pea is an orphan legume that is grown in many places in the world. It is a high-protein, drought-tolerant legume that is capable of surviving extreme environmental challenges and can be a sole food source during famine. However, grass pea produces the neurotoxin ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a neurological disease. This crop is promising as a food source for both animals and humans if ß-ODAP levels and other antinutritional factors such as protease inhibitors are lowered or removed. To understand more about these proteins, a proteomic analysis of grass pea was conducted using three different extraction methods to determine which was more efficient at isolating antinutritional factors. Seed proteins extracted with Tris-buffered saline (TBS), 30% ethanol, and 50% isopropanol were identified by mass spectrometry, resulting in the documentation of the most abundant proteins for each extraction method. Mass spectrometry spectral data and BLAST2GO analysis led to the identification of 1376 proteins from all extraction methods. The molecular function of the extracted proteins revealed distinctly different protein functional profiles. The majority of the TBS-extracted proteins were annotated with nutrient reservoir activity, while the isopropanol extraction yielded the highest percentage of endopeptidase proteinase inhibitors. Our results demonstrate that the 50% isopropanol extraction method was the most efficient at isolating antinutritional factors including protease inhibitors.

Jabuticaba (Plinia jaboticaba) skin extracts as inhibitors of phospholipases A2 and proteases.

The phenolic extracts of jabuticaba skin flour (JSF) were characterized by HPLC, and evaluated for their modulating action upon phospholipases A2 and proteases of snake venom, aiming at their possible use in the treatment of the various diseases associated with the action of venom toxins. Two types of extracts were prepared from JSF: aqueous and methanolic. These extracts, evaluated at different ratios, (venom: extract, m/m), significantly inhibited the phospholipase activity induced by the venom of Bothrops moojeni and Crotalus durissus terrificus, except for Bothrops atrox venom. The greatest hemolysis inhibitory action was observed for the methanolic extract, when incubated with venoms of B. moojeni and C. durissus terrificus, with inhibitions between 21 and 100%. Thrombolysis induced by venoms of B. moojeni and C. durissus terrificus was inhibited by both extracts, ranging from 32 to 83% and 51 to 83% for the aqueous and methanolic extracts, respectively. Both extracts extended coagulation time, induced by the venoms of B. moojeni and Lachesis muta muta. Inhibitory actions are related to phenolic compounds, such as gallic, syringic and p-coumaric acids, besides catechin, epigallocatechin gallate, epicatechin; resveratrol and quercetin, present in the extracts of jabuticaba skin flour, confirming their potential for nutraceutical use.

Water-soluble extracts from edible mushrooms (Agaricus bisporus) as inhibitors of hepatitis C viral replication.

Hepatitis C virus (HCV) is the main agent responsible for chronic liver disease. Recent advances in anti-HCV treatment strategies have significantly increased the viral clearance rate (>90%). However, sustained antiviral responses vary in different cohorts, and high costs limit the broad use of direct-acting antivirals (DAAs). The goal of this study is to evaluate the inhibitory ability of well characterized (LC-QTOF-MS/MS) aqueous extracts obtained from edible mushrooms (Agaricus bisporus) to diminish HCV viral replication. Our data have demonstrated an in vitro inhibitory effect of A. bisporus extracts on NS3/4A protease and HCV replication. Fractionation by ultra-filtration and sequential liquid-liquid extraction showed that the compounds responsible for the inhibition are water-soluble with low molecular weights (<3 kDa) and that action could be through the following five compounds: ergothioneine, adenine, guanine, hypoxanthine, and xanthine, which are present in all fractions (UF-3, AqF-3 kDa and organic fractions) showing NS3/4A inhibition. Low molecular weight aqueous extracts (<3 kDa) from A. bisporus have potential applications in the prophylaxis and treatment of HCV, especially for patients who do not have access to the last generation of DAAs. They may be useful as well for other flaviviruses, which also possess a NS3 serine protease.

Purification and characterization of a cystatin like thiol protease inhibitor from Brassica nigra.

Phytocystatins or plant cystatins belong to a group of thiol protease inhibitors present ubiquitously in living system. They play a crucial role in cellular protein turnover thereby showing involvement in a wide array of physiological processes in plants. With wide importance and tremendous potential applications in the fields of genetic engineering, medicine, agriculture, and food technology, it is imperative to identify and isolate such protease inhibitors from different cheap and easily available plant sources. Present study focuses on the isolation, purification and characterization of a cystatin like thiol protease inhibitor from the seeds of Brassica nigra (rai mustard) following a simple two-step method using ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with 51.85% yield and 151.50 fold purification. Rai seed cystatin (RSC) gave a molecular mass of ~19.50 kDa as determined by SDS PAGE and gel filtration behaviour. Stokes radius and diffusion coefficient of RSC were 19.80 Šand 11.21 × 10-7 cm2 s-1 respectively. Kinetic analysis revealed a reversible and non-competitive mode of inhibition with RSC showing highest inhibition towards papain (Ki = 1.62 × 10-7 M) followed by ficin and bromelain. Purified RSC possessed an α helical content of 35.29% as observed by far-UV CD spectroscopy. UV, fluorescence, CD and FTIR spectral studies revealed a significant conformational alteration in one or both the proteins upon RSC-papain complex formation. Isothermal Titration Calorimetry (ITC) analysis further revealed the values for different thermodynamic parameters involved in complex formation, indicating the process to be enthalpically as well as entropically driven with forces involved in binding the proteins to be electrostatic in nature. Additionally binding stoichiometry (N) of 0.95 ±â€¯0.08 sites indicates that each molecule of RSC is surrounded by nearly one papain molecule.

A Bowman-Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage.

Staphylococcus aureus is a multidrug-resistant bacterium responsible for several cases of hospital-acquired infections, which constitute a global public health problem. The introduction of new healthcare strategies and/or the discovery of molecules capable of inhibiting the growth or killing S. aureus would have a huge impact on the treatment of S. aureus-mediated diseases. Herein, a Bowman-Birk protease inhibitor ( LzaBBI), with strong in vitro antibacterial activity against S. aureus, was purified to homogeneity from Luetzelburgia auriculata seeds. LzaBBI in its native form is a 14.3 kDa protein and has a pI of 4.54, and its NH2-terminal sequence has high identity with other Bowman-Birk inhibitors. LzaBBI showed a mixed-type inhibitory activity against both trypsin and chymotrypsin, respectively, and it remained stable after both boiling at 98 °C for 120 min and incubation at various pHs. Scanning electron microscopy revealed that LzaBBI disrupted the S. aureus membrane integrity, leading to bacterial death. This study suggests that LzaBBI is a powerful candidate for developing a new antimicrobial to overcome drug resistance toward reducing hospital-acquired infections caused by S. aureus.

Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.

Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.

Biochemical characterization of the YBPCI miniprotein, the first carboxypeptidase inhibitor isolated from Yellow Bell Pepper (Capsicum annuum L). A novel contribution to the knowledge of miniproteins stability.

The cystine-knot metallocarboxypeptidase inhibitors (MCPIs) are peptides that contribute to control proteolytic activity, involved in storage, growth and maintenance of plants. Lately studies reported several MCPIs with potential use in biomedical applications; as anti-cancer, anti-thrombotic, anti-malaric and anti-angiogenic agents. We report the isolation, purification, chemical stability and biochemical characterization of a novel carboxypeptidase A inhibitor (YBPCI) isolated from Capsicum annuum L. var. Yellow Bell Pepper, the first cystine-knot miniprotein (CKM) of the species. We demonstrate the stability of YBPCI (IC50 = 0.90 µg/ml) to high temperatures, high salt concentration and extreme pH values. MALDI-TOF/MS analysis detected a molecular weight of 4057 Da, and peptide mass fingerprint resulted in no matches with other protease inhibitors. In vitro gastrointestinal digestion subjecting YBPCI to pH 2 incubation and proteolytic attack resulted in complete inhibitory activity. To summarize, there are no reports to date of carboxypeptidase inhibitors in C. annuum species, giving our report much more relevance.

Antioxidant and Proteinase Inhibitory Activities of Selected Poppy (Papaver somniferum L.) Genotypes.

Poppy seeds (Papaver somniferum L.) belong to tasty food ingredients however, they should be considered also as valuable source of biologically active compounds. Content of selected metabolites, antioxidant and proteinase inhibitory activities were analyzed in vitro in extracts from seeds of fifteen poppy genotypes. Considerable variation in all parameters was detected within the set of analyzed poppy genotypes. The genotype Major expressed the highest antioxidant activity determined by all four methodological approaches (DPPH, ABTS, FRAP, RP). The genotype MS 423 exhibited the highest inhibitory activities against trypsin, thrombin and collagenase. Very specific position among all had the genotype Redy. Its grain extract reached significantly high levels in 9 out of 14 measured parameters (TPC, TFC, TTC, TAC, FRAP, RP, inhibitory activities against trypsin, thrombin, collagenase). Edible grains of poppy are valuable source of natural compounds which may be beneficial in pathological states associated with oxidative stress or increased proteinase activities.

Biochemical, immunological and kinetic characterization and partial sequence analysis of a thiol proteinase inhibitor from Bubalus bubalis kidney: An attempt targeting kidney disorders.

In the present study a thiol proteinase inhibitor was isolated from buffalo kidney making use of ammonium sulphate precipitation and gel filtration chromatography on Sephacryl S-100HR column. Purified inhibitor is homogeneous as it displayed a single band in gel electrophoresis both under reducing and non-reducing environment and is of 65KDa as revealed by gel filtration and SDS PAGE. Kinetic studies revealed the presence of reversible accompanied with competitive mode of inhibition; showing maximum efficacy against papain (Ki=2.90×10-4). It was maximally active at pH 8.0 and was stable for a period of 30, 60 and 90 days at 37, 4 and -20°C respectively. Immunological studies confirmed its purity of epitopes as a single precipitin line is obtained in immunodiffusion. N-terminal analysis revealed that it shared a good homology with mouse kidney cystatin as well as with Human Cys C and Cys E thereby advocating its use as a model for various human oriented studies which targets how the kidney cystatin level varies in accordance with various drugs that are currently being used as a target for variety of diseases.

Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity.

BACKGROUND: The plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study. METHODS: E. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC-MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico. RESULTS: The bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A(2), hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187% to 107%. Significant inhibition (93%) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom. CONCLUSION: For the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity. GENERAL SIGNIFICANCE: Exploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available.

The Daiokanzoto (TJ-84) Kampo Formulation Reduces Virulence Factor Gene Expression in Porphyromonas gingivalis and Possesses Anti-Inflammatory and Anti-Protease Activities.

Kampo formulations used in Japan to treat a wide variety of diseases and to promote health are composed of mixtures of crude extracts from the roots, bark, leaves, and rhizomes of a number of herbs. The present study was aimed at identifying the beneficial biological properties of Daiokanzoto (TJ-84), a Kampo formulation composed of crude extracts of Rhubarb rhizomes and Glycyrrhiza roots, with a view to using it as a potential treatment for periodontal disease. Daiokanzoto dose-dependently inhibited the expression of major Porphyromonas gingivalis virulence factors involved in host colonization and tissue destruction. More specifically, Daiokanzoto reduced the expression of the fimA, hagA, rgpA, and rgpB genes, as determined by quantitative real-time PCR. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to evaluate the anti-inflammatory properties of Daiokanzoto. Daiokanzoto attenuated the P. gingivalis-mediated activation of the NF-κB signaling pathway. It also reduced the secretion of pro-inflammatory cytokines (IL-6 and CXCL8) by lipopolysaccharide-stimulated oral epithelial cells and gingival fibroblasts. Lastly, Daiokanzoto, dose-dependently inhibited the catalytic activity of matrix metalloproteinases (-1 and -9). In conclusion, the present study provided evidence that Daiokanzoto shows potential for treating and/or preventing periodontal disease. The ability of this Kampo formulation to act on both bacterial pathogens and the host inflammatory response, the two etiological components of periodontal disease, is of high therapeutic interest.

Some properties and possible biological role of peptidase inhibitors from the entomopathogenic fungus Tolypocladium cylindrosporum.

The activities of secreted and mycelial inhibitors of proteolytic enzymes from fungi of the order Hypocreales have been investigated. Inhibitors of bromelain, papain, and trypsin of low molecular mass (about 1 kDa) and a subtilisin proteinaceous inhibitor with molecular mass of 45 kDa were revealed in the culture liquid of the fungus Tolypocladium cylindrosporum. The subtilisin inhibitor from T. cylindrosporum has antibiotic properties, significantly decreased the activity of purified bacterial enzymes, and prevented the growth of the bacterium Pseudomonas sp. Data suggesting the existence in fungi of the Hypocreales order of two pools of peptidase inhibitors have been obtained.

Isolation of cholinesterase and ß-secretase 1 inhibiting compounds from Lycopodiella cernua.

Three new serratene-type triterpenoids (1-3) and a new hydroxy unsaturated fatty acid (13) together with nine known compounds (4-12) were isolated from Lycopodiella cernua. The chemical structures were established using NMR, MS, and Mosher's method. Compound 13 showed the most potent inhibitory activity against acetylcholinesterase (AChE) with an IC50 value of 0.22µM. For butyrylcholinesterase (BChE) inhibitory activity, 5 showed the most potent activity with an IC50 value of 0.42µM. Compound 2 showed the most potent activity with an IC50 of 0.23µM for BACE-1 inhibitory activity. The kinetic activities were investigated to determine the type of enzyme inhibition involved. The types of AChE inhibition shown by compounds 4, 5, and 13 were mixed; BChE inhibition by 5 was competitive, while 2 and 6 showed mixed-types. In addition, molecular docking studies were performed to investigate the interaction of these compounds with the pocket sites of AChE. The docking results revealed that the tested inhibitors 3, 4, and 13 were stably present in several pocket domains of the AChE residue.