In vitro Evaluation of Skin Related Enzyme Inhibitory Effect and Emulgel Formulation Development Studies of Onobrychis Argyrea subsp. Argyrea with Phytochemical Analysis.

Species of Onobrychis have been used to treat skin disorders such as wounds and cuts in folk medicine and Onobrychis argyrea subsp. argyrea (OA) commonly known as 'silvery sainfoin', is a member of this genus. In this study, it was aimed to investigate the skin-related biological activities and phytochemical characterization of OA. Moreover, an emulgel formulation was developed from the main methanolic extract of the plant (OAM). Initially, to identifiy of the active fractions, aerial parts of the plant material was extracted with methanol and fractionated by n-hexane, chloroform, ethyl acetate and n-butanol, respectively. Antioxidant activity was determined by CUPRAC, TOAC, FRAP and DPPH assays. Thereafter, the inhibition potential of OAM, novel formulation and all fractions was measured against elastase, collagenase, tyrosinase and hyaluronidase enzymes. OAM was analyzed and characterized by LC/MS-MS. The major bioactive flavonoids which are rutin and isoquercetin were measured and compared as qualitative and quantitative via high performance thin layer chromatography (HPTLC) analysis in OAM and fractions. The results showed that extracts of OA can be a potential cosmeceutical agent for skin related problems.

Chemical Profiling and Enzyme Inhibitory Activities of Essential Oil Isolated from Pistacia khinjuk Leaves: Insights On GC-MS Analysis and Skin Aging-Relevant Enzymes.

Pistacia khinjuk is a species of flowering plants belonging to family Anacardiaceae, with promising pharmacological activities like antioxidants, anti-inflammatory, antiviral, and antimicrobial. This study aimed to investigate the GC-MS chemical composition of essential oil isolated from Pistacia khinjuk leaves and its inhibitory properties against aging-relevant enzymes such a collagenase and elastase. The isolated oil showed predominance of ß-cadinene (15.34 %), γ-amorphene (8.50 %), α-cadinol (8.14 %), τ-cadinol (7.57 %), (E)-ß-caryophyllene (5.77 %), α-pinene (4.70 %), phytol (4.57 %), α-muurolene (3.30 %), (+)-epi-bicyclosesquiphellandrene (3.21 %), and cubenene (3.16 %). Further, it showed remarkable inhibitory activities against collagenase and elastase with IC50 values of 15.61±0.69 and 41.12±2.09 µg/mL, respectively compared to epigallocatechin gallate (IC50=29.52±1.3 µg/mL and 26.86±1.37 µg/mL). as a conclusion, the leaf oil is recommended for topical cosmetic preparations to retard skin aging symptoms such as wrinkles. However, the bioavailability assessment and toxicological profile should be considered in the future studies.

Anti-cancer, Anti-collagenase and Anti-elastase Potentials of Some Natural Derivatives: In vitro and in silico Studies.

The anti-cancer activities of the compounds were evaluated against KYSE-150, KYSE-30, and KYSE-270 cell lines and also on investigated esophageal line HET 1 A as a standard. Modified inhibitory impact on enzymes of collagenase and elastase were used Thring and Moon methods, respectively. Among both compounds, both of them recorded impact on cancer cells being neutral against the control, both had IC50 lower than 100 µM and acted as a potential anticancer drug. The chemical activities of Skullcapflavone I and Skullcapflavone II against elastase and collagenase were investigated utilizing the molecular modeling study. IC50 values of Skullcapflavone I and Skullcapflavone II on collagenase enzyme were obtained 106.74 and 92.04 µM and for elastase enzyme were 186.70 and 123.52 µM, respectively. Anticancer effects of these compounds on KYSE 150, KYSE 30, and KYSE 270 esophageal cancer cell lines studied in this work. For Skullcapflavone I, IC50 values for these cell lines were obtained 14.25, 19.03, 25.10 µM, respectively. Also, for Skullcapflavone II were recorded 20.42, 34.17, 22.40 µM, respectively. The chemical activities of Skullcapflavone I and Skullcapflavone II against some of the expressed surface receptor proteins (CD44, EGFR, and PPARγ) in the mentioned cell lines were assessed using the molecular docking calculations. The calculations showed the possible interactions and their characteristics at an atomic level.

Pistacia lentiscus L. Distilled Leaves as a Potential Cosmeceutical Ingredient: Phytochemical Characterization, Transdermal Diffusion, and Anti-Elastase and Anti-Tyrosinase Activities.

The present work was performed to investigate the phenolic composition of P. lentiscus L. distilled leaves (PDL) and examine its potential against certain key enzymes related to skin aging. High-pressure liquid chromatography coupled to mass spectrometry (HPLC-MS) and various separation procedures combined with nuclear magnetic resonance (NMR) and MS analysis were performed to isolate and identify compounds present in the ethyl acetate extract (EAE) of PDL. A high amount of flavonol glycoside was detected in EAE. Indeed, quercetin-3-O-rhamnoside (FC), myricetin-3-O-rhamnoside (FM2), and kaempferol-3-O-rhamnoside (FB2) were isolated from EAE, and are present in high quantities of 10.47 ± 0.26, 12.17 ± 0.74, and 4.53 ± 0.59 mg/g dry weight, respectively. A transdermal diffusion study was carried out to determine the EAE-molecules that may transmit the cutaneous barrier and showed that FM2 transmits the membrane barrier with a high amount followed by FC. EAE, FM2, and FC were tested against tyrosinase and elastase enzymes. Moreover, intracellular tyrosinase inhibition and cytotoxicity on skin melanoma cells (B16) were evaluated. The results indicated that EAE, FC, and FM2 have important inhibitory activities compared to the well-known standards, at non-cytotoxic concentrations. Therefore, they could be excellent agents for treating skin pigmentation and elasticity problems.

Elastase inhibitory activity of secondary metabolites from the fungus Virgaria nigra CF-231658.

Three known compounds were isolated from Virgaria nigra CF-231658; 2,7-dihydroxy naphthalene (1), virgaricin B (2) and virgaricin (3). The isolated compounds was obtained from liquid-state and agar-supported fermentation using Amberlite XAD-16 solid-phase extraction during the cultivation step. Their structures were elucidated on the basis of 1D and 2D NMR as well as HRMS spectroscopic analyses. The isolated compounds were examined for their ability to inhibit elastase using normal human diploid fibroblasts. Compound 2 displayed the most potent activity with 76.7 ± 2.12% inhibition of the enzyme activity at 5 µM concentration.

Molecular elucidation of pancreatic elastase inhibition by baicalein.

The serine protease, elastase exists in various forms and plays diverse roles in the human body. Pharmacological inhibition of elastase has been investigated for its therapeutic role in managing conditions such as diabetes, pneumonia and arthritis. Sivelestat, a synthetic molecule, is the only elastase inhibitor to have been approved by any major drug regulatory authority (PMDA, in this case) - but still has failed to attain widespread clinical usage owing to its high price, cumbersome administration and obscure long-term safety profile. In order to find a relatively better-suited alternative, screening was conducted using plant flavonoids, which yielded baicalein, a molecule that showed robust inhibition against Pancreatic Elastase inhibition (IC50: 3.53 µM). Other than having a considerably lower IC50than sivelestat, baicalein is also cheaper, safer and easier to administer. While MicroScale Thermophoresis validated baicalein-elastase interaction, enzyme-kinetic studies, molecular docking and molecular dynamic simulation revealed the mode of inhibition to be non-competitive. Baicalein exhibited binding to a distinct allosteric site on the enzyme. The current study demonstrates the elastase inhibition properties of baicalein in an in-vitro and in-silico environment.Communicated by Ramaswamy H. Sarma.

Constituents from the Fruiting Bodies of Trametes cubensis and Trametes suaveolens in Vietnam and Their Anti-Inflammatory Bioactivity.

It is reported that various fungi have been used for medicine and edible foods. The tropical Trametes genus is popular and well-known in Vietnam for its health effects and bioactivities. In this study, the fruiting bodies of the edible fungi T. cubensis and T. suaveolens were collected in Vietnam. The preliminary bioactivity screening data indicated that the methanol extracts of the fruiting bodies of T. cubensis and T. suaveolens displayed significant inhibition of superoxide anion generation and elastase release in human neutrophils. Therefore, the isolation and characterization were performed on these two species by a combination of chromatographic methods and spectrometric analysis. In total, twenty-four compounds were identified, and among these (1-3) were characterized by 1D-, 2D-NMR, and HRMS analytical data. In addition, the anti-inflammatory potentials of some purified compounds were examined by the cellular model for the inhibition of superoxide anion generation and elastase release in human neutrophils. Among the isolated compounds, (5,14), and (19) displayed significant anti-inflammatory potential. As the results suggest, the extracts and isolated compounds from T. cubensis and T. suaveolens are potential candidates for the further development of new anti-inflammatory lead drugs or natural healthy foods.

Chemical and Skincare Property Characterization of the Main Cocoa Byproducts: Extraction Optimization by RSM Approach for Development of Sustainable Ingredients.

Methylxanthines and polyphenols from cocoa byproducts should be considered for their application in the development of functional ingredients for food, cosmetic and pharmaceutical formulations. Different cocoa byproducts were analyzed for their chemical contents, and skincare properties were measured by antioxidant assays and anti-skin aging activity. Musty cocoa beans (MC) and second-quality cocoa beans (SQ) extracts showed the highest polyphenol contents and antioxidant capacities. In the collagenase and elastase inhibition study, the highest effect was observed for the SQ extract with 86 inhibition and 36% inhibition, respectively. Among cocoa byproducts, the contents of catechin and epicatechin were higher in the SQ extract, with 18.15 mg/100 g of sample and 229.8 mg/100 g of sample, respectively. Cocoa bean shells (BS) constitute the main byproduct due to their methylxanthine content (1085 mg of theobromine and 267 mg of caffeine/100 g of sample). Using BS, various influencing factors in the extraction process were investigated by response surface methodology (RSM), before scaling up separations. The extraction process developed under optimized conditions allows us to obtain almost 2 g/min and 0.2 g/min of total methylxanthines and epicatechin, respectively. In this way, this work contributes to the sustainability and valorization of the cocoa production chain.

Antibacterial, antifungal and enzyme inhibitory effects of selected plants from Turkey.

In this study, antibacterial, antifungal, antihyaluronidase, anticollagenase and antielastase activity of Hypericum bithynicum, Malva neglecta, Morus alba, Rubus discolor, Sambucus ebulus and Smilax excelsa were investigated. Methanol extracts of M. neglecta and R. discolor and all extracts of H. bithynicum were more active against Staphylococcus epidermidis. Similarly, water extracts of M. alba and S. ebulus were more active against Streptococcus pneumonia. Additionally, S. ebulus and S. excelsa had prominent antifungal activity on Candida albicans. Besides, methanol extract of M. neglecta and n-hexane extract of H. bithynicum were determined to have significant antihyaluronidase activity. Only R. discolor showed significant antielastase effect.

Two triterpenoids from Rubus fraxinifolius leaves and their tyrosinase and elastase inhibitory activities.

Numerous therapeutic compounds have been isolated from naturally abundant organic resources, which may offer economical and sustainable sources of compounds with safe and efficacious biological activities. In the cosmetics industry, natural compounds with anti-aging activities are eagerly sought. Thus, we prepared various extracts from Rubus fraxinifolius leaves and used enzyme inhibition assays to isolate compounds with protective effects against skin aging. Two triterpenoids were isolated from Rubus fraxinifolius Poir. leaves. The structures were characterized by spectroscopic analyses (LC-ESI-MS, 1D/2D NMR) and comparison to reported data. Compound 1 and 2 were determined as 2,3-O-ethyleneglycol, 19-hydroxyurs-12-en-23,28-dioic acid and 2,3-O-propanediol,19-hydroxyurs-12-en-28-oic acid. Methanol extract and isolates were assessed for their inhibitory effects on elastase and tyrosinase. Compounds 1 and 2 inhibited elastase with IC50 122.199 µg/mL and 98.22 µg/mL, and also inhibited tyrosinase with IC50 207.79 µg/mL and 221.51 µg/mL, respectively. The molecular docking proved that both compounds have affinities toward the enzymes.

Topical Delivery of Geranium/Calendula Essential Oil-Entrapped Ethanolic Lipid Vesicular Cream to Combat Skin Aging.

The present study deals with the evaluation of the age-defying potential of topical cream formulations bearing Geranium essential oil/Calendula essential oil-entrapped ethanolic lipid vesicles (ELVs). Two types of cream formulations were prepared, viz., conventional and ELVs spiked o/w creams. Essential oil- (EO-) loaded ELVs were characterized by vesicle size, polydispersity index, encapsulation efficiency, and scanning electron microscopy. The cream formulations were evaluated for homogeneity, spreadability, viscosity, pH, in vitro antioxidant capacity, sun protection factor, and in vitro collagenase and elastase inhibition capacity. Confocal laser scanning microscopy (CLSM) was performed to ascertain skin permeation of conventional and vesicular cream. The results of in vitro antioxidant studies showed that GEO-/CEO-loaded vesicular creams have notable antioxidant capacity when compared to nonvesicular creams. GEO- or CEO-loaded vesicular creams exhibited the highest SPF value 10.26 and 18.54, respectively. Both the EO-based vesicular creams showed in vitro collagenase and elastase enzyme inhibition capacity. CLSM images clearly depicted that vesicular cream deep into the skin layers. From the research findings, the age-defying potential and photoprotective effects of GEO and CEO were confirmed. It can be concluded that ELVs are able to preserve the efficiency of EOs and have the potential to combat skin aging.

Antioxidant and anti-aging carbon quantum dots using tannic acid.

Overexpression of collagenase, elastase, and tyrosinase is caused by external factors such as ultraviolet (UV) radiation and stress, resulting in wrinkle formation and freckles through the loss of skin elasticity and skin pigmentation. In this study, we developed novel carbon quantum dots (CQDs) with antioxidant and anti-aging properties using tannic acid as a carbon source through a simple microwave-assisted pyrolysis method. The synthesized tannic acid-derived CQDs (T-CQDs) showed bright blue fluorescence (QY = 28.2 ± 4.0%), exhibiting maximum emission at 430 nm under 350 nm excitation. Even though small amount of the T-CQDs (3µg ml-1) was used, they exhibited excellent free radical scavenging ability (82.8 ± 4.3%). Also, the T-CQDs (10µg ml-1) revealed remarkable inhibitory activity against skin aging-related collagenase (77.6 ± 4.8%), elastase (52.6 ± 1.0%), and tyrosinase (44.2 ± 1.3%), demonstrating their antioxidant and anti-aging effects. Furthermore, their antioxidant and anti-aging properties were superior to those of tannic acid, L-ascorbic acid, and quercetin used as positive controls. Finally, the T-CQDs effectively suppressed UV-induced reactive oxygen species generation by 30% at the cellular levels and showed high cell viability (99.7 ± 0.8%) even at 500µg ml-1. These results demonstrate that the T-CQDs with superior antioxidant, anti-aging properties, and low cytotoxicity can be utilized as novel anti-aging materials in cosmetic and nanomedicine fields.

Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis.

A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (Ki) of 1.69 × 10-8 mol·L-1. Further study showed that HMEI-A inhibited the formation of neutrophil extracellular trap (NET). These results suggested that HMEI-A from the leech of H. manillensis is a novel elastase inhibitor which can suppress NET formation. It may play a significant role in blood-sucking of leeches and is a potential candidate as an anti-inflammatory agent.

Antioxidant and Cytoprotective Properties of Plant Extract from Dry Flowers as Functional Dyes for Cosmetic Products.

Nowadays, natural dyes are expected by the cosmetic and food industries. In contrast to synthetic dyes, colorants derived from natural sources are more environmentally friendly and safer for human health. In this work, plant extracts from Gomphrena globasa L., Clitoria ternatea L., Carthamus tinctorius L., Punica granatum L. and Papaver rhoeas L. as the natural and functional dyes for the cosmetics industry were assessed. Cytotoxicity on keratinocyte and fibroblast cell lines was determined as well as antioxidant and anti-aging properties by determining their ability to inhibit the activity of collagenase and elastase enzymes. In addition, the composition of the extracts was determined. The obtained extracts were also applied in face cream formulation and color analyses were performed. It has been shown that the obtained extracts were characterized by no cytotoxicity and a high antioxidant potential. The extracts also show strong ability to inhibit the activity of collagenase and moderate ability to inhibit elastase and provide effective and long-lasting hydration after their application on the skin. Application analyses showed that the extracts of P. rhoeas L., C. ternatea L. and C. tinctorius L. can be used as effective cosmetic dyes that allow for attainment of an intense and stable color during the storage of the product. The extracts of P. granatum L. and G. globasa L., despite their beneficial effects as active ingredients, did not work effectively as cosmetic dyes, because cosmetic emulsions with these extracts did not differ significantly in color from emulsions without the extract.

The recombinant plant Bauhinia bauhinioides elastase inhibitor reduces rat thrombus without alterations in hemostatic parameters.

The anti-inflammatory effects of the plant protease inhibitor BbCI (Bauhinia bauhinioides cruzipain inhibitor), which blocks elastase, cathepsin G, and L, and proteinase 3 has been demonstrated. Here, we investigated the recombinant rBbCI-His(6) (containing a histidine tail) in an experimental venous thrombosis model of vena cava (VC) ligature in rats, comparing to heparin. We evaluate the effects of the inhibitors (native or recombinant) or heparin on the activated partial thromboplastin time (aPTT) and prothrombin time (PT) in human and rat plasmas. The rats undergoing treatment received a saline solution or increasing concentrations of rBbCI-His(6), heparin, or a mixture of both. After 4 h of ligature VC, thrombus, if present was removed and weighed. aPTT, PT, and cytokines were measured in blood collected by cardiac puncture. aPTT, PT, and bleeding time (BT) were also measured at the time of VC (vena cava) ligature. rBbCI-His(6) (0.45 or 1.40 mg/kg) does not alter aPTT, PT or BT. No differences in coagulation parameters were detected in rBbCI-His(6) treated rats at the time of VC ligature or when the thrombus was removed. There was a significant decrease in the weight of thrombus in the animals of the groups treated with the rBbCI-His(6) (1.40 mg/kg), with the rBbCI-His(6) mixture (1.40 mg/kg) + heparin (50 IU/kg) and heparin (100 IU/kg) in relation to control group (saline). The growth-related oncogene/keratinocyte chemoattractant (GRO/KC) serum levels in rats treated with rBbCI-His(6) (1.40 mg/kg) or heparin (200 IU/kg) were reduced. In the experimental model used, rBbCI-His(6) alone had an antithrombotic effect, not altering blood clotting or bleeding time.

Possible involvement of progranulin in the protective effect of elastase inhibitor on cerebral ischemic injuries of neuronal and glial cells.

In a previous study, we demonstrated that neutrophil elastase is activated in the brain parenchyma after cerebral ischemia, which enzyme cleaves progranulin (PGRN), an anti-inflammatory factor. In that study, we also found that sivelestat, a selective neutrophil elastase inhibitor, attenuates ischemia-induced inflammatory responses. However, it was not clear whether this anti-inflammatory effect was due to the direct effect of sivelestat. In this study, we evaluated the effects of sivelestat or recombinant PGRN (rPGRN) on cell injuries in cultured neurons, astrocytes, and microglia under oxygen/glucose deprivation (OGD) conditions. We demonstrated that OGD-induced neuronal cell injury, astrocyte activation, and increased proinflammatory cytokines caused by microglial activation, were suppressed by rPGRN treatment, whereas sivelestat had no effect on any of these events. These results indicate that the anti-inflammatory responses after in vivo cerebral ischemia were not due to the direct action of sivelestat but due to the suppression of PGRN cleavage by inhibition of elastase activity. It was also suggested that the pleiotropic effect of rPGRN could be attributed to the differentiation of M1 microglia into anti-inflammatory type M2 microglia. Therefore, the inhibition of PGRN cleavage by sivelestat could contribute to the establishment of a new therapeutic approach for cerebral ischemia.

Effects of a High-Molecular-Weight Polysaccharides Isolated from Korean Persimmon on the Antioxidant, Anti-Inflammatory, and Antiwrinkle Activity.

Persimmon (Diospyros kaki), a familiar and widespread fruit worldwide, is known to exhibit several physiological effects because of the presence of pharmacologically active compounds called phytochemicals. However, its high-molecular-weight compounds, particularly polysaccharides, have not been extensively studied. In this study, D. kaki extract (DK) was fractionated into low- and high-molecular-weight fractions (DK-L and DK-H, respectively) through ethanol fractionation, and their effects on antioxidant, anti-inflammatory, and antiwrinkle activities were investigated by an in vitro system. DK-H contained significantly higher contents of neutral sugar, uronic acid, and polyphenols compared to DK and DK-L. Furthermore, DK-H exhibited significantly improved pharmacological activities, such as antioxidant, anti-inflammatory, and antiwrinkle properties, compared to those of DK and DK-L, demonstrating that DK-H may play an important role in mediating the beneficial effects of persimmon. Sugar composition analysis and molecular characterization indicated that DK-H consisted of a galacturonic acid (GalA)-rich polysaccharide with a molecular weight of >345 kDa that mainly comprised GalA and small amounts of neutral sugar and polyphenol residues. These results suggest that the bioactive fraction DK-H is likely to be a GalA-rich pectic polysaccharide containing a small number of polyphenol residues, which may be a novel candidate in the pharmaceutical and cosmeceutical industries.

Pseudomonas aeruginosa elastase (LasB) as a therapeutic target.

Why is P. aeruginosa LasB elastase an attractive target for antivirulence therapy and what is the state-of-the art in LasB inhibitor design and development?

Effect of the dichloro-substitution on antiproliferative activity of phthalimide-thiazole derivatives. Rational design, synthesis, elastase, caspase 3/7, and EGFR tyrosine kinase activity and molecular modeling study.

Phthalimide derivatives are a promising group of anticancer drugs, while aminothiazoles have great potential as elastase inhibitors. In these context fourteen phthalimido-thiazoles containing a dichloro-substituted phenyl ring with high antiproliferative activity against various cancer cell lines were designed and synthesized. Among the screened derivatives, compounds 5a-5e and 6a-6f showed high activity against human leukemia (MV4-11) cells with IC50 values in the range of 5.56-16.10 µM. The phthalimide-thiazoles 5a, 5b and 5d showed the highest selectivity index (SI) relative to MV4-11 with 11.92, 10.80 and 8.21 values, respectively. The antiproliferative activity of compounds 5e, 5f and 6e, 6f against human lung carcinoma (A549) cells is also very high, with IC50 values in the range of 6.69-10.41 µM. Lead compounds 6e and 6f showed elastase inhibition effect, with IC50 values about 32 µM with mixed mechanism of action. The molecular modeling studies showed that the binding energies calculated for all set of compounds are strongly correlated with the experimentally determined values of IC50. The lead compound 6e also increases almost 16 times caspase 3/7 activity in A549 cells compared to control. We have also demonstrated that compound 6f reduced EGFR tyrosine kinase levels in A549 cells by approximately 31%. These results clearly suggest that 3,4-dichloro-derivative 6e and 3,5-dichloro-derivative 6f could constitute lead dual-targeted anticancer drug candidates.

Targeting Necrosis: Elastase-like Protease Inhibitors Curtail Necrotic Cell Death Both In Vitro and in Three In Vivo Disease Models.

Necrosis is the main mode of cell death, which leads to multiple clinical conditions affecting hundreds of millions of people worldwide. Its molecular mechanisms are poorly understood, hampering therapeutics development. Here, we identify key proteolytic activities essential for necrosis using various biochemical approaches, enzymatic assays, medicinal chemistry, and siRNA library screening. These findings provide strategies to treat and prevent necrosis, including known medicines used for other indications, siRNAs, and establish a platform for the design of new inhibitory molecules. Indeed, inhibitors of these pathways demonstrated protective activity in vitro and in vivo in animal models of traumatic brain injury, acute myocardial infarction, and drug-induced liver toxicity. Consequently, this study may pave the way for the development of novel therapies for the treatment, inhibition, or prevention of a large number of hitherto untreatable diseases.