Enzyme Hyaluronidase Research Articles

Inhibition of the Hyaluronidase Enzyme Produced by Staphylococcus Bacteria Using Vasicine Alkaloids of the Vasica Plant

Hyaluronidase is an enzyme that breaks down primarily hyaluronic acid, thereby disrupting the structural integrity of the extracellular matrix (ECM) found in connective tissues, which is considered a virulence factor. The aim of this research was to study the effect of a plant-active compound (vasicine) that was separated from a plant sample as an inhibitor agent for the hyaluronidase enzyme that was isolated from Staphylococcus aureus, which is considered a virulence factor. 120 samples were randomly isolated from infected burns. The isolates showing highly hydrolyzed zones were identified. Cell growth as well as hyaluronidase activity are measured spectrophotometrically by the turbidity reduction assay. The results showed that 100 out of 120 clinical samples grew bacteria when cultured on blood agar medium. Also, it was found that of the 100 isolates, 56 (56%) belonged to S. aureus and had an inhibitory zone of hyaluronidase of 5–26 mm, and the inhibitory zone was increasing over time.

Stachys byzantina K. Koch (Lamiaceae) as a potential ingredient for delaying skin ageing and treating hyperpigmentation disorders in pharmaceutical products.

This study aimed to investigate whether the plant species Stachys byzantina produces bioactives with the potential to delay the skin ageing process and treat hyperpigmentation conditions. The antioxidant action was assessed by 2,2-diphenyl-1-picrylhydrazylradical scavenging, Griess reaction, oxygen radical absorption capacity, and β-carotene bleaching assays. Inhibitory activities for tyrosinase, hyaluronidase, and elastase enzymes were tested. The antiglycation activity, the sun protection factor (SPF), and the toxicity to skin cells by MTT(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)assay were also evaluated. The ethanolic extract of S. byzantina aerial parts and all fractions obtained by solvent partition inhibited the tyrosinase enzyme at different levels. The dichloromethane fraction (DF) demonstrated the highest inhibition (IC50 = 63.5±10.9 µg/ml). DF also inhibited the hyaluronidase enzyme with IC50 = 369±11.64 μg/ml and elastase by 40% at 500 μg/ml. This fraction showed prominent antioxidant and antiglycation activities, high SPF, and no cytotoxicity at concentrations lower than 50 μg/ml. The phenolic and flavonoid contents were 116.30±6.7 (mgTAE/g) and 66.38±13.5 (mgQE/g), respectively. Chlorogenic acid (23.54±2.46mg/g) and verbascoside (203.97±19.8mg/g) were identified and quantified. Stachys byzantina is a potential source of cosmetic and therapeutic ingredients to reduce hyperpigmentation and the impacts caused by free radicals, advanced glycation end products, and sun radiation in skin ageing.

In vitro study of the gel cohesivity and persistence to hyaluronidase degradation of a novel stabilized composition of 26 mg/mL of high molecular weight HA

Aim: Hyaluronic acid (HA) is extensively used in injectable skin quality products due to its documented role in skin rejuvenation. The rapid in vivo degradation of HA by the enzyme hyaluronidase necessitates the development of advanced formulations to ensure the efficacy and longevity of the treatments. In this context, a novel 2.6% high molecular weight HA (H-HA)/3.2% sorbitol composition has been introduced, featuring stabilization through hydrogen bonds rather than traditional crosslinking. Methods: The stabilized composition was evaluated through two in vitro enzymatic degradation tests. In the first test, the efficiency on the gel degradation was followed by rheology and compared with two crosslinked HA products available on the market. In the second test, the effect on the gel structure of a less diluted hyaluronidase dose was followed by rheological and cohesivity measurements. Results: In vitro study demonstrates that, before its complete degradation into a liquid-like state, the stabilized composition exhibits high elasticity and cohesivity during the enzymatic degradation process, surpassing traditional crosslinked HA products. Conclusion: The stabilization provided by the sorbitol in the stabilized composition effectively enhances product properties and protects them during gel degradation. These attributes indicate significant potential for improved clinical outcomes in skin quality treatments.

From small‐scale studies to an encompassing view: Inhibiting inflammation and clinically relevant enzymes with various extracts of Primula vulgaris using in vitro and in silico techniques

AbstractThe genus Primula holds great importance as a source of traditional remedies in various folk medicine systems. In the present study, we investigated the chemical composition and biological properties of different extracts (ethyl acetate, ethanol, ethanol/water, and water) of aerial parts and rhizomes of Primula vulgaris. To determine the chemical profile, the extracts were analyzed using ultrahigh performance liquid chromatography‐high‐resolution mass spectrometry (UHPLC‐HRMS) technique and flavonoids were a major group in this profile. The antioxidant capacity was demonstrated by in vitro chemical tests and in general the ethanol/water extract was found to be the most potent. Enzyme inhibition was studied against various enzymes and ethanol and ethanol/water extracts were more active than others. To assess the anti‐inflammatory potential of the extracts at the molecular level, human dermal fibroblasts (HDF) were treated with lipopolysaccharide (LPS). In vitro experiments showed that the levels of nuclear factor kappa B (NFKB), receptor for advanced glycation endproducts (RAGE), activator protein‐1 (AP‐1), interleukin 6 (IL‐6), interleukin 17 (IL‐17) and tumor necrosis factor (TNF)‐alpha were significantly reduced after treatment with the tested extracts. In addition, the extracts showed an inhibitory effect on the enzymes collagenase, elastase, and hyaluronidase, which are involved in the inflammatory process and destabilization of the extracellular matrix (ECM). P. vulgaris has been observed to modulate matrix metalloproteinase (MMP) synthesis by decreasing the concentration of cellular reactive oxygen species (ROS) during LPS‐induced inflammation. This study also examined the molecular binding and dynamic behavior of various enzymes and proteins associated with skin and infections using ligands derived from the tested extracts. Simulations with AutoDock Vina V1.1.2 and GROMACS 2023.1 showed that isoquercetin in particular showed superior performance in interactions with hyaluronidase. These findings are important for the development of potential therapeutic strategies for skin health and infection control. In summary, P. vulgaris can be considered as an important source of natural bioactive compounds for the development of effective health‐promoting applications in nutraceuticals, pharmaceuticals, and cosmetics

A novel method to quantify TSG‐6 protein levels in synovial fluid from osteoarthritis patients

AbstractTSG‐6, the protein product of Tumor Necrosis Factor‐stimulated gene‐6, is a potential biomarker of disease activity/progression in osteoarthritis (OA). By ELISA, TSG‐6 is elevated in synovial fluid (SF) from OA patients and its catalytic activity, for the covalent transfer of inter‐α‐inhibitor heavy chains onto hyaluronan (HA), is reported to correlate with progression to total knee replacement. Here, quantitative western blot (qWB) analysis, following sequential digestion with hyaluronidase and chondroitinase enzymes, was developed for the accurate determination of TSG‐6 levels in SFs. qWB analysis of 125 OA SFs yielded values of 1.0–45.2 μg/mL (median: 3.9 μg/mL), 100–1000‐fold higher than those previously reported; TSG‐6 concentration was positively correlated with age and pain/outcome scores in male patients. The values determined, by ELISA or a heavy chain transfer activity assay, following the same digestion conditions (in 23 SFs), were both ≥10‐fold lower. Apparent TSG‐6 catalytic activity was significantly affected by the concentration of HA present in SF or added exogenously. Thus, the interactions of TSG‐6 with components of OA SF prevent accurate determination of its concentration/activity when using existing assays. However, the quantitative western blot method developed here appears more suitable for further evaluation of the utility of TSG‐6 as a biomarker in OA.

Evaluation of potential anti-aging effects of Achillea phrygia Boiss. & Balansa (Asteraceae)

This study aims to determine the anti-aging effects of Achillea phrygia, an endemic plant, by evaluating its sun protection factor (SPF) level, antioxidant activity, total phenolic content, extracellular matrix-degrading enzymes (ECM) inhibition, genotoxic/anti-genotoxic, and cytotoxic activities. The SPF level was assessed using an in vitro quantitative method, while antioxidant capacity was determined through DPPH, β-carotene, and hydroxyl-radical (H2O2) scavenging assays. The total phenolic content was quantitatively conducted using the Folin Ciocalteu reagent. The inhibition of ECM-degrading enzymes was determined using matrix metalloproteinase-1 (MMP-1), hyaluronidase, and elastase enzymes. Genotoxic/anti-genotoxic properties were assessed using the AMES Salmonella/microsome assay, and cytotoxicity effects were assessed through the MTT assay. The results indicated that A. phrygia showed moderate SPF activity (SPF = 4.013) and exhibited IC50 values of 0.183 ± 0.03, 0.079 ± 0.51, and 1.18 ± 0.35 mg/mL for DPPH, β-carotene, and hydroxyl-radicals, respectively. The total phenolic content was measured to be 23.56 ± 1.42 mg GAE/g dry extract. Furthermore, the extract demonstrated inhibition of MMP-1 (47.98%) and elastase (39.2%) activities. Importantly, it did not induce DNA damage and showed antigenotoxic activity ranging from 10% to 65.6%. The cytotoxicity assay revealed an IC50 value of 42.41±4.05 µg/mL. These findings suggest that A. phrygia could be utilized as a cosmetic ingredient in skincare products due to its ability to protect against UV radiation, exhibit antioxidant properties, prevent extracellular matrix degradation, and inhibit DNA damage.

Elderberry Leaves with Antioxidant and Anti-Inflammatory Properties as a Valuable Plant Material for Wound Healing.

Sambuci folium (elderberry leaves) have been used in traditional medicine, mainly externally, to treat skin diseases and wounds. Therefore, the aim of this study was to screen the biological activity of elderberry leaves (antioxidant potential and possibility of inhibition of tyrosinase and hyaluronidase enzymes) combined with phytochemical analysis. For this purpose, a phytochemical analysis was carried out. Elderberry leaves of 12 varieties ("Sampo", "Obelisk", "Dwubarwny", "Haschberg", "Haschberg 1", "Koralowy", "Sambo", "Black Beauty", "Black Tower", "Golden hybrid", "Samyl", "Samyl 1") in two growth stages. The compounds from the selected groups, phenolic acids (chlorogenic acid) and flavonols (quercetin), were chromatographically determined in hydroalcoholic leaf extracts. All tested elderberry leaf extracts showed antioxidant effects, but the most promising potential: very high compounds content (TPC = 61.85 mg GAE/g), antioxidant (e.g., DPPH IC50 = 1.88 mg/mL; CUPRAC IC0.5 = 0.63 mg/mL) and optimal anti-inflammatory (inhibition of hyaluronidase activity 41.28%) activities were indicated for older leaves of the "Sampo" variety. Additionally, the extract obtained from "Sampo" and "Golden hybrid" variety facilitated the treatment of wounds in the scratch test. In summary, the best multidirectional pro-health effect in treating skin inflammation was specified for "Sampo" leaves II extract (leaves during the flowering period); however, wound treatment was noted as rich in chlorogenic acid younger leaf extracts of the "Golden hybrid" variety.

Unveiling the potential of ursolic acid modified hyaluronate nanoparticles for combination drug therapy in triple negative breast cancer

Triple negative breast cancer (TNBC) represents the most aggressive and heterogenous disease, and combination therapy holds promising potential. Here, an enzyme-responsive polymeric prodrug with self-assembly properties was synthesized for targeted co-delivery of paclitaxel (PTX) and ursolic acid (UA). Hyaluronic acid (HA) was conjugated with UA, yielding an amphiphilic prodrug with 13.85 mol% UA and a CMC of 32.3 μg/mL. The HA-UA conjugate exhibited ∼14 % and 47 % hydrolysis at pH 7.4 and in tumor cell lysate. HA-UA/PTX NPs exhibited a spherical structure with 173 nm particle size, and 0.15 PDI. The nanoparticles showed high drug loading (11.58 %) and entrapment efficiency (76.87 %) of PTX. Release experiments revealed accelerated drug release (∼78 %) in the presence of hyaluronidase enzyme. Cellular uptake in MDA-MB-231 cells showed enhanced uptake of HA-UA/PTX NPs through CD44 receptor-mediated endocytosis. In vitro, HA-UA/PTX NPs exhibited higher cytotoxicity, apoptosis, and mitochondrial depolarization compared to PTX alone. In vivo, HA-UA/PTX NPs demonstrated improved pharmacokinetic properties, with 2.18, 2.40, and 2.35-fold higher AUC, t1/2, and MRT compared to free PTX. Notably, HA-UA/PTX NPs exhibited superior antitumor efficacy with a 90 % tumor inhibition rate in 4T1 tumor model and low systemic toxicity, showcasing their significant potential as carriers for TNBC combination therapy.

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.

Towards a zero-waste sustainable biorefinery of Codium sp. seaweed: From bioactives application to soil enhancement materials

In the pursuit of sustainability and circularity, this study aimed to develop a multi-purpose biorefinery pipeline using extracts from the green seaweed Codium sp. and transforming its post-extracted biomass into biochars, emphasizing holistic resource utilization. Five extracts from Codium sp. were evaluated, testing their antioxidant potential, cytotoxicity, photoprotective effects, anti-inflammatory activity, and inhibitory capabilities against enzymes hyaluronidase, collagenase, and elastase.The ethyl acetate extract showed the highest values in the ORAC assay, indicating antioxidant activity. However, no relevant antioxidant activity was observed in the FRAP and DPPH assays for the other extracts. The hydroethanolic extract exhibited the best anti-enzymatic capacity, inhibiting elastase by 88% at 200 μg/mL. It also demonstrated a photoprotective effect and reduced ROS production (30%) induced by UVB exposure. The hydroethanolic and ethanol extracts significantly reduced the concentration of the tumour necrosis factor-alpha (TNF-α) in response to LPS stimulation, indicating anti-inflammatory activity.The hydroethanolic post-extracted biomass was converted into biochars using conventional slow pyrolysis, hydrothermal carbonization (HTC), and acid-mediated carbonization (AMC). The biochars contained 56–75% C and 22–30% O and the one synthesized by HTC had the highest textural development. The biochar obtained by AMC showed better performance in improving soil water retention capacity, suggesting that acid surface chemistry plays a more significant role than porosity.The Codium sp. extracts and biochars produced from the post-extracted biomass have demonstrated potential as a source of valuable bioactive compounds and biomaterials for a circular economy framework and biorefinery pipeline with wide-ranging applications.

Electrospun Nanofibers Loaded with Marigold Extract Based on PVP/HPβCD and PCL/PVP Scaffolds for Wound Healing Applications.

Marigold flower is a traditionally used plant material topically applied on the skin due to its anti-inflammatory properties and antibacterial activity. This potential of action justifies the implementation of marigold extract in nanofiber scaffolds based on poly-vinylpyrrolidone/hydroxypropyl-β-cyclodextrin (PVP/HPβCD) and polycaprolactone/polyvinylpyrrolidone (PCL/PVP) obtained by electrospinning for wound treatment. Using SEM, the morphology of electrospun scaffolds showed a fiber diameter in the range of 298-527 nm, with a uniform and bead-free appearance. ATR-FTIR spectroscopy confirmed the presence of marigold extracts in nanofibrous scaffolds. The composition of the nanofibers can control the release; in the case of PVP/HPβCD, immediate release of 80% of chlorogenic acid (an analytical and functional marker for marigold extract) was achieved within 30 min, while in the case of PCL/PVP, the controlled release was achieved within 24 h (70% of chlorogenic acid). All systems showed weak antibacterial activity against skin and wound-infecting bacteria Staphylococcus aureus (MIC 100 mg/mL), and Pseudomonas aeruginosa (MIC 200 mg/mL) and yeasts Candida albicans (MIC 100 mg/mL). Analysis of the effect of different scaffold compositions of the obtained electrofibers showed that those based on PCL/PVP had better wound healing potential. The scratch was closed after 36 h, compared to the 48 h required for PVP/HPβCD. Overall, the study shows that scaffolds of PCL/PVP nanofibers loaded with classic marigold extract have the best potential as wound dressing materials because of their ability to selectively modulate inflammation (via inhibition of hyaluronidase enzyme) and supportive antimicrobial properties, thereby aiding in the early stages of wound healing and repair.

Nanoemulsion containing Jatropha gossypiifolia leaf extract reduces dermonecrosis induced by Bothrops erythromelas venom and accelerates wound closure

Ethnopharmacological relevanceThe species Jatropha gossypiifolia, popularly known as “pinhão-roxo”, is distributed throughout Brazil, is commonly employed for topical or oral administration in treating wounds, inflammations, and snake bites. Given the significant impact of snakebites on public health and the limitations of antivenom, coupled with the diverse molecular composition of this plant species, investigating its healing and antidermonecrotic capacities is relevant. Aim of the studyThis study aimed to develop a topical nanoemulsion incorporating the hydroethanolic extract of J. gossypiifolia leaves, to evaluate its therapeutic potential, particularly in terms of its efficacy in wound healing and inhibition of dermonecrosis induced by B. erythromelas venom (BeV). Material and methodsThe extract of J. gossypiifolia (JgE) leaves was obtained by maceration and remaceration. The phytochemical analysis was conducted and J. gossypiifolia nanoemulsion (JgNe) was obtained, characterized and assessed for stability. The cytotoxicity was determined in normal cells (erythrocytes and 3T3) using hemolytic assay and cell viability assay using crystal violet staining. The antioxidant activity was evaluated by the reduction of ABTS and DPPH radicals. The evaluation of wound healing was conducted in vivo following treatment with JgNe, wherein the percentage of wound closure and inflammatory mediators. The skin irritation test was assessed in vivo by applying JgNe directly to the animal's skin. In vitro, the antivenom capacity was evaluated through enzymatic inhibition assays (phospholipase A2 and hyaluronidase) of BeV. Additionally, the in vivo antidermonecrotic activity of JgNe was evaluated by measuring the reduction of the dermonecrotic halo. ResultsThe HPLC-DAD analysis identified flavonoids, specifically vitexin, luteolin derivatives and apigenin derivatives. In addition, 95.08 ± 5.46 mg of gallic acid/g of extract and 137.92 ± 0.99 mg quercetin/g extract, was quantified. JgNe maintained stability over a 4-week period. Moreover, JgE and JgNe demonstrated no cytotoxicity in human erythrocytes and murine fibroblasts at tested concentrations (32.25–250 μg/mL). Additionally, exhibited significant antioxidant activity by reducing ABTS and DPPH radicals. The treatment with JgNe did not induce skin irritation and accelerated wound healing, with significant wound closure observed from 5th day and reduction in nitrite levels, myeloperoxidase activity, and cytokine. Both JgE and JgNe demonstrated in vitro inhibition of the phospholipase and hyaluronidase enzymes of BeV. Moreover, JgNe exhibited antidermonecrotic activity by reducing the dermonecrotic halo caused by BeV after 24 h. ConclusionsJgNe and JgE exhibited no cytotoxicity at the tested concentrations. Additionally, our findings demonstrate that JgNe has the ability to accelerate wound closure and reduce dermonecrosis caused by BeV, indicating to be promising formulation for complementary therapy to antivenom treatment.

Garcinol: A novel and potent inhibitor of hyaluronidase enzyme

Extracellular matrix (ECM) contains hyaluronic acid (HA) as its integral part that is involved in numerous functional activities within the body. Degradation of HA by hyaluronidase enzyme involved in many pathophysiological conditions such as asthma, arthritis, COPD and in venom spreading during envenomation. Inhibitor of hyaluronidase enzyme has a wide range of application along with the hyaluronan-hyaluronidase system. In this present study, we have evaluated the inhibitory effect of garcinol against hyaluronidase from Hippasa partita spider venom (HPHyal), bovine testicular hyaluronidase (BTH) and human serum hyaluronidase. Garcinia indica fruit rind has been used to isolate the active component garcinol. Garcinol has been used in treatment of diverse ailments. Garcinol has exhibited anti-oxidant, anti-inflammatory, HAT inhibition and miRNA deregulator in development and progression of cancers. Experimental data have shown that garcinol completely inhibited all the three tested hyaluronidase enzymes. The inhibition was found to be non-competitive pattern with reversible type. In the docking study, garcinol with hyaluronidase enzyme has been stabilized by hydrogen bonding and hydrophobic interactions. Thus, garcinol could be a potent novel inhibitor of hyaluronidase enzyme which can be further used for pharmacotherapeutic applications.

3D-Printed Chitosan-Based Scaffolds with Scutellariae baicalensis Extract for Dental Applications.

The plant material Scutellariae baicalensis radix, which is rich in flavones (baicalin), possesses antibacterial, antifungal, antioxidant, and anti-inflammatory properties. This work aimed to develop a 3D-printed chitosan-based hydrogel rich in Scutellariae baicalensis extract as an innovative approach for the personalized treatment of periodontal diseases. Chitosan-based hydrogels were prepared, and the printability of the prepared hydrogels was determined. The hydrogel with 2.5% w/v of high molecular-weight chitosan (CS), 2% w/v gelatin (Gel), and 10% w/w of extract (Ex) presented the best printability, producing smooth and uniform scaffolds. It was proved that the CS/Gel/Ex hydrogel was stabilized by hydrogen bonds and remained in amorphous dispersion in the 3D-printed structures (confirmed by ATR-FTIR and XRPD). Due to the amorphization of the active substance, a significant increase in the release of baicalin in vitro was observed. It was demonstrated that there was an initial burst release and a continuous release profile (n = 3). Higuchi kinetic was the most likely baicalin release kinetic. The second fit, the Korsmeyer-Peppas kinetics model, showed coupled diffusion of the active ingredient in the hydrated matrix and polymer relaxation regulated release, with n values ranging from 0.45 to 0.89. The anti-inflammatory properties of 3D-printed scaffolds were assessed as the ability to inhibit the activity of the hyaluronidase enzyme. Activity was assessed as IC50 = 63.57 ± 4.98 mg hydrogel/mL (n = 6). Cytotoxicity tests demonstrated the biocompatibility of the material. After 24 h of exposure to the 2.5CS/2Gel/10Ex scaffold, fibroblasts migrated toward the scratch, closed the "wound" by 97.1%, and significantly accelerated the wound healing process. The results render the 3D-printed CS/Gel/extract scaffolds as potential candidates for treating periodontal diseases.

Synergistic quorum sensing inhibition and mild-temperature photothermal therapy of integrated nanoplatform for implant-associated biofilm infections

In current clinical practice, the presence of biofilms poses a significant challenge in the effective elimination of bacterial infections because of the physical and chemical barriers formed by biofilms, which offer persistent protection to bacteria. Here, we developed hollow mesoporous polydopamine (hMP) nanoparticles (NPs) loaded with luteolin (Lu) as a quorum sensing inhibitor, which were further coated with hyaluronic acid (HA) shells to create hMP-Lu@HA NPs. We observed that upon reaching the infection site, the HA shells underwent initial degradation by the hyaluronidase enzyme present in the bacterial infection's microenvironment to expose the hMP-Lu NPs. Subsequently, Lu was released in response to the acidic conditions characteristic of bacterial infections, which effectively hindered and dispersed the biofilm. Moreover, when subjected to near-infrared irradiation, the robust photothermal conversion effect of hMP NPs accelerated the release of Lu and disrupted the integrity of the biofilms by localized heating. This dual action enhanced the eradication of the biofilm infection. Importantly, hMP-Lu@HA NPs also promoted tissue regeneration and healing at the implantation site, concurrently addressing biofilm infection. Taken together, this nanosystem, combined with mild-temperature photothermal therapy and quorum sensing inhibition strategy, holds significant potential for applications in the treatment of implantation-associated infections.

Enhancing hyaluronidase enzyme activity: Insights from advancement in bovine and ovine testicular hyaluronidase purification

This essay investigates the use of an affinity resin named Capto lentil lectin for the purification of bovine and ovine testicular hyaluronidase. Hyaluronidase, an enzyme that degrades hyaluronic acid, is used widely in medical fields like dermatology, orthopedics, and ophthalmology. The research highlights the importance of optimizing the purification process to increase enzyme activity and purity. A new purification method is proposed, which begins with ammonium sulfate precipitation, followed by Blue Sepharose and Capto Lentil Lectin chromatography. This novel approach significantly increases the yield, purity, and activity of the enzyme. This study paves the way for further research into improving the purification process. The study further discusses challenges in identifying hyaluronidase bands using SDS-PAGE and highlights the necessity of using Western blotting for precise results.

Chemical profiling and dermatological and anti-aging properties of Syzygium jambos L. (Alston): evidence from molecular docking, molecular dynamics, and in vitro experiments.

The phytoconstituents of the aqueous extract from Syzygium jambos L. (Alston) leaves were defined using HPLC-PDA-MS/MS and the antioxidant, anti-aging, antibacterial, and anti-biofilm activities of the extract were in silico and in vitro investigated. The antioxidant activities were performed using in vitro DPPH and FRAP assays as well as H2-DCFDA assay in HaCaT cells in which oxidative stress was induced by UVA radiation. Anti-aging activity was tested in vitro, using aging-related enzymes. The antibacterial, anti-biofilm and inhibitory effects on bacterial mobilities (swarming and swimming) were assessed against Pseudomonas aeruginosa. Results showed that S. jambos aqueous extract contained 28 phytochemicals belonging to different metabolite classes, mainly phenolic acids, gallic acid derivatives, flavonoids, and ellagitannins. Mineral content analysis showed that S. jambos leaves contained moderate amounts of nitrogen, potassium, manganese, magnesium, and zinc, relatively low amounts of phosphorus and copper, and high concentration of calcium and iron. The extract displayed strong antioxidant activities in vitro and inhibited UVA-induced oxidative stress in HaCaT cells. Docking the major compounds identified in the extract into the four main protein targets involved in skin aging revealed an appreciable inhibitory potential of these compounds against tyrosinase, elastase, hyaluronidase, and collagenase enzymes. Moreover, molecular dynamic simulations were adopted to confirm the binding affinity of some selected compounds towards the target enzymes. The extract exhibited pronounced in vitro anti-aging effects, compared to kojic acid and quercetin (the reference compounds). It also inhibited the growth of P. aeruginosa, counteracted its ability to form biofilm, and impeded its swarming and swimming mobilities. Altogether, these findings strongly propose S. jambos leaves as a promising source of bioactive metabolites for the development of natural cosmeceutical and dermatological agents.

In-silico analysis of TMEM2 as a pancreatic adenocarcinoma and cancer-associated fibroblast biomarker, and functional characterization of NSC777201, for targeted drug development.

Pancreatic adenocarcinoma (PAAD), known as one of the deadliest cancers, is characterized by a complex tumor microenvironment, primarily comprised of cancer-associated fibroblasts (CAFs) in the extracellular matrix. These CAFs significantly alter the matrix by interacting with hyaluronic acid (HA) and the enzyme hyaluronidase, which degrades HA - an essential process for cancer progression and spread. Despite the critical role of this interaction, the specific functions of CAFs and hyaluronidase in PAAD development are not fully understood. Our study investigates this interaction and assesses NSC777201, a new anti-cancer compound targeting hyaluronidase. This research utilized computational methods to analyze gene expression data from the Gene Expression Omnibus (GEO) database, specifically GSE172096, comparing gene expression profiles of cancer-associated and normal fibroblasts. We conducted in-house sequencing of pancreatic cancer cells treated with NSC777201 to identify differentially expressed genes (DEGs) and performed functional enrichment and pathway analysis. The identified DEGs were further validated using the TCGA-PAAD and Human Protein Atlas (HPA) databases for their diagnostic, prognostic, and survival implications, accompanied by Ingenuity Pathway Analysis (IPA) and molecular docking of NSC777201, in-vitro, and preclinical in-vivo validations. The result revealed 416 DEGs associated with CAFs and 570 DEGs related to NSC777201 treatment, with nine overlapping DEGs. A key finding was the transmembrane protein TMEM2, which strongly correlated with FAP, a CAF marker, and was associated with higher-risk groups in PAAD. NSC777201 treatment showed inhibition of TMEM2, validated by rescue assay, indicating the importance of targeting TMEM2. Further analyses, including IPA, demonstrated that NSC777201 regulates CAF cell senescence, enhancing its therapeutic potential. Both in-vitro and in-vivo studies confirmed the inhibitory effect of NSC777201 on TMEM2 expression, reinforcing its role in targeting PAAD. Therefore, TMEM2 has been identified as a theragnostic biomarker in PAAD, influenced by CAF activity and HA accumulation. NSC777201 exhibits significant potential in targeting and potentially reversing critical processes in PAAD progression, demonstrating its efficacy as a promising therapeutic agent.

Enzyme-triggered on-demand release of a H2O2-self-supplying CuO2@Fe3O4 nanoagent for enhanced chemodyamic antimicrobial therapy and wound healing.

Nanoagents for chemodynamic therapy (CDT) hold a promising future in the field of antimicrobials, especially copper peroxide (CuO2) (CP) nanomaterials which have garnered significant attention due to their ability to self-supply H2O2. Nevertheless, the poor stability of CuO2 remains a critical challenge which restricts its practical application in the antibacterial field. In this study, an advanced nano-antimicrobial system HA-CP@Fe3O4 with enzyme-responsive properties is developed by coating hyaluronic acid (HA) on CuO2-loaded iron tetraoxide nanoparticles. The coating of HA not only stabilizes the CuO2 nanomaterials but also provides responsiveness towards the enzyme hyaluronidase, which is typically secreted by some bacteria. The outer layer of HA in HA-CP@Fe3O4 undergoes decomposition in the presence of hyaluronidase-secreting bacteria, resulting in the release of CuO2@Fe3O4. The released CuO2@Fe3O4 then self-supplies H2O2 and generates reactive oxygen species (ROS) within the infected microenvironment through Fenton and Russell effects, to ultimately achieve effective and precise antimicrobial activity. Simultaneously, the magnetic property provided by Fe3O4 allows the substance to be directed towards the infection site. Both in vitro and in vivo tests demonstrated that HA-CP@Fe3O4 exhibited excellent antimicrobial capabilities at low concentration (30 μg mL-1), exceptional biocompatibility and the ability to accelerate wound healing. The findings of this work offer a new and promising approach for targeted and precise CDT.

α-ketoglutarate produced by lactic acid bacteria inhibits hyaluronidase activity.

In Japan, the growing interest in anti-aging skin care is associated with the unprecedented aging society. Skin aging can be attributed to various factors, including the activation of hyaluronidase enzyme in subcutaneous tissues exposed to ultraviolet radiation. This enzyme breaks down hyaluronic acid, leading to skin sagging. Therefore, hyaluronidase inhibitors can effectively prevent skin aging. Previously, food components have been actively explored to search for hyaluronidase inhibitors considering the high safety of these materials. Although lactic acid bacteria (LAB)-fermented foods inhibit this enzyme, their active compounds responsible for hyaluronidase inhibition remain unknown. Thus, in this study, we aimed to explore the mechanism underlying the LAB-mediated inhibition of hyaluronidase activity. Supernatants of a LAB-fermented milk-based beverage were subjected to a hyaluronidase inhibition assay, followed by purification and separation using hydrophobic adsorbents and high-performance liquid chromatography, respectively. Subsequently, liquid chromatograph time-of-flight mass analysis was performed, revealing α-ketoglutarate (AKG) as the inhibitor of this enzyme. The half-maximal inhibitory concentration (IC50) of AKG was approximately 0.13-fold that of the known strong hyaluronidase inhibitor disodium cromoglycate (DSCG). To the best of our knowledge, this is the first report on hyaluronidase inhibition mediated by AKG, a metabolic product of LAB. Additionally, Lactobacillus acidophilus JCM1132 was identified as a highly effective AKG-producing LAB (63.9 µg/mL) through LC-MS/MS-based quantitative analyses using various LAB-fermented milk samples. We anticipate that the findings of this study will potentially support the development of functional foods and cosmetics enriched with AKG.