Optimization of novel single-step gelatin extraction method using RSM model for valorization of surimi industry by-products

Surimi industry produces large quantity of by-products as a combination of skin, bones, and scale, which due to technical difficulty in separation, are being currently utilized for production of low- value products such as biofertilizers and fish feed. Present paper focuses on utilization of combined skin, bones, and scale from Pink Perch (Nemipterus japonicus) obtained from surimi industry for gelatin extraction using single step process. Single step extraction method with acetic acid and water was optimized using Response Surface Methodology (RSM) to maximize yield and gel strength so that the process can be applied for sustainable utilization. Parameters such as pH (A), extraction temperature (B) and extraction time (C) with respect to yield and L-hydroxyproline content were optimized. Highest gelatin yield was obtained at pH 3, 75°C extraction temperature, and 30 min extraction time. Gelatin yield and L-hydroxyproline content under optimum condition were 16.2% and 41.62 mg.g−1. The chemical composition, functional, rheological, and structural properties of gelatin were examined and compared with commercial bovine gelatin. Gelatin thus obtained at optimized condition exhibited high gel strength (793g) and higher imino acid content (18.1%) than bovine gelatin. FTIR spectra depicted high similarities between both gelatin sample. Thus, the optimized method can be utilized for gelatin extraction from Pink Perch by-products for development of high value products such as food application.

GC-MS Analysis and Wound Healing Efficiency of Ethanolic Leaves Extract of Cassia occidentalis and Pithecellobium dulce in Wistar Albino Rats: Comparative Study

Aim: To identify and compare the bioactive compounds in the ethanolic leaves extracts of Cassia occidentalis and Pithecellobium dulce and to evaluate the wound healing efficiency in Wistar Albino rats. Study Design: The leaves ethanolic extracts was analysed by GC-MS and the extract was prepared in the form of a cream by ethanolic leaves extracts of C. occidentalis and P. dulce at 5% (w/v), 10% (w/v), and also in combination, a simple ointment base was developed with a composition of (1:1) Topical application of 5% (w/v) and 10% (w/v) was utilised in excision wound models. For excision wound models, the treatment duration was ten days. The day on which the wound was inflicted was designated as day '0'. Wound healing Activity: Excision wound Model: The animals were randomly separated into eight groups of six rats each: Group I: Control.; Group II: Standard group, treated with Framycetin sulfate cream (Soframycin, Aventis);. Group III: Treated with ethanolic extract of C. occidentalis (ELCO) (5% w/v); Group IV: Treated with ethanolic extract of C. occidentalis (ELCO) (10% w/v);Group V: Treated with ethanolic extract of P. dulce (ELPD)(5% w/v); Group VI: Treated with ethanolic extract of P. dulce (ELPD) (10 % w/v);Group VII: Treated with ethanolic extract of C. occidentalis and P. dulce (ELCO & ELPD 1:1) (5% w/v); Group VIII: Treated with ethanolic extract of C. occidentalis and P. dulce (ELCO & ELPD 1:1) (10 % w/v) till complete epithelization. Next dead space wound model and histology was studied. Place and Duration of Study: The GC-MS was carried out at Lab in Chennai. The extraction procedures were done at Department of Microbiology, Acharya Nagarjuna University, Guntur and treatment of wound healing activities were conducted at Ratnam Institute of Pharmacy in Nellore, Andhra Pradesh, India, and housed in the Department of Pharmacology between October to January 2016. Methodology: To study bioactive compounds, GC-MS was adopted, for wound healing activity: Excision wound Model, Dead space wound model and histology procedures was applied. Results: In the current study, ethanol leaves extract (EL) of Cassia occidentalis and Pithecellobium dulce were compared using GC-MS and their wound healing efficacy in wistar rats was examined. The GC-MS analysis of EL from both plants revealed 14-16 distinct bioactive phytochemical components with varying molecular weights and retention duration (RT). Excision and dead space wound models were utilised to assess the wound healing activities of EL extracts on rats. Wound concentration, full epithelialzation time, granulation, tissue weight, and hydroxyproline content were used to measure healing. In the excision wound model, the standard group (Framycetin sulphate cream) and group-VII (10% w/v; 1:1) combination EL treatment exhibited 98.5 ± 0.54 % and 98.4 ± 0.46 % wound healing activity, respectively. When compared to the control, the granulation tissue weight and hydroxyproline content in the dead space wound rose considerably. Histological examination revealed fewer inflammatory cells and more collagen, indicating a role in accelerating wound healing activity. Conclusion: The results of our investigation indicate unequivocally that ethanolic leaf extracts of these plant species are effective at encouraging wound healing. The 10% (ELCO+ELPD) tropical treatment drastically reduced the wound as compared to standard and also increased granulation and hydroxyproline content. However, it requires more clinical examination before being considered for wound therapy.

Formulation and Evaluation of Helichrysum italicum Essential Oil-Based Topical Formulations for Wound Healing in Diabetic Rats

As proper wound management is crucial to reducing morbidity and improving quality of life, this study evaluated for the first time the wound healing potential of H. italicum essential oil (HIEO) prepared in the form of ointment and gel in streptozotocin-induced diabetic wound models in rats. After creating full-thickness cutaneous wounds, forty-eight diabetic rats were divided into six groups: (1) negative control; (2) positive control; (3) ointment base; (4) gel base; (5) 0.5% HIEO ointment (6) 0.5% HIEO gel. Wound healing potential was determined by the percentage of wound contraction, hydroxyproline content, redox status, and histological observation. A significant decrease in the wound size was observed in animals treated with HIEO formulations compared with other groups. The HIEO groups also showed a higher level of total hydroxyproline content, and more pronounced restitution of adnexal structures with only the underlying muscle defect indicating the incision site. Hence, our results legitimate the traditional data of the pro-healing effect of HIEO because HIEO in both formulations such as gel and ointment exhibited the significant wound repairing effect in the incision wound model.

Mechanical Properties of Porcine and Fish Skin-Based Collagen and Conjugated Collagen Fibers

Collagen is a protein that is a major component of animal skins and tendons. It is used in various medical, cosmetic, and food products through extraction and purification. The fibrous products of purified collagen fibers extracted from raw mammal materials have relatively excellent mechanical properties and are used for high-end medical products. In this study, we examined collagen materials produced from porcine and fish skins, which are major sources of collagen raw materials. We examined a method for spinning collagen fibers from fish skin-based collagen and analyzed the physical properties of those collagen fibers. In addition, we examined the characteristics and advantages of conjugated fibers according to their porcine- and/or fish skin-based compositions. The spinnability and mechanical properties of these conjugated fibers were analyzed according to their compositions. The mechanical properties of collagen structure are determined by hydroxyproline content and can be manipulated by the composition of collagen in the conjugated fibers.

OP0245 ANTI-S100A4 MONOCLONAL ANTIBODY TREATMENT AMELIORATES SKIN FIBROSIS IN INFLAMMATORY AND NON-INFLAMMATORY PRE-CLINICAL MODELS OF SYSTEMIC SCLEROSIS

Background:AX-202 is a monoclonal antibody that inhibits the bioactivity of S100A4. S100A4 is an alarm signal that is released from cells in response to stress or injury and functions as an amplifying mechanism of inflammation and fibrosis in the diseased tissue microenvironment. Previous in vitro studies have found that S100A4 induces fibroblast activation, sensitizes fibroblasts to the effects of TGFβ, drives epithelial-mesenchymal transition, and stimulates monocyte cytokine release (1-3). Moreover, S100A4-/- mice are protected from fibrosis in several animal models (1). In patients with systemic sclerosis (SSc), S100A4 is elevated both in lesional tissue and systemically and correlates with skin involvement, disease activity, and pulmonary function.Objectives:The aim of this study was to assess the antifibrotic effects of murine AX-202 in two pre-clinical models of SSs reflecting both inflammation-mediated and inflammation non-mediated fibrosis and confirm the in vivo activity of humanized AX-202.Methods:We first evaluated the effects of murine AX-202 in the bleomycin-induced skin fibrosis model and the tight-skin 1 (Tsk-1) model. In the bleomycin (BLM) model, fibrosis was induced by 3 weeks of BLM s.c. injections followed by 3 weeks of AX-202 treatment in parallel with continued BLM s.c. injections. The control groups included NaCl s.c. injections for 6 weeks, BLM s.c. injections for 6 weeks, or BLM s.c. injections for 3 weeks, followed by NaCl s.c. injections for 3 weeks. Three dosing regimens of AX-202 were tested: 3.75, 7.5, or 12.5 mg/kg i.p. every 3rd day. In the Tsk-1 model, treatment with 7.5 mg/kg i.p. every 3rd day was administered from week 5 until week 10. The control groups included pa mice, Tsk-1 mice, and Tsk-1 mice treated i.p. with isotype IgG. We subsequently evaluated the effects of humanized AX-202 in the model of BLM-induced skin fibrosis in a similar design as used for the murine AX-202 study. Three dosing regimens were tested: 8 mg/kg and 16 mg/kg i.p. every 3rd day and 24 mg/kg i.v. once weekly.Results:In the BLM model, murine AX-202 (7.5 mg/kg) was effective both in the prevention of progression of pre-established skin fibrosis and in the induction of regression of fibrosis as assessed by the dermal thickness (-55%, p<0.0001 vs BLM for 6 weeks, and -23%, p<0.0001 vs BLM for 3 weeks), myofibroblast count and hydroxyproline content. Murine AX-202 also ameliorated fibrosis in the Tsk-1 model as assessed by the hypodermal thickness (-24%, p=0.01 vs Tsk-1 isotype control), myofibroblast count, and hydroxyproline content. In both models, the antifibrotic effects were associated with a reduction in pSMAD3 expression. Humanized AX-202 was effective in the prevention of progression of pre-established skin fibrosis in all doses tested across all endpoints (dermal thickness, myofibroblast counts, hydroxyproline content). In the two groups treated with 16 mg/kg i.p. and 24 mg/kg i.v., humanized AX-202 also induced regression of fibrosis (-83%, p<0.001, and -61%, p<0.001 vs BLM for 3 weeks, respectively). Both murine and humanized AX-202 were well tolerated in all study groups in both models.Conclusion:We demonstrate that AX-202 confers potent antifibrotic effects in complementary models of SSc. These results confirm and expand previous data showing that inhibition of S100A4 by AX-202 is a promising potential therapeutic candidate for disease modification in SSc or other fibrotic conditions.References:[1]Tomcik M et al. S100A4 amplifies TGF-beta-induced fibroblast activation in systemic sclerosis. Ann Rheum Dis. 2015;74(9):1748-55.[2]Cerezo LA et al. The metastasis-associated protein S100A4 promotes the inflammatory response of mononuclear cells via the TLR4 signalling pathway in rheumatoid arthritis. Rheumatology (Oxford). 2014;53(8):1520-6.[3]Fei F, et al. Role of metastasis-induced protein S100A4 in human non-tumor pathophysiologies. Cell Biosci. 2017;7:64.Acknowledgements:The study was supported by Arxx Therapeutics and MHCR 023728.Disclosure of Interests:Michal Tomčík: None declared, Thuong Trinh-Minh: None declared, Cuong Tran Manh: None declared, Hana Štorkánová: None declared, Lenka Štorkánová: None declared, Ladislav Šenolt: None declared, Jörg Klingelhöfer Employee of: Arxx Therapeutics, Rizwan I Hussain Employee of: Arxx Therapeutics, Jonas Hallén Employee of: Arxx Therapeutics, Jörg H.W. Distler Shareholder of: the stock owner of 4D Science, Consultant of: Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Grant/research support from: Anamar, Active Biotech, Array Biopharma, ARXX, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB

In silico Study of Trianthema portulacastrum Embedded Iron Oxide Nanoparticles on Glycogen Synthase Kinase-3β: A Possible Contributor to its Enhanced in vivo Wound Healing Potential

Rich amount of phenolic compounds are available in Trianthema portulacastrum L. (TP) leaves and are traditionally utilized as a wound dressing material. Oxidative stress and inflammation affect the Wnt/β-catenin pathway by modulating the glycogen synthase kinase-3β (GSK) activity subjected to delay in wound healing. The objective of the current study was to explore the wound healing effect of ferric oxide nanoparticles biosynthesized with fractionated TP extract (FeTP). The ability of TP active components (polyphenols) to inhibit the GSK was explored by using molecular docking studies. FeTP were synthesized, characterized, utilized to prepare an ointment and its efficacy was investigated against full-thickness dermal wounds. Different wound healing parameters, level of enzymatic antioxidants, hydroxyproline content and tissue cytokines level were analyzed. Histopathology was performed to confirm the healing by newly formed tissue architecture. Rats treated with FeTP showed significantly swift healing with faster wound contraction rate, high tensile strength and hydroxyproline content along with the utilization of less time for epithelialization. Histopathological study also validated the potential wound healing effect of FeTP with complete re-epithelialization. The results of the present study cumulatively revealed that the green synthesized FeTP ointment approach may serve as a potential tool for dermal wound healing.

The Non-Steroidal FXR Agonist Cilofexor Improves Portal Hypertension and Reduces Hepatic Fibrosis in a Rat NASH Model

Background: The farnesoid X receptor (FXR) influences hepatic metabolism, inflammation and liver fibrosis as key components of non-alcoholic steatohepatitis (NASH). We studied the effects of the non-steroidal FXR agonist cilofexor (formerly GS-9674) on portal pressure and fibrosis in experimental NASH. Methods: NASH was induced in Wistar rats using a choline-deficient high-fat diet plus intraperitoneal sodium nitrite injections. First, a dose-finding study was performed with 10 mg/kg and 30 mg/kg of cilofexor, focusing on histological readouts. Liver fibrosis was assessed by Picro-Sirius-Red, desmin staining and hepatic hydroxyproline content. Gene expression was determined by RT-PCR. In a subsequent hemodynamic study, rats received 30 mg/kg cilofexor with or without propranolol (25 mg/kg). Portal pressure, systemic hemodynamics and splanchnic blood flow were measured. Results: Cilofexor dose-dependently induced FXR target genes shp, cyp7a1 and fgf15 in hepatic and ileal tissues, paralleled by a dose-dependent reduction in liver fibrosis area (Picro-Sirius-Red) of −41% (10 mg/kg) and −69% (30 mg/kg), respectively. The 30 mg/kg cilofexor dose significantly reduced hepatic hydroxyproline content (−41%), expression of col1a1 (−37%) and pdgfr-β (−36%), as well as desmin area (−42%) in NASH rats. Importantly, cilofexor decreased portal pressure (11.9 ± 2.1 vs. 8.9 ± 2.2 mmHg; p = 0.020) without affecting splanchnic blood-flow or systemic hemodynamics. The addition of propranolol to cilofexor additionally reduced splanchnic inflow (−28%) but also mean arterial pressure (−25%) and heart rate (−37%). Conclusion: The non-steroidal FXR agonist cilofexor decreased portal hypertension and reduced liver fibrosis in NASH rats. While cilofexor seems to primarily decrease sinusoidal resistance in cirrhotic portal hypertension, the combination with propranolol additionally reduced mesenteric hyperperfusion.

Liver Biopsy Hydroxyproline Content Is a Diagnostic for Hepatocellular Carcinoma in Murine Models of Nonalcoholic Steatohepatitis

There is increasing evidence that nonalcoholic steatohepatitis (NASH) is a risk factor for hepatocellular carcinoma (HCC) in the absence of cirrhosis, a phenomenon termed noncirrhotic HCC. Early diagnosis of HCC is critical to a favorable prognosis. We tested the hypothesis that hydroxyproline content of liver biopsy samples is diagnostic for HCC in murine models of NASH induced by diet or by diet and chemicals. The training set comprised mice fed a standard diet or a fast-food diet with or without administration of thioacetamide. At harvest, livers from the modified diet cohort exhibited NASH with a subset of NASH livers exhibiting HCC. Hydroxyproline content was measured in liver biopsy samples with tissue in the NASH+HCC cohort sampled from the remote, nontumor parenchyma. Plotting the receiver operating characteristics (ROC) with hydroxyproline as the continuous variable against the absence or presence of HCC yielded an area under ROC of 0.87, a threshold of >0.18 μg hydroxyproline/mg liver and sensitivity of 91% with a specificity of 83.3%. The use of liver hydroxyproline content as a diagnostic for HCC in a test set comprising healthy, NASH and NASH+HCC livers proved 87% accurate.

Investigating the healing effect of the hydroalcoholic extract of pomegranate seed (Punica granatum) on the full thickness wound in rabbit

Objective: This research designed to investigate the wound healing process with pomegranate hydroalcoholic seed extract (Punica granatum) in comparison with no-treatment, betamethasone, phenytoin and eucerin in rabbits. Methods: The positive group including groups that received phenytoin cream (1%) and topical eucerin, respectively, twice a day to complete wound healing. negative Control group did not obtain any treatment. Treatment groups were received cream of PSE (2, 5,7,10 w/w) in eucerin and 75% w/w as purified extract twice daily. In order to measure the percentage of wound healing, the zone of the wound was evaluated daily. Histological studies were done on the 7th and 15th days of treatment. Next, hydroxyproline content of wounds healed and tensile strength of wound tissue samples were measured. Results: The results demonstrated between PSE treatment groups and eucerin animals were statistically significant aadifferences (P<0.05) in most of the days reviewed. Treatment of Rabbits with 10% PSE had the best results (complete wound recovery in 12 days). Also, this treatment showed higher hydroxyproline content and higher tissue strength. Conclusion: This research reveals that the extract of 10% PSE administrated topically has the proper potential to induce wound recovery in the wound model of rabbits. In addition, 10% PSE accelerates the healing of the wound. Further study needs to clarify the results of this research.