Proposed molecular mechanism of non-competitive inhibition using molecular dynamics simulations between α-glucosidase enzyme and mangostin compound as antidiabetic.

CONTEXT: Further understanding of the molecular mechanisms is necessary since it is important for designing new drugs. This study aimed to understand the molecular mechanisms involved in the design of drugs that are inhibitors of the α-glucosidase enzyme. This research aims to gain further understanding of the molecular mechanisms underlying antidiabetic drug design. The molecular docking process yielded 4 compounds with the best affinity energy, including γ-Mangostin, 1,6-dimethyl-ester-3-isomangostin, 1,3,6-trimethyl-ester-α-mangostin, and 3,6,7-trimethyl-ester-γ-mangostin. Free energy calculation with molecular mechanics with generalized born and surface area solvation indicated that the 3,6,7-trimethyl-γ-mangostin had a better free energy value compared to acarbose and simulated maltose together with 3,6,7-trimethyl-γ-mangostin compound. Based on the analysis of electrostatic, van der Waals, and intermolecular hydrogen interactions, 3,6,7-trimethyl-γ-mangostin adopts a noncompetitive inhibition mechanism, whereas acarbose adopts a competitive inhibition mechanism. Consequently, 3,6,7-trimethyl-ester-γ-mangostin, which is a derivative of γ-mangostin, can provide better activity in silico with molecular docking approaches and molecular dynamics simulations. METHOD: This research commenced with retrieving protein structures from the RCSB database, generating the formation of ligands using the ChemDraw Professional software, conducting molecular docking with the Autodock Vina software, and performing molecular dynamics simulations using the Amber software, along with the evaluation of RMSD values and intermolecular hydrogen bonds. Free energy, electrostatic interactions, and Van der Waals interaction were calculated using MM/GBSA. Acarbose, used as a positive control, and maltose are simulated together with test compound that has the best free energy. The forcefields used for molecular dynamics simulations are ff19SB, gaff2, and tip3p.

Nanoparticle-Based Antioxidants in Stress Signaling and Programmed Cell Death in Breast Cancer Treatment.

Breast cancer (BC) is a complex and heterogeneous disease, and oxidative stress is a hallmark of BC. Oxidative stress is characterized by an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense mechanisms. ROS has been implicated in BC development and progression by inducing DNA damage, inflammation, and angiogenesis. Antioxidants have been shown to scavenge ROS and protect cells from oxidative damage, thereby regulating signaling pathways involved in cell growth, survival, and death. Plants contain antioxidants like ascorbic acid, tocopherols, carotenoids, and flavonoids, which have been found to regulate stress signaling and PCD in BC. Combining different antioxidants has shown promise in enhancing the effectiveness of BC treatment. Antioxidant nanoparticles, when loaded with antioxidants, can effectively target breast cancer cells and enhance their cellular uptake. Notably, these nanoparticles have shown promising results in inducing PCD and sensitizing breast cancer cells to chemotherapy, even in cases where resistance is observed. This review aims to explore how nanotechnology can modulate stress signaling and PCD in breast cancer. By summarizing current research, it underscores the potential of nanotechnology in enhancing antioxidant properties for the treatment of breast cancer.

Effect of Drug-Polymer Interaction in Amorphous Solid Dispersion on the Physical Stability and Dissolution of Drugs: The Case of Alpha-Mangostin.

Improving drug solubility is necessary for formulations of poorly water-soluble drugs, especially for oral administration. Amorphous solid dispersions (ASDs) are widely used in the pharmaceutical industry to improve the physical stability and solubility of drugs. Therefore, this study aims to characterize interaction between a drug and polymer in ASD, as well as evaluate the impact on the physical stability and dissolution of alpha-mangostin (AM). AM was used as a model of a poorly water-soluble drug, while polyvinylpyrrolidone (PVP) and eudragit were used as polymers. The amorphization of AM-eudragit and AM-PVP was confirmed as having a halo pattern with powder X-ray diffraction measurements and the absence of an AM melting peak in the differential scanning calorimetry (DSC) curve. The solubility of amorphous AM increased in the presence of either eudragit or PVP due to amorphization and interactions of AM-polymer. Furthermore, FT-IR spectroscopy and in silico studies revealed hydrogen bond interactions between the carbonyl group of AM and the proton of eudragit as well as PVP. AM-eudragit with a ratio of 1:1 recrystallized after 7 days of storage at 25 °C and 90% RH, while the AM-PVP 1:4 and 1:10 samples retained the X-ray halo patterns, even under humid conditions. In a dissolution test, the presence of polymer in ASD significantly improved the dissolution profile due to the intermolecular interaction of AM-polymer. AM-eudragit 1:4 maintained AM supersaturation for a longer time compared to the 1:1 sample. However, a high supersaturation was not achieved in AM-PVP 1:10 due to the formation of large agglomerations, leading to a slow dissolution rate. Based on the results, interaction of AM-polymer in ASD can significantly improve the pharmaceutical properties of AM including the physical stability and dissolution.

Preparation and molecular interaction of organic solvent-free piperine pro-liposome from soy lecithin.

Pro-liposome is a type of drug delivery system (DDS) with numerous advantages as a stable material with various applicability for several pharmaceutical dosage forms, to effectively deliver the material to reach its target in the human body. Nevertheless, it is mostly designed by employing an organic solvent hence giving rise to safety issues. We have developed a method for the preparation of organic solvent-free liposomes composed of soy lecithin and cholesterol by highlighting the importance of temperature during the initial mixing process, a self-hydration of a thin layer spread film, and a spray-drying technique with a suitable excipient as the carrier. The method was successfully applied to prepare a stable pro-liposome containing 0.17% (w/w) of piperine with an encapsulation efficiency of 95.58 ± 2.91%. Moreover, the study revealed that a piperine molecule forms hydrophobic interaction with six of the adjacent phospholipids in the liposome structure, this information can be useful for researchers designing similar studies. In conclusion, organic solvent-free pro-liposome can be an alternative method in the development of DDS, and several factors could be continuously improved to fulfill the intended pro-liposome characteristic.

Nano Delivery Chitosan-Protein/Hydrolysate of Green Peas Bromelain (PHGPB) Synthesized by Colloidal-Spray Drying Method.

Patients with chronic kidney disease (CKD) suffer persistent decreased kidney function. Previous study of protein hydrolysate of green pea (Pisum sativum) bromelain (PHGPB) has shown promising results as an antifibrotic in glucose-induced renal mesangial culture cells, by decreasing their TGF-ß levels. To be effective, protein derived from PHGPB must provide adequate protein intake and reach the target organs. This paper presents a drug delivery system for the formulation of PHGPB using chitosan as polymeric nanoparticles. A PHGPB nano delivery system was synthesized by precipitation with fixed chitosan 0.1 wt.%, followed by a spray drying process at different aerosol flow rates of 1, 3, and 5 L/min. FTIR results showed that the PHGPB was entrapped in the chitosan polymer particles. Homogeneous size and spherical morphology of NDs were obtained for the chitosan-PHGPB with a flow rate of 1 L/min. Our in vivo study showed that the highest entrapment efficiency, solubility, and sustained release were achieved by the delivery system method at 1 L/min. It was concluded that the chitosan-PHGPB delivery system developed in this study improves pharmacokinetics compared to pure PHGPB.

Structure-based computational screening of 470 natural quercetin derivatives for identification of SARS-CoV-2 Mpro inhibitor.

Coronavirus disease 2019 (COVID-19) is a global pandemic infecting the respiratory system through a notorious virus known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to viral mutations and the risk of drug resistance, it is crucial to identify new molecules having potential prophylactic or therapeutic effect against SARS-CoV-2 infection. In the present study, we aimed to identify a potential inhibitor of SARS-CoV-2 through virtual screening of a compound library of 470 quercetin derivatives by targeting the main protease-Mpro (PDB ID: 6LU7). The study was carried out with computational techniques such as molecular docking simulation studies (MDSS), molecular dynamics (MD) simulations, and molecular mechanics generalized Born surface area (MMGBSA) techniques. Among the natural derivatives, compound 382 (PubChem CID 65604) showed the best binding affinity to Mpro (-11.1 kcal/mol). Compound 382 interacted with LYS5, TYR126, GLN127, LYS137, ASP289, PHE291, ARG131, SER139, GLU288, and GLU290 of the Mpro protein. The SARS-CoV-2 Mpro-382 complex showed acceptable stability during the 100 ns MD simulations. The SARS-CoV-2 Mpro-382 complex also showed an MM-GBSA binding free energy value of -54.0 kcal/mol. The binding affinity, stability, and free energy results for 382 and Mpro were better than those of the native ligand and the standard inhibitors ledipasvir and cobicistat. The conclusion of our study was that compound 382 has the potential to inhibit SARS-Cov-2 Mpro. However, further investigations such as in-vitro assays are recommended to confirm its in-silico potency.

A Review of Fibraurea tinctoria and Its Component, Berberine, as an Antidiabetic and Antioxidant.

Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia caused by resistance to insulin action, inadequate insulin secretion, or excessive glucagon production. Numerous studies have linked diabetes mellitus and oxidative stress. People with diabetes usually exhibit high oxidative stress due to persistent and chronic hyperglycemia, which impairs the activity of the antioxidant defense system and promotes the formation of free radicals. Recently, several studies have focused on exploring natural antioxidants to improve diabetes mellitus. Fibraurea tinctoria has long been known as the native Borneo used in traditional medicine to treat diabetes. Taxonomically, this plant is part of the Menispermaceae family, widely known for producing various alkaloids. Among them are protoberberine alkaloids such as berberine. Berberine is an isoquinoline alkaloid with many pharmacological activities. Berberine is receiving considerable interest because of its antidiabetic and antioxidant activities, which are based on many biochemical pathways. Therefore, this review explores the pharmacological effects of Fibraurea tinctoria and its active constituent, berberine, against oxidative stress and diabetes, emphasizing its mechanistic aspects. This review also summarizes the pharmacokinetics and toxicity of berberine and in silico studies of berberine in several diseases and its protein targets.

In Silico Study of Mangostin Compounds and Its Derivatives as Inhibitors of α-Glucosidase Enzymes for Anti-Diabetic Studies.

Diabetes is a chronic disease with a high mortality rate worldwide and can cause other diseases such as kidney damage, narrowing of blood vessels, and heart disease. The concomitant use of drugs such as metformin, sulfonylurea, miglitol, and acarbose may cause side effects with long-term administration. Therefore, natural ingredients are the best choice, considering that their long-term side effects are not significant. One of the compounds that can be used as a candidate antidiabetic is mangostin; however, information on the molecular mechanism needs to be further analyzed through molecular docking, simulating molecular dynamics, and testing the in silico antidiabetic potential. This study focused on modeling the protein structure, molecular docking, and molecular dynamics simulations and analyses. This process produces RMSD values, free energies, and intermolecular hydrogen bonding. Based on the analysis results, all molecular dynamics simulations can occur under physiological conditions, and γ-mangostin is the best among the test compounds.

Inhibition of Crystal Nucleation and Growth in Aqueous Drug Solutions: Impact of Different Polymers on the Supersaturation Profiles of Amorphous Drugs-The Case of Alpha-Mangostin.

The polymer used in supersaturated solutions plays a critical role in maintaining supersaturation levels of amorphous drugs. The prevention of drug crystallization in the supersaturated solutions by adding polymers depends on their ability to inhibit nucleation and crystal growth of drugs. This showed that understanding the mechanism of nucleation inhibition by polymers is necessary to develop the drug formulation in supersaturated solutions. Therefore, this study aims to evaluate the impact of water-soluble polymers on the supersaturation behavior of drugs and elucidate the mechanism of maintaining the supersaturation levels in an aqueous solution. It was carried out using alpha-mangostin (AM) as a model of the poorly water-soluble drug, while hypromellose (HPMC), polyvinylpyrrolidone (PVP), and eudragit were used as polymers. Their ability to inhibit the nucleation and crystal growth of AM was also evaluated. The supersaturation profiles of AM were measured in biorelevant dissolution media, while the crystal growth rate of AM was evaluated from the decrease in dissolved drug concentration by determining the induction time for AM nucleation. The interaction of AM with each polymer was evaluated and predicted by FT-IR, NMR measurement, and an in silico study, respectively. Based on observation, the PVP effectively maintained AM in a supersaturated state for the long term while eudragit conserved for 15 min. Meanwhile, an inhibitory effect of HPMC on the AM crystal nucleation was not observed. It was also \]-+discovered that the effectiveness of the various polymers depends on the interaction between the polymer and the drug. FT-IR and in silico studies demonstrated that the interaction of PVP-AM had the best polymer compared to eudragit and HPMC. NMR analysis suggested that the interaction between the methyl group from PVP with the carbonyl group of AM occurred in the PVP solution. The viscosity measurement revealed that the inhibition of nucleation and crystal growth of AM was not caused by increasing the viscosity. These results indicated that polymer-AM interactions could contribute to the crystallization inhibition and maintenance of AM in a supersaturated state. Therefore, an investigation of the mechanism of drug nucleation inhibition by polymers is recommended in the selection of crystallization inhibitors and a planned strategy to develop supersaturated formulations of drugs.

The Impact of Water-Soluble Chitosan on the Inhibition of Crystal Nucleation of Alpha-Mangostin from Supersaturated Solutions.

The use of an amorphous drugs system to generate supersaturated solutions is generally developed to improve the solubility and dissolution of poorly soluble drugs. This is because the drug in the supersaturation system has a high energy state with a tendency to precipitate. In the amorphous solid dispersion (ASD) formulation, it was discovered that polymer plays a critical role in inhibiting nucleation or crystal growth of the drugs. Therefore, this study aimed to evaluate the crystallization inhibition of water-soluble chitosan (WSC) on nucleation as well as crystal growth from alpha-mangostin (AM) and elucidate its inhibition mechanism in the supersaturated solutions. During the experiment, WSC was used as a polymer to evaluate its ability to inhibit AM nucleation. The interaction between WSC and AM was also estimated using FT-IR, NMR, and in silico study. The result showed that in the absence of polymer, the concentration of AM rapidly decreased due to the precipitation in one minute. Meanwhile, the addition of WSC effectively inhibited AM crystallization and maintained a supersaturated state for the long term. FT-IR measurement also revealed that the shift in the amine primer of WSC occurred because of the interaction between WSC and AM. In the 1H NMR spectra, the proton peaks of WSC showed an upfield shift with the presence of AM, indicating the intermolecular interactions between AM and WSC. Moreover, in silico study revealed the hydrogen bond interaction between the carbonyl group of AM with hydrocarbon groups of WSC. This indicated that WSC interacted with AM in the supersaturated solution and suppressed their molecular mobility, thereby inhibiting the formation of the crystal nucleus. Based on these results, it can be concluded that the interaction between drug polymers contributed to the maintenance of the drug supersaturation by inhibiting both nucleation and growth.

Complexed Polymer Film-Forming Spray: An Optimal Delivery System for Secretome of Mesenchymal Stem Cell as Diabetic Wound Dressing?

Diabetes-related wounds have physiological factors that make healing more complicated. High sugar levels can increase microbial infection risk while limiting nutrition and oxygen transfer to the wound area. The secretome of mesenchymal stem cells has been widely known for its efficacy in regenerative therapy. However, applying the secretome directly to the wound can reduce its effectiveness. In this review, we examined the literature on synthesizing the combinations of carboxymethyl chitosan, hyaluronic acid, and collagen tripeptides, as well as the possibility of physicochemical properties enhancement of the hydrogel matrix, which could potentially be used as an optimal delivery system of stem cell's secretome for diabetic wound healing.

Preparation of Mangosteen Peel Extract Microcapsules by Fluidized Bed Spray-Drying for Tableting: Improving the Solubility and Antioxidant Stability.

Mangosteen fruit has been widely consumed and used as a source of antioxidants, either in the form of fresh fruit or processed products. However, mangosteen peel only becomes industrial waste due to its bitter taste, low content solubility, and poor stability. Therefore, this study aimed to design mangosteen peel extract microcapsules (MPEMs) and tablets to overcome the challenges. The fluidized bed spray-drying method was used to develop MPEM, with hydroxypropyl methylcellulose (HPMC) as the core mixture and polyvinyl alcohol (PVA) as the coating agent. The obtained MPEM was spherical with a hollow surface and had a size of 411.2 µm. The flow rate and compressibility of MPEM increased significantly after granulation. A formula containing 5% w/w polyvinyl pyrrolidone K30 (PVP K30) as a binder had the best tablet characteristics, with a hardness of 87.8 ± 1.398 N, friability of 0.94%, and disintegration time of 25.75 ± 0.676 min. Microencapsulation of mangosteen peel extract maintains the stability of its compound (total phenolic and α-mangosteen) and its antioxidant activity (IC50) during the manufacturing process and a month of storage at IVB zone conditions. According to the findings, the microencapsulation is an effective technique for improving the solubility and antioxidant stability of mangosteen peel extract during manufacture and storage.

Refolding of bioactive human epidermal growth factor from E. coli BL21(DE3) inclusion bodies & evaluations on its in vitro & in vivo bioactivity.

Human epidermal growth factor (hEGF) is a mitogenic protein widely used in pharmaceutical and cosmetic industries, thus recombinant DNA technology has been applied to meet the high demand for hEGF. The overexpression of recombinant protein in E. coli often leads to the formation of inclusion bodies (IBs). Mild solubilisation preserves the native secondary protein structure in IBs, thereby the high recovery of active protein from IBs. The redox system also plays a pivotal role in the formation of disulphide bonds during refolding of disulphide bond-containing protein. This study aimed to recover hEGF from bacterial IBs through freeze-thawing solubilisation and glutathione-based oxidative refolding. CBD-Ssp DnaB-hEGF fusion protein was expressed as IBs in E. coli, washed with Triton X-100 and urea to remove most protein contaminants, then the solubilised fusion protein was obtained by freeze-thawing with the addition of 2 M urea. The solubilised protein was subsequently refolded by intein cleavage via a glutathione-based redox system. The refolded hEGF demonstrated heat-resistant properties, interacted with specific antibodies on ELISA, stimulated keratinocyte proliferation and possessed significant in vivo wound healing properties on the 8th day, confirming that hEGF was correctly folded. In summary, the protocol described is suitable for the recovery of refolded hEGF from bacterial IBs by mild solubilisation and oxidative refolding.

Film-Forming Spray of Water-Soluble Chitosan Containing Liposome-Coated Human Epidermal Growth Factor for Wound Healing.

Human epidermal growth factor (hEGF) has been known to have excellent wound-healing activity. However, direct application to the wound area can lead to low hEGF bioavailability due to protease enzymes or endocytosis. The use of liposomes as coatings and carriers can protect hEGF from degradation by enzymes, chemical reactions, and immune reactions. Sustained release using a matrix polymer can also keep the levels of hEGF in line with the treatment. Therefore, this study aimed to develop a film-forming spray of water-soluble chitosan (FFSWSC) containing hEGF-liposomes as a potential wound dressing. The hEGF-liposomes were prepared using the hydration film method, and the preparation of the FFSWSC was achieved by the ionic gelation method. The hydration film method produced hEGF-liposomes that were round and spread with a Z-average of 219.3 nm and encapsulation efficiency of 99.87%, whereas the film-forming solution, which provided good sprayability, had a formula containing 2% WSC and 3% propylene glycol with a viscosity, spray angle, droplet size, spray weight, and occlusion factor of 21.94 ± 0.05 mPa.s, 73.03 ± 1.28°, 54.25 ± 13.33 µm, 0.14 ± 0.00 g, and 14.57 ± 3.41%, respectively. The pH, viscosity, and particle size of the FFSWSC containing hEGF-liposomes were stable during storage for a month in a climatic chamber (40 ± 2 °C, RH 75 ± 5%). A wound healing activity test on mice revealed that hEGF-liposomes in FFSWSC accelerated wound closure significantly, with a complete wound closure on day 6. Based on the findings, we concluded that FFSWSC containing hEGF-liposomes has the potential to be used as a wound dressing.

Characterization and acute oral toxicity of concentrated minerals of Pamekasan Madura seawater.

Indonesia is a maritime country with abundant seawater mineral content. One of the regions with the highest salt production is Pamekasan Madura. Minerals are known to have many roles and benefits for our bodies, such as regulating fluid balance and metabolism. Therefore, this study aimed to characterize the physicochemical and microbial properties of concentrated minerals obtained through solvent evaporation and salt deposition for ± 60 days. Acute oral toxicity examination was performed as a first step in determining the safety of concentrated minerals to be used as a raw material for drugs. Based on the test results, the concentrated mineral has a clear yellow color, salty taste, and a bit bitter, odorless, with a pH of 6.6 ± 0.21. Concentrated minerals have high mineral content with levels of potassium, sodium, magnesium, boron, and calcium being 44734.1598 ± 12950.4633, 33192.1198 ± 2699.3419, 8738.1388 ± 100.4894, 2092.5715 ± 60.3224, and 276.9704 ± 13.1133 mg/Kg, respectively. The results of microbiological analysis of untreated concentrated minerals (without antimicrobials or sterilization) showed that the total plate count was within limits, including coliform and Salmonella. However, the total mold and yeast levels exceed the threshold. Based on the results of acute oral toxicity testing, the concentrated mineral is practically nontoxic. With high mineral content and low toxicity, it can be concluded that the concentrated minerals from Pamekasan Madura seawater is potential to be used as a raw medicinal material.

Activity and Effectiveness of Recombinant hEGF Excreted by Escherichia coli BL21 on Wound Healing in Induced Diabetic Mice.

CONTEXT: Human epidermal growth factor (hEGF) has biological activities and can be used in medicines and cosmetics. A high level of effectiveness of hEGF can be obtained when three disulfide bonds fold perfectly. Extracellular secretion from E. coli BL21 using the PelB signal peptide is a new way to obtain hEGF with a structure that folds appropriately. OBJECT: This study aimed to determine the activity and effectiveness of recombinant hEGF excreted by E. coli BL21 on wound healing in induced diabetic mice. METHODS: Cell proliferation and migration tests were performed on NIH3T3 cells, followed by wound healing tests in induced diabetic mice, along with histological and endotoxin test at various hEGF concentrations (25, 50, and 75 µg/mL). RESULTS: Based on the results, hEGF at a level of 50 µg/mL showed optimal proliferation and migration activities. Wound healing in induced diabetic mice showed faster-wound closure within 12 days at hEGF 50 and 75 µg/mL with a percentage wound closure of 95% and 98.5%, respectively, which was significant versus control. In the histology test, the number of fibroblasts showed an increase and was significant at hEGF 75 µg/mL compared to the control group. The single test vial (STV) showed that hEGF solution was free of endotoxin. CONCLUSION: Recombinant hEGF produced by extracellular secretion using E. coli BL21 has optimal diabetic wound healing activity through increased fibroblast proliferation.

Film-Forming Sprays for Topical Drug Delivery.

Film-forming sprays offer many advantages compared to conventional topical preparations because they can provide uniform drug distribution and dose, increased bioavailability, lower incidence of irritation, continuous drug release, and accelerated wound healing through moisture control. Film-forming sprays consist of polymers and excipients that improve the characteristics of preparations and enhance the stability of active substances. Each type of polymer and excipient will produce films with different features. Therefore, the various types of polymers and excipients and their evaluation standards need to be examined for the development of a more optimal form of film-forming spray. The selected literature included research on polymers as film-forming matrices and the application of these sprays for medical purposes or for potential medical use. This article discusses the types and concentrations of polymers and excipients, sprayer types, evaluations, and critical parameters in determining the sprayability and film characteristics. The review concludes that both natural and synthetic polymers that have in situ film or viscoelastic properties can be used to optimise topical drug delivery.

Stabilization of eye drops containing autologous serum and recombinant human epidermal growth factor for dry eye syndrome.

Human epidermal growth factor (hEGF) and autologous serum are considered safer and more effective in treating dry eye syndrome. However, suitable formulas and preparation methods are needed to obtain eye drop containing autologous serum and hEGF, which are stable during storage and use. Therefore, this study aimed to develop a stable and effective eye drops containing autologous serum and hEGF. Stabilization of autologous serum and hEGF was done by adding lyoprotectant and antioxidant agents, and then prepared using the freeze-drying method. The clarity, pH, sterility, and endotoxin content of the preparation were evaluated. The effectiveness of the preparation was assessed by a cell viability test using a WST-8 reagent. Based on the results, all formulas produce preparations that are isotonic, clear, sterile, stable, and free from endotoxins. Cell viability test shows the addition of 25 µg/mL hEGF increased epithelial cell proliferation by up to 197%. It can be concluded that eye drops containing autologous serum and 25 µg/mL hEGF can be a promising therapy for dry eye syndrome.

Optimization of Secreted Recombinant Human Epidermal Growth Factor Production Using Pectate Lyase B from Escherichia Coli BL21(DE3) by Central Composite Design and Its Production in High Cell Density Culture.

CONTEXT: Human Epidermal Growth Factor (hEGF) is a potential therapeutic protein that has been widely used as a healing agent for various chronic wounds. It induces the proliferation and metabolism of epithelial cells, regenerates skin cells, and validates skin elasticity. In the previous study, recombinant hEGF (rhEGF) had been successfully expressed extracellularly in Escherichia coli (E. coli) BL21 (DE3) using pectate lyase B (PelB) signal peptide. The previous study has shown that the medium concentration and the induction time influenced the production of rhEGF. AIMS: Therefore, this study was conducted to optimize the induction time and medium concentration for rhEGF extracellular secretion then followed by scale-up production. SETTINGS AND DESIGN: This experiment was carried out using E. coli BL21 (DE3) which contains pD881 plasmid that carries hEGF and PelB gene. Optimization design of induction time and medium concentration were obtained using Central Composite Design (CCD). METHODS AND MATERIAL: The method of study started by the rejuvenation of E. coli culture, extracellular secretion, and optimization in the flask scale then followed by scaled-up production with high-cell density culture in the fermenter. STATISTICAL ANALYSIS USED: The optimization was carried out using Response Surface Methodology (RSM) and multi regression analysis. RESULTS: This work showed that the multiplication of 1.5-fold medium concentration with induction time 3h after the culture started gave the best result among another condition in this study. Additionally, the rhEGF production in the fermenter scale was identified by SDS-PAGE Tricine and quantified by ELISA, which showed 122.40 µg of the rhEGF per milliliter medium. CONCLUSIONS: In respect of the result, we conclude that the optimized condition of extracellular secretion was successfully obtained, and gives higher result before the previous study.