Heni Rachmawati

Turmeric curcumin inhibits entry of all hepatitis C virus genotypes into human liver cells

Objective Hepatitis C virus (HCV) infection causes severe liver disease and affects more than 160 million individuals worldwide. People undergoing liver organ transplantation face universal re-infection of the graft. Therefore, affordable antiviral strategies targeting the early stages of infection are urgently needed to prevent the recurrence of HCV infection. The aim of the study was to determine the potency of turmeric curcumin as an HCV entry inhibitor. Design The antiviral activity of curcumin and its derivatives was evaluated using HCV pseudo-particles (HCVpp) and cell-culture-derived HCV (HCVcc) in hepatoma cell lines and primary human hepatocytes. The mechanism of action was dissected using R18-labelled virions and a membrane fluidity assay. Results Curcumin treatment had no effect on HCV RNA replication or viral assembly/release. However, co-incubation of HCV with curcumin potently inhibited entry of all major HCV genotypes. Similar antiviral activities were also exerted by other curcumin derivatives but not by tetrahydrocurcumin, suggesting the importance of α,β-unsaturated ketone groups for the antiviral activity. Expression levels of known HCV receptors were unaltered, while pretreating the virus with the compound reduced viral infectivity without viral lysis. Membrane fluidity experiments indicated that curcumin affected the fluidity of the HCV envelope resulting in impairment of viral binding and fusion. Curcumin has also been found to inhibit cell-to-cell transmission and to be effective in combination with other antiviral agents. Conclusions Turmeric curcumin inhibits HCV entry independently of the genotype and in primary human hepatocytes by affecting membrane fluidity thereby impairing virus binding and fusion.

Curcumin nanoemulsion for transdermal application: formulation and evaluation

Abstract The aim of this work is to develop a curcumin nanoemulsion for transdermal delivery. The incorporation of curcumin inside a nanoglobul should improve curcumin stability and permeability. A nanoemulsion was prepared by the self-nanoemulsification method, using an oil phase of glyceryl monooleate, Cremophor RH40 and polyethylene glycol 400. Evaluation of the nanoemulsion included analysis of particle size, polydispersity index, zeta potential, physical stability, Raman spectrum and morphology. In addition, the physical performance of the nanoemulsion in Viscolam AT 100P gel was studied. A modified vertical diffusion cell and shed snake skin of Python reticulatus were used to study the in vitro permeation of curcumin. A spontaneously formed stable nanoemulsion has a loading capacity of 350 mg curcumin/10 g of oil phase. The mean droplet diameter, polydispersity index and zeta potential of optimized nanoemulsion were 85.0 ± 1.5 nm, 0.18 ± 0.0 and −5.9 ± 0.3 mV, respectively. Curcumin in a nanoemulsion was more stable than unencapsulated curcumin. Furthermore, nanoemulsification significantly improved the permeation flux of curcumin from the hydrophilic matrix gel; the release kinetic of curcumin changed from zero order to a Higuchi release profile. Overall, the developed nanoemulsion system not only improved curcumin permeability but also protected the curcumin from chemical degradation.

Construction of synthetic open reading frame encoding human interferon alpha 2b for high expression in Escherichia coli and characterization of its gene product.

The aim of this research was to obtain recombinant human interferon alpha 2b (rhIFNalpha2b) from a synthetic open reading frame (ORF) overexpressed in Escherichia coli. For gene assembly, oligonucleotides were designed by Thermodynamically Balanced Inside Out (TBIO) method using the published synthetic codon optimized hIFNalpha2b ORF for high expression in E. coli. The synthetic ORF was assembled by a two-step Polymerase Chain Reaction (PCR) and cloned into a pGEM-T vector. The two-step PCR resulted in a DNA band of 522 base pairs (bp) corresponding to the size of hIFNalpha2b ORF. Fifteen recombinant pGEM-Ts were obtained and the sequencing results showed that the ORFs contained one to ten mutations with an error rate of 8.3 per kilo base. An ORF carrying one mutation was cloned into a pET32b vector and site-directed mutagenesis was performed to correct the mutation. The hIFNalpha2b ORF was overexpressed as a thioredoxin-his-tag fusion protein in E. coli BL21. The rhIFNalpha2b fusion protein was isolated from inclusion bodies (IB), renatured, and purified using Nickel columns, and all steps were monitored by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). A rhIFNalpha2b fusion protein of 37kDa in size was produced in high expression levels relative to total protein, renatured and purified from IB with a yield of 3.46mg/l without any further optimization. The purified rhIFNalpha2b was confirmed by peptide analysis with nano-LC-MS/MS2 mass spectrometry. Our current research demonstrates for the first time that by using the TBIO method a synthetic ORF encoding hIFNalpha2b gene can be expressed at high levels in E. coli.

The Influence of Polymer Structure on the Physical Characteristic of Intraoral Film Containing BSA-loaded Nanoemulsion

Therapeutic protein has limitation, both in stability and invasive route of administration. Therefore, formulation to optimize the use of therapeutic protein becomes the research focus of interest. The purpose of this study was to develop nanoemulsion as a carrier system for a standard-model protein drug – bovine serum albumin (BSA). BSA-loaded nanoemulsion was then formulated into hydrophilic film for intraoral route. The influence of polymers was evaluated in relation to physical property of the film. Nanoemulsion was prepared using self assembly method with composition of Glyceryl monooleate (GMO), Cremophor RH-40 and Polyethylene glycol (PEG 400), with ratio of 1:8:1. The film was prepared using different polymers and plasticizers with solvent casting technique. Standard characterization of BSA nanoemulsion was including droplets size, size distribution, zeta potential, morphology, and entrapment efficiency. Physical properties of the film containing BSA nanoemulsion was included macroscopic of film, film thickness, film weight uniformity, folding endurance, tear resistance, Tensile strength (TS), percentage of elongation (PE), Elasticity modulus (EM) and film morphology. Based on all evaluated parameters, Carboxy methyl chitosan (CMCs) produced better film suitable for intraoral dosge form. Critical factors such as type and ratio of film-forming polymer, plasticizer, process and BSA concentration influenced the film characteristics.

Intermolecular Interactions and the Release Pattern of Electrospun Curcumin-Polyvinyl(pyrrolidone) Fiber

An electrospun fiber of polyvinyl(pyrrolidone) (PVP)-Tween 20 (T20) with curcumin as the encapsulated drug has been developed. A study of intermolecular interactions was performed using Raman spectroscopy, Fourier transform infrared (FT-IR), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). The Raman and FT-IR studies showed that curcumin preferrably interacted with T20 and altered PVP chain packing, as supported by XRD and physical stability data. The hydroxyl stretching band in PVP shifted to a lower wavenumber with higher intenstity in the presence of curcumin and PVP, indicating that hydrogen bond formation is more intense in a curcumin or curcumin-T20 containing fiber. The thermal pattern of the fiber did not indicate phase separation. The conversion of curcumin into an amorphous state was confirmed by XRD analysis. An in vitro release study in phosphate buffer pH 6.8 showed that intermolecular interactions between each material influenced the drug release rate. However, low porosity was found to limit the hydrogen bond-mediated release.

Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies.

DLBS1033 is a bioactive protein fraction isolated from Lumbricus rubellus that tends to be unstable when exposed to the gastrointestinal environment. Accordingly, appropriate pharmaceutical development is needed to maximize absorption of the protein fraction in the gastrointestinal tract. In vitro, ex vivo, and in vivo stability assays were performed to study the stability of the bioactive protein fraction in gastric conditions. The bioactive protein fraction DLBS1033 was found to be unstable at low pH and in gastric fluid. The “enteric coating” formulation showed no leakage in gastric fluid–like medium and possessed a good release profile in simulated intestinal medium. DLBS1033 was absorbed through the small intestine in an intact protein form, confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) analysis. This result confirmed that an enteric coating formula using methacrylic acid copolymer could protect DLBS1033 from the acidic condition of the stomach by preventing the release of DLBS1033 in the stomach, while promoting its release when reaching the intestine. From the blood concentration–versus-time curve, 99mTc-DLBS1033 showed a circulation half-life of 70 minutes. This relatively long biological half-life supports its function as a thrombolytic protein. Thus, an enteric delivery system is considered the best approach for DLBS1033 as an oral thrombolytic agent.

Curcumin-Loaded PLA Nanoparticles: Formulation and Physical Evaluation.

Curcumin is a polyphenolic compound derived from Curcuma domestica (Zingiberaceae) that possesses diverse pharmacological effects including anti-inflammatory, antioxidant, antimicrobial, and anticarcinogenic activities. Although phase I clinical trials have shown curcumin as a safe drug even at high doses (12 g/day) in humans, poor bioavaibility largely limits its pharmacological activity. Nanoencapsulation in biodegradable polymers is a promising alternative to improve curcumin bioavaibility. In this study, curcumin was encapsulated in biodegradable polymer poly-(lactic acid) (PLA) nanoparticles via the emulsification-solvent evaporation method. Optimization of selected parameters of this method including the type of solvent, surfactant concentration, drug loading, sonication time, and centrifugation speed, were performed to obtain polymeric nano-carriers with optimum characteristics. Dichloromethane was used as the solvent and vitamin E polyethylene glycol succinate (TPGS) was used as the surfactant. Four minutes of sonication time and centrifugation at 10500 rpm were able to produce spherical nanoparticles with average size below 300 nm. The highest encapsulation efficiency was found on PLA nanoparticles containing 5% of curcumin at 89.42 ± 1.04%. The particle size, polydispersity index, zeta potential of 5% curcumin-PLA nanoparticles were 387.50 ± 58.60 nm, 0.289 ± 0.047, and −1.12 mV, respectively. Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD) studies showed partial interaction between the drug and polymer.

In Silico Study to Develop a Lectin-Like Protein from Mushroom Agaricus bisporus for Pharmaceutical Application

A lectin-like protein of unknown function designated as LSMT was recently discovered in the edible mushroom Agaricus bisporus. The protein shares high structural similarity to HA-33 from Clostridium botulinum (HA33) and Ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which have been developed as drug carrier and anti-cancer, respectively. These homologous proteins display the ability to penetrate the intestinal epithelial cell monolayer, and are beneficial for oral administration. As the characteristics of LSMT are unknown, a structural study in silico was performed to assess its potential pharmaceutical application. The study suggested potential binding to target ligands such as HA-33 and CNL although the nature, specificity, capacity, mode, and strength may differ. Further molecular docking experiments suggest that interactions between the LSMT and tested ligands may take place. This finding indicates the possible use of the LSMT protein, initiating new research on its use for pharmaceutical purposes.

A novel immune-tolerable and permeable lectin-like protein from mushroom Agaricus bisporus

A lectin like protein designated as LSMT is recently discovered in Agaricus bisporus. The protein adopts very similar structure to Ricin-B like lectin from Clitocybe nebularis (CNL) and HA-33 from Clostridium botulinum (HA-33), which both recognize sugar molecules that decorate the surface of the epithelial cells of the intestine. A preliminary study in silico pointed out potential capability of LSMT to perform such biological activity. Following that hypothesis, we demonstrated that LSMT is indeed capable of penetrating out from a dialysis tube of the mice intestine origin. Furthermore, the protein appeared not to evoke the immune response upon introduction into mice, unlike its structural homologs. This is the first report on the biological implication of LSMT that might lead to its application.

The Influence of Formula and Process on Physical Properties and the Release Profile of PVA/BSA Nanofibers Formed by Electrospinning Technique

Electrospinning is a simple versatile process to produce nanofibers. However, it requires careful approach to form appropriates fibers for different purposes. This report describes aspects influencing successful development of nanofiber containing BSA using electrospinning method. Optical and scanning electron microscopy, energy dispersive X-Ray and Fourier transformed infrared spectroscopy, differential scanning calorimetric, and X-Ray diffraction analysis of nanofiber were performed. Modification of PVA/BSA nanofiber with Eudragit L-100 was conducted by dip coating method. The presence of BSA increased the diameter of the fibers. Modification of PVA/BSA nanofiber with Eudragit L-100 delayed the release of BSA in acidic medium but promoting its release in intestinal mimicking medium.