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Background: Inflammation is the response to the reaction of any type of bodily injury by elevating cellular metabolism and releasing soluble mediators. It is also a contributing factor of pain. Predimenol, which has previously been known as DLBS1442, is a bioactive extract from Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae). It can be an alternative treatment for pain relief, especially for long-term use. Objective: The objective of this study is to evaluate the anti-inflammatory activities of predimenol through the evaluation of several parameters involved in the inflammatory pathway. Methods: Cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), and nuclear factor κB (NF-κB) were observed after 24 h exposure of predimenol (0-180 μg/mL) to lipopolysaccharides (LPS)-activated RAW 264.7 cell. The inflammatory markers were measured using nitric oxide (NO) assay and enzyme-linked immunosorbent assay (ELISA) for COX-2 inhibitor assay. The gene expressions of TNF-α, IL-1β, IL-2 and IL-6 were quantified using the polymerase chain reaction (PCR) method. Western blotting was applied to detect phosphorylated IκB kinase (IKK) protein to confirm the activation of NF-κB. Results: Our study showed a similar mechanism with most non-steroidal anti-inflammatory drugs (NSAIDs). Predimenol consistently downregulated the expression of iNOS and inhibited COX-2 activity. Moreover, predimenol significantly inhibited the LPS-induced production of NO, TNF-α, IL-1β, IL-2 and IL-6. Down-regulation of these markers was suggested due to the reduction of NF- κB transcription level and activation by predimenol. Conclusion: Predimenol exhibits anti-inflammatory activities through NF-kB inactivation-mediated COX-2 suppression, which may suggest that predimenol is a potential analgesic and anti-inflammatory agent.

Agung H Karsono,1 Olivia M Tandrasasmita,1 Guntur Berlian,1 Raymond R Tjandrawinata2,3 1Section of Molecular Pharmacology, Research Innovation and Invention, Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Cikarang, West Java 17550, Indonesia; 2Dexa Laboratories of Biomolecular Sciences, Dexa Medica, Cikarang, West Java 17550, Indonesia; 3Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Tangerang, 15345, IndonesiaCorrespondence: Raymond R TjandrawinataDexa Laboratories of Biomolecular Sciences, Dexa Medica, Industri Selatan V Block PP 7, Jababeka Industrial Estate II, Cikarang, West Java 17550, IndonesiaTel +62-21-898-41-901Fax +62-21-89841905Email raymond@dexa-medica.comPurpose: Centella asiatica is a traditional medicinal plant, especially for wound healing and as a neuroprotective agent. DLBS1649 is a bioactive extract from C. asiatica, and was studied to investigate its benefits as an antiaging agent.Methods: DLBS1649 was administered to HEK293 and 3T3L1 mammalian cells cultured in a time- or dose-dependent manner. Telomere length analysis was performed. TERT, CMYC, SIRT1, SIRT2, and KL expression were observed using reverse-transcription qPCR. Telomerase protein was studied with ELISA, while calorie restriction was observed using Oil Red O. In vivo study was conducted using Drosophila melanogaster with restricted mean survival time as the statistical method of analysis.Results: DLBS1649 50 μg/mL showed an effect in the prevention of telomere shortening by 50% and decrease in telomerase activity by 28% compared to the controls (70% and 40%, respectively) in the HEK293 cell cultures. TERT-, CMYC-, SIRT1-, SIRT2-, and KL-expression degression was also reduced (29%, 9%, 18%, 25%, 9%, and 30%, respectively) compared to the controls (46%, 40%, 56%, 44%, and 46%, respectively) after ten serial passages. Calorie-restriction activity from DLBS1649 50 μg/mL was seen, with lower fat droplet counts being detected in the treated samples (37%) than the controls (28%) in 3T3L1 cells. DLBS1649 2 mg/mL increased restricted mean survival time in male and female D. melanogaster (23.87% [p< 0.05] and 12.58%, respectively).Conclusion: The results revealed DLBS1649’s potential as an antiaging agent based on telomere-length preservation, decreased expression of aging-related genes, increased calorie restriction in vitro, and mortality reduction in D. melanogaster in vivo.Keywords: herbal, TERT, SIRT, klotho, fruit flies, mortality

Dicky A Wibowo,1 Florensia Nailufar,1 Raymond R Tjandrawinata2 1Animal Pharmacology Section, Dexa Laboratories of Biomolecular Sciences (DLBS), PT Dexa Medica, Cikarang, 17550, Indonesia; 2Dexa Laboratories of Biomolecular Sciences (DLBS), PT Dexa Medica, Cikarang, 17550, IndonesiaCorrespondence: Raymond R TjandrawinataDexa Laboratories of Biomolecular Sciences (DLBS), PT Dexa Medica, Industrial Estate Jababeka II, Industri Selatan V Block PP No. 7, Cikarang, 17550, IndonesiaTel +62-21-89841901Fax +62-21-89841905Email raymond@dexa-medica.comBackground: Diarrhea is a common health problem worldwide, especially in developing countries. It is the second leading cause of mortality for children. Uncaria gambir Roxb. extract has been used to treat diarrhea and dysentery, and as an astringent medicine, in Asian countries. Here, we investigated the antidiarrheal effect of DLBS1Y62, which is the bioactive fraction of dried sap extract from U. gambir, using castor oil-induced diarrhea and castor oil-induced enteropooling in rats.Methods: DLBS1Y62 was obtained by crushing and milling the dried sap extract of U. gambir leaves. Male Wistar rats, 2– 3 months old, weighing 200– 250 g (n=30), were used for this study. Negative controls received 0.05 mL purified water. Positive controls were treated with 2 mg/kg BW loperamide orally as a suspension. Groups I, II, and III received 6.25, 12.5, and 25 mg/kg BW DLBS1Y62, respectively. Group IV received a combination of 6.25 mg/kg BW DLBS1Y62 and 20 mg/kg BW attapulgite. Diarrheal onset and frequency were observed; then, the weight and volume of intestinal contents were measured.Results: DLBS1Y62 at all dose levels and in combination with attapulgite could inhibit the formation of further fecal forms of diarrhea, without delaying the onset of diarrhea. The rats that received DLBS1Y62 25 mg/kg BW had the lowest frequency of diarrhea and average intestinal contents compared with the treatment and negative control groups. DLBS1Y62 at a dose of 25 mg/kg BW also gave similar results to 2 mg/kg BW loperamide as a positive control in reducing diarrheal frequency and intestinal content.Conclusion: The results of this study suggest that DLBS1Y62, particularly at a dose of 25 mg/kg BW, containing tannin as a compound, may become an alternative treatment for diarrhea.Keywords: Uncaria gambir Roxb., DLBS1Y62, diarrhea, rats

Agaricus bisporus mannose-binding protein (Abmb) was discovered as part of mushroom tyrosinase (PPO3) complex. Apart from its presence, nothing is known about its function or activity in the mushroom. The protein is evolutionarily related to lectins with β-trefoil fold, which are glucose or galactose (and their derivatives) binding proteins. Abmb is also recently showed to display the typical agglutination activity of lectin when in complex with PPO3; this further supports Abmb similarity to its structural homologs from lectin with β-trefoil fold. However, Abmb has no affinity towards glucose or galactose but for mannose, thus its binding to the sugar may be different from its homologs. To date, the natural ligand of Abmb is unknown and the structure of Abmb in the presence of a ligand is not available. Therefore, the mannose-binding site of Abmb was predicted using molecular docking, which was consulted with the information from its structural homologs. This conservative approach would prevent over-speculation. The mannose-binding site of Abmb is likely located in the same region to that of Abmb structural homologs but with a shift in position due to the presence of additional surface loop. In addition, benefiting from the information from an in vitro study on Abmb sugar specificity, the mannose poses suggested that the sugar might interact with the side chains of Arg15, Thr45, Gln48, Asp49, Asp51 and Arg51. Most of these residues were equally present in Abmb structural homologs despite variation of their positions in the amino acid sequence. The variation probably originates from alteration of its amino acid sequence during evolution.

Metabolic syndrome has become one of the major health issues worldwide. Cider beverages have several functional properties for health, such as antioxidant, antimicrobial, anti-inflammatory, and antidiabetic effects. In this study, we formulated cider beverages from bignay fruits (Antidesma bunius), identified their compounds, and evaluated their functional effects for metabolic syndrome protection. Ciders were produced from the aqueous extract of bignay fruit powder, fermented using Saccharomyces cerevisiae and Acetobacter xylinum for 3, 7, and 14 days. Compound identification in bignay ciders was done using gas chromatography-mass spectrometry (GC/MS). Antioxidant activity was done by the 1,1-diphenyl-2-picrylhydrazyl assay, while enzymatic inhibitory assays were tested against lipase, α-glucosidase, α-amylase, and angiotensin converting enzyme (ACE). GC/MS profiling showed that most bignay ciders contained major organic acids and amino acids. All ciders exerted high antioxidant activity (>60%). Bignay ciders fermented from A. xylinum demonstrated significant inhibition (>90%) against lipase and α-glucosidase activities. However, ciders had no functional effect on a-amylase and angiotensin-converting enzyme inhibition. These data suggest that bignay ciders may have potential as functional beverages with antioxidant, antiobesity, and antidiabetic effects for management of metabolic syndrome.

Background: Hyperinsulinemia, a common feature in PCOS, have been found to contribute to metabolic disturbance, such as dyslipidaemia and diabetes mellitus type 2. Oral anti-diabetic medications have been prescribed to alleviate this effect. We sought to fnd whether DLBS3233, an insulin sensitizer, could alleviate dyslipidaemia in women with PCOS with high BMI. Aim: This study aimed to investigate the effect of DLBS3233, an herbal combination of C burmanii and L spesiosa extract, on lipid profle, insulin resistance, and free testosterone of women with PCOS with high BMI. Study Setting and Design: This was a controlled trial conducted in Dr. Cipto Mangunkusumo Hospital, Jakarta, and Dr Hasan Sadikin Hospital, Bandung, Indonesia.Materials and Methods: A controlled trial was conducted on 62 volunteers diagnosed with PCOS according to Rotterdam criteria and exhibited insulin resistance as signifed by HOMA-IR > 2.0; baseline lipid profile (LDL, HDL, Triglyceride and Total cholesterol) and free testosterone concentration were obtained. Participants were given 100 mg of DLBS3233 in the morning, and volunteers were followed up monthly, with laboratory tests conducted at the third and sixth months. Data were analysed through intention-to-treat analysis, separating high BMI (≥25 kg/m2) subjects. Statistical Analysis: Repeated-measures model. Results: DLBS3233 improved lipid profle and insulin sensitivity by reducing triglycerides, HOMA-IR, and free testosterone in subjects with high BMI. Limitations and Implications: The current study does not compare the effect of DLBS3233 with a control group. A larger study with a proper control group would have to be conducted to have more conclusive results. Conclusion: This study showed that DLBS3233 holds promise as a novel therapy to improve lipid profle for women with PCOS.

The main bitter constituents of sambiloto (Androgaphis paniculata (Burm., F) Ness) are diterpene lactones, namely andrographolide and deoxyandrographolide which have been reported to have antidiabetic, cytotoxic, antiatherosclerosis, anti-inflammatory, and antimicrobial activity. There are many studies that performed the isolation of deoxyandrographolide and andrographolide from A. paniculata herbs, but most of them included several steps that make them not efficient. This research was conducted to do an isolation of deoxyandrographolide and andrographolide through liquid-liquid fractionation (LLF) due to its simplicity, low cost, and time efficient. The extraction of deoxyandrographolide and andrographolide from the herbs was carried out using chloroform as the solvent by using Soxhlet apparatus, and LLF was performed to isolate the compounds. The identities of the compounds were confirmed by TLC scanner compared to its standard references. Hence, these present methods were successfully isolated and determined deoxyandrographolide and andrographolide of A. paniculata. The compounds were also showed relatively moderate cytotoxicity on 3T3-L1 cell lines using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, with LC50 of deoxyandrographolide and andrographolide; 29.3173 µg/mL and 37.7011 µg/mL, respectively.

Background The study was carried out to evaluate the anti-ulcerative and gastroprotective effect of DLBS2411, a bioactive fraction from Cinnamomum burmannii (Nees & T. Nees) Blume, in Wistar rats (Rattus norvegicus). Methods The rats were divided into five treatment groups, which were the Normal control group, Negative control group (ethanol-induced) and two treatment groups: DLBS2411 at the doses of 25 mg/kg body weight (BW) and 50 mg/kg BW, and the Positive control group treated with sucralfate at the dose of 100 mg/kg BW. Gastroprotective effect was measured by the ulcerative lesion index, ulcer surface area, percentage of lesion area, and cure ratio. Hematological and histopathological analyses were also conducted to gain additional data regarding the gastroprotective effect of DLBS2411 in the rats' stomachs. Results DLBS2411 was found to contain not less than 15% of total phenolic compounds. Treatment with DLBS2411 at doses of 25 mg/kg BW and 50 mg/kg BW significantly reduced the percentage of ulcer area in rats. The percentage of ulcer area for the Negative control group and both doses in the DLBS2411 treatment group reached 22.64±6.82%, 6.75±4.41%, and 6.18±4.63%, respectively. Ulcer surface area in the treatment groups and Positive control group also decreased. Histopathological data showed that gastric epithelial cells in the Negative control group were more severely ulcerated than in the treatment group of DLBS2411 and the Positive control group. Conclusion This study showed that DLBS2411 at the dose of 50 mg/kg BW was more effective in protecting the stomach lining than DLBS2411 at the dose of 25 mg/kg BW, as measured by percentage of ulceration inhibition and the ulcerative lesion index.

Fibrinogen supplies the primary building block of the blood clot or thrombus after α-thrombin converts fibrinogen to fibrin during the final phases of coagulation. When the homeostasis system is disrupted, blood clots that aggregate in the blood vessels can lead to thrombosis. Fibrin-degrading enzyme from Bacillus subtilis K2 (Subtilisin K2) of Indonesian moromi has many excellent characteristics apart from its strong fibrinolytic activity. Bioinformatic analysis using the CDART webserver indicated that the enzyme appeared to share a conserved domain with the peptidase s8 superfamily also known as the subtilase family. This study used molecular docking between these fibrin-degrading enzymes and specific substrates (fibrin and fibrinogen) using the HADDOCK webserver and aimed to predict the enzyme mechanism of action. This analysis revealed that the enzyme interlocked with the two substrates; however, it suggested no productive interactions between Subtilisin K2 and fibrinogen. A hydrolysis reaction is suggested between Subtilisin K2 and the fibrin substrate. There was a strong indication that amino acids Asp19, His51, and Ser208 in Subtilisin K2’s active site interacts with Leu168, Ile171, and Leu172 of the fibrin substrate with a ∆G of -19.4 kcal/mol. Subtilisin K2 tends to act more as a fibrin-degrading enzyme than as a fibrinogen-degrading enzyme.

Agaricus bisporus mannose binding protein (Abmb) demonstrates permeability to epithelial monolayer barrier of the intestine, resistance to gastrointestinal tract conditions and to proteolysis therefore it holds potential as a drug carrier for oral route administration. Abmb also display antiproliferative activity to breast cancer cells and stimulation of immune system thus could potentially be also developed for therapeutic purpose. It is not immunogenic or toxic thereby safe for use. In this paper we further provide evidence that Abmb also lacks of agglutinating activity despite sharing high structural homology to lectins. Abmb is thereby the only mannose specific binding protein that is not member of lectin family. This evidence provides further support on the use of Abmb as pharmaceutical or medicinal agent. Its molecular globularity that may contribute to its lack of agglutination capacity was also evaluated.

Objective: A new method of cocrystallization based on the use of supercritical carbon dioxide (CO2) as an anti-solvent was explored. In the present study, we investigate and analyze paracetamol (PCA)-5-nitroisophthalic acid (5NIP) cocrystal produced using supercritical anti-solvent (SAS) process. Methods: PCA-5NIP cocrystals prepared by SAS cocrystallization were compared to those produced using traditional solvent evaporation by rapid evaporation (RE) process. The cocrystals produced were characterized using powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), polarized light microscopy (PLM), Fourier Transform Infrared (FTIR) spectroscopy, particle size analysis and scanning electron microscopy (SEM). Results: The products obtained from SAS and RE process exhibited identical PXRD spectra and were distinguishable from the individual compounds, indicating the formation of a new phase. DSC analysis revealed that PCA-5NIP cocrystals from each method possess similar melting point which lies between the melting points of the parent compounds. Cocrystal particles with a mean diameter of 4.66 µm were produced from SAS process, which was smaller than those produced by traditional solvent evaporation method with a mean diameter of 38.09 μm. Conclusion: This study demonstrates the ability of SAS process to produce the submicron size of PCA-5NIP cocrystal with altered physicochemical properties in a single step process.

Trigonella foenum-graceum atau fenugreek digunakan secara luas sebagai obat tradisional untuk pengobatan diabetes tetapi mekanisme kerjanya masih belum jelas. Penelitian bertujuan memprediksi senyawa dalam fenugreek yang berkhasiat sebagai antidiabetes secara in-silico dengan menggunakan perangkat lunak Molegro Virtual Docking . Docking dilakukan 10 Senyawa uji dalam fenugreek yaitu 4-hidroxyisoleucine, coumarine, diosgenin, galactomannan, isovitexin, quarcetin, tigogenin, trigoneline, vitexin dan yamogenin dengan 3 reseptor yaitu sugar beet alpha-glucosidase- (PDB ID : 3W37), human dipeptidyl peptidase-4 (PDB ID : 1X70), human peroxisome proliferator activated gamma (PDB: 2PRG) serta senyawa pembanding acarbose, sitagliptin dan rosiglitazone. Dari 10 senyawa uji, galactomanann memiliki nilai Rerank Score/RS paling rendah di dua reseptor yaitu alpha glucosidase dan peroxisome proliferator activated gamma dengan nilai berturut turut -116.56 kcal/mol dan -131.18 kcal/mol dan nilai RS acarbose -113.60 kcal/mol dan rosiglitazone -124.54 kcal/mol . Dari 10 senyawa uji, tigogenin memiliki nilai RS paling rendah direseptor dipeptidyl peptidase-4 dengan nilai RS -86.54 kcal/mol dan nilai RS sitagliptin -87.02 kcal/mol. Berdasarkan nilai RS, galactomannan diprediksi memiliki aktivitas antidiabetes yang bekerja pada reseptor alpha-glucosidase dan peroxisome proliferator activated gamma sedangkan tigogenin diprediksi memiliki aktivitas antidiabetes yang bekerja pada reseptor dipeptidyl peptidase-4.

Background: Obesity has become a risk factor for metabolic diseases. One of the cellular characteristics of obesity is the occurrence of adipose cells hyperplasia. Lagerstroemia speciosa is a plant which has been used for the treatment of diabetes. Furthermore, some studies also indicated that L. speciosa possesses antiobesity activity. Its antiobesity activity was examined in the present study through adipogenesis, lipogenesis, and lipolysis pathways. Aim: DLBS3733, a bioactive fraction of L. speciosa, was explored for its potential benefits to alter obesity through adipogenesis and lipogenesis inhibition and lipolysis induction activity. Materials and methods: This study was performed using 3T3-L1 cells. mRNA level and protein expressions related to adipogenesis, lipogenesis, and lipolysis pathways were assayed in this study. Results: Antiadipogenic effects of DLBS3733 (15 µg/mL) were found to be mediated by a significant downregulation of mRNA level of multicomponents involved in adipogenesis which include C/EBPα (CCAAT/enhancer-binding protein alpha) and PPAR-γ (peroxisome proliferator-activated receptor gamma) by 75% and 80.1% (p

Objective: Extraction of Curcuma mangga (C. mangga) using supercritical carbon dioxide (SC-CO2) was investigated to provide information about the optimum extraction condition. Methods: A Taguchi method with L9 orthogonal array design was used to determine the optimum extraction conditions. Effects of extraction pressure, temperature, CO2 flow rate and dynamic extraction time on C. mangga oil yield were investigated at levels ranging between 150-350 bar, 40-60 °C, 10-20 g/min and 120-240 min, respectively. Results: The highest C. mangga oil yield (5.223%) from SC-CO2 extraction was obtained at a pressure of 350 bar, temperature of 60 °C, CO2 flow rate of 20 g/min and dynamic extraction time of 240 min. The experimental C. mangga oil yield at optimum condition was in a good agreement with the values predicted by computational process using Taguchi method. Based on S/N ratio calculation, the most influencing parameters in maximizing C. mangga oil yield is extraction temperature, followed by extraction pressure, dynamic extraction time and CO2 flow rate. Conclusion: In this study, Taguchi method was successfully applied to optimize SC-CO2 extraction of C. mangga. Taguchi method was able to simplify the experimental procedure of SC-CO2 extraction.

Background. There are still some unmet needs for stroke management and safety. DLBS1033 is a protein fraction extracted from the earthworm Lumbricus rubellus that has shown fibrinolytic and fibrinogenolytic activities, reduces blood viscosity, and inhibits platelet aggregation that it can be considered an add-on therapy and potential medical breakthrough in acute ischemic stroke management. Objective. This study is aimed at measuring the benefit of DLBS1033 in acute ischemic stroke management. Methods. This was a randomized, open-label trial at a referral stroke center from November 2019 to December 2020. Subjects who met the inclusion criteria were randomly divided into a control group and an experimental group. The control group received standard therapy consisting of aspirin 100 mg once daily, atorvastatin 20 mg once daily, and vitamin B12 100 mg three times daily. The experimental group received standard therapy and DLBS1033 three times daily. The functional outcomes were measured using the National Institutes of Health Stroke Scale (NIHSS), Barthel Index (BI), and modified Rankin Scale (mRS) at baseline, hospital discharge, and day 30. Results. Collected data from 180 subjects was analyzed. The NIHSS scores’ improvements were significantly greater in the experimental group compared to the control group at both hospital discharge ( − 5.57 ± 2.16 vs. − 3.64 ± 2.65 ; p < 0.001 ) and day 30 ( − 6.62 ± 2.64 vs. − 5.14 ± 2.41 ; p = 0.001 ). Compared with the control group, the improvements in the BI scores were significantly better in the experimental group, at both hospital discharge ( 10.69 ± 5.36 vs. 6.64 ± 5.04 ; p < 0.001 ) and day 30 ( 10.9 ± 8.19 vs. 8.56 ± 7.45 ; p = 0.003 ). The distribution of mRS scores was improved in both groups during 30 days of follow-up and was more favorable in the experimental group. In both groups, a favorable outcome ( mRS < 2 ) was achieved better at day 30 (86.7% vs. 80%; p = 0.302 ) than at baseline (0% vs. 6.7%; p = 0.028 ) and at hospital discharge (58.9% vs. 43.3%; p = 0.085 ). There was no clinically significant adverse event related to the study product. Conclusions. DLBS1033 in addition to the standard care was more effective in improving functional status compared to standard care alone in acute ischemic stroke patients with a similar safety profile.

AIM: This study was aimed to determine the antifibrotic activity of Phaleria macrocarpa (PM) extract in liver fibrosis (LF) and its possible mechanism in the rat model. METHODS: Sprague Dawley male rats were injected with 2 mL/kg BW of carbon tetrachloride intraperitoneally twice a week for 2 weeks, followed by 1 mL/kg BW for 6 weeks. Afterward, the treatments began from the 3rd week: Silymarin 100 mg/kg BW/day, standardized PM extract (Proliverenol) 75 or 150 mg/kg BW/day orally. Rats were sacrificed in the 8th week. Blood and liver were collected to analyze liver function, liver damage and fibrosis marker, oxidative stress markers, pro-fibrogenic cytokine, and antifibrotic marker. RESULTS: Our study showed that the treatment of silymarin and PM resulted in the normalized activity of liver function, followed by the amelioration of oxidative stress, demonstrated by the decreased malondialdehyde levels and an increased ratio of glutathione and glutathione disulfide. All markers examined showed that PM extract has antioxidant activity due to decreased hepatic stellate cell activation. We also found a decrease in tumor growth factors-β1 and protein expressions of matrix metalloproteinases-13 in all treatment groups compared to the carbon tetrachloride group. There were tendencies of the decreased fibrotic area following improvements of biochemical parameters. CONCLUSION: PM extracts ameliorate carbon tetrachloride-induced LF. The proposed mechanism is by overcoming oxidative stress and regulating pro-fibrogenic cytokine and antifibrotic markers.

Fibrinogen supplies the primary building block of the blood clot or thrombus after α-thrombin converts this fibrinogen to fibrin during the final phases of coagulation. When the homeostasis system is disrupted, blood clots that aggregate in the blood vessels can lead to thrombosis. Fibrin degrading enzyme from Bacillus subtilis K2 (Subtilisin K2) has many excellent characteristics with strong fibrinolytic activity. Bioinformatic analysis indicated that the enzyme molecule appeared to share conserved domain, with peptidase s8 super family also known as the subtilase family with its motif: Asp subtilase (motif: VAVIDSGIDSsH), His subtilase (motif: HGTHVAGTIAA) and Ser subtilase (motif: GTSMATPHVAG). Study on molecular docking between this fibrin degrading enzymes and the specific substrates, fibrin and fibrinogen was aimed to predict the mechanism of action. This analysis, revealed no productive interaction between Subtilisin K2 and fibrinogen. However, hydrolysis reaction is indicated strongly between Subtilisin K2 and fibrin substrate. Amino acid Asp19, His51, and Ser208 in the Subtilisin K2’s active site interacted with Leu168, Ile171, and Leu172 of the fibrin substrate with ∆G of –19.4 kcal/mol that showed suitable substrate specificity. The fibrin degrading enzyme Subtilisin K2 tend to act more as fibrin degrading enzyme than as fibrinogen degrading enzyme.

The screening of proteolytic and fibrinolytic bacteria from moromi (an Indonesian soybean-based fermented food) yielded a number of isolates. Based on morphological and biochemical analyses and sequencing of the 16S rRNA gene, the isolate that exhibited the highest proteolytic and fibrinolytic activity was identified as Bacillus subtilis K2. The study was performed to analyze molecular characteristic of a fibrin-degrading enzyme from B. subtilis K2. BLASTn analysis of the nucleotide sequence encoding this fibrinolytic protein demonstrated 73.6% homology with the gene encoding the fibrin-degrading enzyme nattokinase of the B. subtilis subsp. natto, which was isolated from fermented soybean in Japan. An analysis of the putative amino-acid sequence of this protein indicated that it is a serine protease enzyme with aspartate, histidine, and serine in the catalytic triad. This enzyme was determined to be a 26-kDa molecule, as confirmed with a zymogram assay. Further bioinformatic analysis using Protparam demonstrated that the enzyme has a pI of 6.02, low instability index, high aliphatic index, and low GRAVY value. Molecular docking analysis using HADDOCK indicated that there are favorable interactions between subtilisin K2 and the fibrin substrate, as demonstrated by a high binding affinity (ΔG: − 19.4 kcal/mol) and low Kd value (6.3E-15 M). Overall, the study concluded that subtilisin K2 belong to serine protease enzyme has strong interactions with its fibrin substrate and fibrin can be rapidly degraded by this enzyme, suggesting its application as a treatment for thrombus diseases.

The mushroom Agaricus bisporus secretes biologically active compounds and proteins with benefits for human health. Most reported proteins from A. bisporus are tyrosinases and lectins. Lectins are of therapeutic or pharmaceutical interest. To date, only limited information is available on A. bisporus lectins and lectin-like proteins. No therapeutic products derived from A. bisporus lectin (ABL) are available on the market despite its extensive exploration. Recently, A. bisporus mannose-binding protein (Abmb) was discovered. Its discovery enriches the information and increases the interest in proteins with therapeutic potential from this mushroom. Furthermore, the A. bisporus genome reveals the possible occurrence of other lectins in this mushroom that may also have therapeutic potential. Most of these putative lectins belong to the same lectin groups as ABL and Abmb. Their relationship is discussed. Particular attention is addressed to ABL and Abmb, which have been explored for their potential in medicinal or pharmaceutical applications. ABL and Abmb have anti-proliferative activities toward cancer cells and a stimulatory effect on the immune system. Possible scenarios for their use in therapy and modification are also presented.

Agaricus bisporus mannose-binding protein (Abmb) was discovered as part of the mushroom tyrosinase (PPO3) complex, but its function in the mushroom has remained obscure. The protein has a β-trefoil structure that is common for Ricin-B-like lectins. Indeed, its closest structural homologs are the hemagglutinin components of botulinum toxin (HA-33) and the Ricin-B-like lectin from Clitocybe nebularis (CNL), both of which bind galactose, and actinohivin, a recently discovered mannose-binding lectin from actinomycetes. Here we show that Abmb is evolutionarily related to them, which are lectins with a β-trefoil fold. We also show for the first time that Abmb can exhibit typical lectin agglutination activity but only when in the complex with mushroom tyrosinase. This is unexpected and unique because the two proteins are not evolutionarily related and have different activities. Lectin and tyrosinase major role in defense mechanism as well as Abmb and PPO3 gene regulation during the early stages of the development of mushroom fruiting bodies suggested that Abmb has likely a function in defense against bacterial infection and/or insect-induced damage.

Abrus precatorius Linn. (Fabaceae), also known as “Saga Rambat”, is commonly found in tropical countries, including Indonesia. The leaves of the plant are widely used commercially in Indonesia to treat cough and sprue. The aim of this research is to segregate and identify the chemical compound of A. precatorius leaf methanolic extract originated from Kebumen, Central Java, Indonesia. Dried leaves of A. precatorius were extracted with methanol then partitioned between n-hexane/water, methylene chloride/water, and ethyl acetate/water. The fractionation of the water layer was performed by various stationary phases. Identification of the chemical structure was performed by using data combination of 1D-NMR (1H-NMR and 13C-NMR), infra-red (IR), and mass spectrum (MS). Purification of water fraction from methanolic extract of A. precatorius leaves resulted in isolation of an alkaloid, identified as abrine or N-methyltryptophan. The isolated compound was also identified in five other origins of the plant in Java Island, Indonesia.

We show that a lectin like protein from the mushroom Agaricus bisporus (LSMT) is capable to permeate the epithelial monolayer barrier of the intestine ex vivo. The protein is not toxic or immunogenic upon prolonged administration and elevated dose in mice. Thus, it could be a candidate as a drug carrier for oral administration. However, its permeability should be tested after the protein has been modified, mimicking the condition in which it is used as a drug carrier. The protein was conjugated to captopril, the selected model of a Biopharmaceutical Classification System (BCS) class III drug, with high solubility but poor permeability. The drug was conjugated to LSMT that had been modified with 4-succinimidyloxycarbonyl-alpha-methyl-2-pyridyldithiotoluene (SMPT) as a linker. The success of LSMT modification was confirmed with TLC and MS; the latter also indicated the amount of captopril molecule linked. The modified LSMT could permeate through the intestinal monolayer barrier, and thus could be absorbed in the intestine after modification. The modified protein appears to remain stable after incubation in simulated gastrointestinal fluids. This pioneering work provides an essential basis for further development of the protein as a drug carrier for oral administration.

Raymond R Tjandrawinata,1 Arini Setiawati,2 Dwi Nofiarny,1 Liana W Susanto,1 Effi Setiawati3 1Dexa Laboratories of Biomolecular Sciences Unit, Dexa Medica Group, Cikarang, West Java, Indonesia; 2Department of Pharmacology and Therapeutics, Medical Faculty, University of Indonesia, Jakarta, Indonesia; 3Bioavailability and Bioequivalence Laboratory Unit, PT Equilab International, Jakarta, Indonesia Purpose: The current study aimed to evaluate whether a generic product of etoricoxib 120mg film-coated tablet (the test drug) was bioequivalent to the reference product (Arcoxia® film-coated tablet 120mg).Methods: This was a randomized, open-label, two-sequence, crossover study under fasting condition, with a 14-day washout period, involving 26 healthy adult male and female subjects. Blood samples were taken and analyzed for plasma concentrations of etoricoxib (Chemical Abstracts Service [CAS] 202409-33-4) using a high-pressure liquid chromatography–ultraviolet detector (HPLC-UV) system capable of measuring etoricoxib concentrations ranging from 5.00 to 5002.90 ng/mL, with the lowest limit of quantitation of 5.00 ng/mL. A noncompartmental method was used to determine the pharmacokinetic parameters of a single-dose administration of the drug, including the area under plasma concentration–time curve from time zero to the time of last observed concentration (AUC0-t), the area under plasma concentration–time curve from time zero to infinity (AUC0-∞), the maximum plasma concentration (Cmax), the time to reach the maximum plasma concentration (tmax), and the terminal half-life (t½).Results: After a single-dose administration of etoricoxib 120mg film-coated tablet, the mean (SD) values for the AUC0-72h and Cmax of the test drug were 45913.42 (13142.19) ng·h/mL and 3155.93 (752.81) ng/mL, respectively; the values for the reference drug were 44577.20 (13541.85) ng⋅h/mL and 2915.13 (772.81) ng/mL, respectively. The geometric mean ratios (90% CIs) of the test drug/reference drug were 103.40% (98.70%–108.32%) for AUC0-72h and 109.26% (100.18%–119.18%) for Cmax. No clinically significant differences in tmax and t½values were found between the test drug and the reference drug. No adverse events were experienced by the subjects during this study.Conclusion: The present study demonstrated that the evaluated generic etoricoxib 120mg film-coated tablets were bioequivalent to the reference drug. Keywords: bioavailability, bioequivalence, etoricoxib, nonsteroidal anti-inflammatory drug, selective cyclooxygenase-2 inhibitor&nbsp

Nerve injury occurred due to mechanical, thermal, chemical, or ischemic factors. Nerve regeneration is needed for recovery. Schwann cell proliferation and differentiation are important factors in the nerve regeneration process. Schwann cells release neurotrophins in the nerve regeneration process. In this study, Lumbricus rubellus protein fraction DLBS1033N was used as therapeutic protein candidate for nerve regeneration treatments. DLBS1033N treatments promoted the growth and survival of Schwann cell in free serum and free serum plus minimum O2 conditions. Real-time PCR and ELISA methods revealed that DLBS1033N induced NGF expressions. The growth and survival of Schwann cells were related to NGF expressions in a specific inhibitor TrkA study. Furthermore, in real-time PCR study, DLBS1033N was able to activate phosphatidylinositol-3-kinase (PI3K) pathway. This study showed that L. rubellus protein fraction DLS1033N can promote the growth and survival of Schwann cells by inducing NGF expressions. Cells growth and survival activities are likely achieved via PI3K pathway.

Ethnopharmacological relevance: Lagerstroemia speciosa (L.) Pers., commonly known as banaba and locally known as bungur, is widely used in Indonesia and other countries as a folk remedy for various chronic diseases such as diabetes mellitus and hypertension. L. speciosa (L.) Pers. has been used and evaluated on conditions associated to liver diseases by altering cholesterol absorption, lipid metabolism, as well as the related gene expressions. Aim of the study: The aim of this study is to evaluate the effect of DLBS3733, a standardized bioactive fraction of Lagerstroemia speciosa (L.) Pers. leaves, on ameliorating hepatic steatosis induced by oleic acid, and elucidate its mechanism of action to ameliorate lipid accumulation in HepG2 cells. Materials and methods: Effects of DLBS3733 on expression of genes and proteins associated with lipid metabolism were evaluated in HepG2 cells in this study. Genes associated with lipid metabolism were evaluated using PCR, while the protein levels were revealed using western blot and ELISA. Cellular lipid accumulations and triglyceride (TG) synthesis were measured using ELISA, and antioxidant assay was conducted using DPPH assay. Results: DLBS3733 significantly reduced lipid accumulation and TG synthesis by 51% and 32% (p

The light subunit of mushroom Agaricus bisporus tyrosinase (LSMT) is a protein of unknown function that was discovered serendipitously during the elucidation of the crystal structure of the enzyme. The protein is non-immunogenic and can penetrate the intestinal epithelial cell barrier, and thus, similar to its structural homologue HA-33 from Clostridium botulinum, may be potentially absorbable by the intestine. LSMT also shares high structural homology with the ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which has been shown to display biological activity against leukemic cancer cells and dendritic cells. Therefore, we evaluated the biological activity of LSMT. An in vitro assay suggested that LSMT presentation to most of the cancer cell lines studied has a negligible effect on their proliferation. However, inhibition of cell growth and a slight stimulation of cell proliferation were observed with breast cancer and macrophage cells, respectively. LSMT appeared to be relatively resistant against proteolysis by trypsin and papain, but not bromelain. Challenges with gastric and intestinal juice suggested that the protein is resistant to gastrointestinal tract conditions. This is the first report on the biological characteristics and implication of LSMT. (C) 2017 Elsevier B.V. All rights reserved.