Structure Identification and Risk Assurance of Unknown Impurities in Pramipexole Oral Drug Formulation.

Impurities compounds in any pharmaceutical product or drug substance are inevitable from a chemistry point of view. The quality and safety of a pharmaceutical product are also significantly affected by these impurities content; therefore, impurities need to be identified and characterized through the use of appropriate analytical methods. Pramipexole is a nonergot dopamine agonist used to treat various Parkinson's disease symptoms. Two unknown impurities were detected from a pramipexole dihydrochloride solid dosage form. These impurities were identified and characterized using ultra-performance liquid chromatography coupled with high-resolution mass spectroscopy (UPLC-HRMS). These impurities were found to be enriched when mannitol existed in the formulation. The structure and mechanism involved in the existence of the impurities were proposed. Furthermore, observation of the binding affinity potential risk of these impurities to the pramipexole receptor has also been demonstrated through molecular docking and molecular dynamics simulation study. The binding energy result showed that pramipexole interaction with dopamine receptors D2 and D3 was higher than pramipexole mannose adduct and pramipexole ribose adduct.

An elevated OmpA expression during the production of a recombinant protein in Escherichia coli.

Escherichia coli cells rapidly respond to changes in the environment. Such response must be anticipated upon development of fermentation strategy for commercial purposes. The response may signal changes in cell physiology, which is critical for the cell growth and the level of the target protein production. One of the responses is the elevated expression of membrane proteins to tightly control the trafficking of molecules into and out from the cells. Normally, the expression level of the membrane protein is basal as the fermentation is carried out in physiological conditions. Here, we reported an elevated expression of the outer membrane protein A (OmpA) during a series of fermentation conduct, starting from the shake flask, 1-L to finally 10-L fermentor. The incidence led to a lower expression of the target protein and thereby resulting in lower process efficiency. OmpA expression was concomitant to the bacterial growth and already observed in the early exponential phase. Despite the drawback, this phenomenon actually inspires the observation of OmpA expression as one of the indicators for the E. coli cells response to the fermentation conditions. This auxiliary check would prevent the higher OmpA expression that led to the low expression of the target protein.

Revealing Edible Bird Nest as Novel Functional Foods in Combating Metabolic Syndrome: Comprehensive In Silico, In Vitro, and In Vivo Studies.

Metabolic dysfunction, which includes intra-abdominal adiposity, glucose intolerance, insulin resistance, dyslipidemia, and hypertension, manifests into metabolic syndrome and related diseases. Therefore, the discovery of new therapies in the fight against metabolic syndrome is very challenging. This study aims to reveal the existence of an edible bird nest (EBN) as a functional food candidate that may be a new alternative in fighting metabolic syndrome. The study included three approaches: in silico molecular docking simulation, in vitro, and in vivo in rats fed on cholesterol- and fat-enriched diets. Four terpenoids of Bakuchiol, Curculigosaponin A, Dehydrolindestrenolide, and 1-methyl-3-(1-methyl-ethyl)-benzene in EBN have been identified through LCMS/MS-QTOF. In molecular docking simulations, Bakuchiol and Dehydrolindestrenolide are considered very potent because they have higher inhibitory power on the four receptors (iNOS, ROS1 kinase, FTO, and lipase) than standard drugs. In vitro tests also provide insight into the antioxidant, antidiabetic, and antiobesity activities of EBN, which is quite feasible due to the smaller EC50 value of EBN compared to standard drugs. Interestingly, in vivo studies also showed significant improvements (p < 0.05) in the lipid profile, blood glucose, enzymatic levels, and inflammatory biomarkers in rats given high-dose dietary supplementation of EBN. More interestingly, high-dose dietary supplementation of EBN upregulates PGC-1α and downregulates HMG-CoA reductase. Comprehensively, it has been revealed that EBN can be novel functional foods for combating metabolic syndrome.

The Effect and Safety Assessment of Metformin and DLBS3233 (A Bioactive Fraction of Lagerstroemia speciosa and Cinnamomum burmannii) on Improving Metabolic Parameters in Women with Polycystic Ovary Syndrome.

Background: Polycystic ovary syndrome (PCOS) can lead to compensatory hyperinsulinemia with consequent metabolic abnormalities in women. In this study, DLBS3233 and Metformin were used to be tested. DLBS3233 itself is the new insulin-sensitizing drug, a combination-bioactive-fraction derived from two Indonesian herbals, Lagerstroemia speciosa and Cinnamomum burmannii. DLBS3233 alone and in combination with metformin were evaluated for efficacy and safety in insulin-resistant women with polycystic ovary syndrome (PCOS). Methods: A randomized, double-blind, 3-arm, double-dummy, non-inferiority, and also a controlled clinical study was conducted at the Dr. Kariadi Hospital, Indonesia, between October 2014 and February 2019. The study involved 60 female subjects (with 20 female subjects in each group) that had polycystic ovary syndrome (PCOS).Treatment I consists of one placebo capsule twice per day and one 100 mg DLBS3233 capsule once per day. Treatment II consists of one placebo caplet once per day and one 750 mg Metformin XR caplet twice per day. Treatment III consists of one 750 mg Metformin XR caplet twice per day and one 100 mg DLBS3233 capsule once per day. Results: In treatment I, the homeostatic model assessment for insulin resistance (HOMA-IR) levels were 3.55, 3.59, and 3.80 at pretest, 3 months, and 6 months after intervention, respectively. In treatment II, the HOMA-IR level were 4.00, 2.21, and 4.40 at pretest, 3 months, and 6 months after intervention respectively. In treatment III, the HOMA-IR levels were 3.30, 2.86, and 3.12 at pretest, 3 months, and 6 months after intervention, respectively. There was no apparent difference existed in the fasting plasma glucose (FPG), high-density lipoprotein (HDL), triglycerides, ferriman-gallwey scores (FGS), and safety assessment on vital signs and laboratory examinations (liver function and renal function) in all groups. Conclusion: Either DLBS3233 alone or the DLBS3233/Metformin combination showed no significant efficacy and did not negatively affect cardiovascular function, liver and kidney function in PCOS subjects. ClinicalTrialsgov Identifier: NCT01999686 Date: 3rd of December, 2013.

Purification and proteomic analysis of potent fibrinolytic enzymes extracted from Lumbricus rubellus.

BACKGROUND: Lumbrokinase derived from earthworms, Lumbricus rubellus is known to have fibrinolytic enzymes that have potential as therapeutic drugs due to its ability to dissolve fibrin. The current study is aimed to purify the Lumbrokinase from L. rubellus and identify its protein component. METHODS: Water extract of local earthworm Lumbricus rubellus revealed several proteins. Therefore, to identify its protein component, purification through HiPrep DEAE fast flow and proteomic analysis were conducted prior to identifications. A combination of two-dimension gel electrophoresis (2DE) and electrospray ionization mass spectrometry analysis was used to identify the purified fractions. RESULTS: The purified fractions contain five protein bands, namely F25-1, F25-2, F85-1, F85-2, and F85-3, which displayed strong fibrinogenolytic activity. F25 fractions showed fibrinogenolytic activity of 974.85 U/mg, while F85 fractions showed higher activity of 1,484.11 U/mg. Fractions F85-1, F85-2, and F85-3 showed molecular weights of 42.6 kDa, 27.03 kDa, and 14 kDa, respectively and were identified as Lumbrokinase iso-enzymes. CONCLUSION: This preliminary study indicates that the F25 and F85 fractions are similar to published fibrinolytic protease-1 and lumbrokinase, respectively, in terms of their amino acid sequence.

The effect of Psidium guajava Leaves' extract for mild and asymptomatic corona virus Disease-19.

COVID-19 is an emerging disease that attacks the respiratory and systemic systems. Various treatments have been used for COVID-19, but no antiviral agents seemed to be efficacious. Many medicinal plants are commonly used for viral infections in Indonesia, including the guajava leaf. The study aimed to determine the effects of Psidium guajava extract supplementation on inflammation markers of asymptomatic and mild COVID-19 patients. The conversion time of PCR results was also evaluated. This study was an experimental, single-blinded, randomized clinical trial (ClinicalTrials.gov: NCT04810728) comparing the efficacy of P. guajava extract at the dose of 1000 mg/8h on top of standard treatment with the standard treatment only for asymptomatic and mild COVID-19 subjects. The primary endpoints were neutrophil and lymphocyte percentages as well as the neutrophil/lymphocyte ratio (NLR) on day 7 of the treatment. The secondary endpoints were high-sensitivity c-reactive protein (hs-CRP) level, PCR-based conversion time, and recovery rate at weeks 2 and 4. A total of 90 subjects were enrolled, and there were 40 subjects in the P. guajava (experimental) group and 41 subjects in the control group who completed the research. On day 7, significantly lower percentage of neutrophil (52.4% vs 58.9%, p = 0.002), higher lymphocyte percentage (35.5%, vs 29.7%, p = 0.002), and lower NLR (1.5 vs 2.1, p = 0.001) were demonstrated in the experimental versus control group. The PCR-based conversion time was shorter (14 days vs 16 days, p < 0.001), and recovery rate at 2 and 4 weeks were higher (49% vs 27%, p = 0.03 and 100% vs 82%, p = 0.003, respectively) in the experimental group. There were no differences in the baseline characteristics. P. guajava extract supplementation reduced neutrophil and increased lymphocyte percentages which led to reduced NLR, accelerated PCR-based conversion time, and increased recovery rate in subjects with mild and asymptomatic COVID-19 infection.\.

Anti-inflammatory Effect of Predimenol, A Bioactive Extract from Phaleria macrocarpa, through the Suppression of NF-κB and COX-2.

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-  (TNF-), interleukin-1ß (IL-1ß), interleukin-2 (IL-2), interleukin-6 (IL-6), and nuclear factor B (NF-B) 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-1ß, IL-2 and IL-6 were quantified using the polymerase chain reaction (PCR) method. Western blotting was applied to detect phosphorylated IB 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-1ß, 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.

Potential Antiaging Effects of DLBS1649, a Centella asiatica Bioactive Extract.

PURPOSE: 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.

Antidiarrheal Effect of DLBS1Y62, a Bioactive Fraction of Uncaria gambir Roxb. Dried Sap Extract, in Wistar Rats.

BACKGROUND: 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.

Molecular mechanism of DLBS3733, a bioactive fraction of Lagerstroemia speciosa (L.) Pers., on ameliorating hepatic lipid accumulation in HepG2 cells.

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 < 0.01), respectively, through the significant increment of adiponectin expression by 58% (p < 0.01). Subsequently, adiponectin enhanced PPARα expression and AMPK phosphorylation which further regulate the downstream signaling pathway of lipogenesis and lipolysis. Moreover, 2.5 µg/mL DLBS3733 was found to significantly downregulate the expression of HMGCR, ACC and SREBP by 66%, 61% and 36%, respectively (p < 0.01), as well as significantly upregulate CPT-1 by 300% at the protein level (P < 0.05). DLBS3733 was also found to possess high antioxidant activity, where the highest concentration exhibited DPPH inhibition activity by up to 93% (P < 0.01). CONCLUSIONS: We propose that DLBS3733 may provide a prevention on hepatic steatosis through its activity as anti-lipogenesis, anti-cholesterologenesis and pro-lipolysis in HepG2 cells. This is the first report that revealed the molecular mechanism of L. speciosa (L.) Pers. as a potential treatment of hepatic steatosis-related diseases.

Effect of DLBS1033 on Functional Outcomes for Patients with Acute Ischemic Stroke: A Randomized Controlled Trial.

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.

Molecular analysis of a fibrin-degrading enzyme from Bacillus subtilis K2 isolated from the Indonesian soybean-based fermented food moromi.

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.

Relationship of Agaricus bisporus mannose-binding protein to lectins with ß-trefoil fold.

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 ß-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 ß-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.

Gastroprotective Effect of DLBS2411 Bioactive Fraction from Cinnamomum burmannii Against Ethanol-Induced Gastric Damage in Rats.

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.

Agaricus bisporus mannose binding protein is not an agglutinating protein.

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.

Orf239342 from the mushroom Agaricus bisporus is a mannose binding protein.

A recently discovered lectin-like protein from mushroom tyrosinase designated as orf239342 inhibits proliferation of the MCF-7 breast cancer cells. This characteristic is likely derived from its ability to recognize sugar entity on the cell surface. Thereby, the binding specificity of orf239342 to sugars was studied. Orf239342 was found to bind specifically to mannose upon analysis with the surface plasmon resonance technique. Finally, our in vitro study showed that mannose impeded orf239342 ability to inhibit proliferation of the MCF-7 breast cancer cells, providing further evidence for the mannose binding onto the protein. Our finding is a breakthrough to characterise orf239342 i.e. to define its functioning in the mushroom, association to the tyrosinase, or even possible application in breast cancer therapy. In addition, the finding allows the more appropriate designation of the protein as Agaricus bisporus mannose binding-protein (AbMb).

Green isolation and physical modification of pineapple stem waste starch as pharmaceutical excipient.

The waste of inedible parts of pineapple, particularly in tropical countries, contributes to environmental burden. This study aimed to utilize pineapple stem waste as a source of starch-based pharmaceutical excipient. The starch was isolated from pineapple stem waste using a simple process without applying harsh chemicals. The isolated starch (PSS) was then physically modified through gelatinization and spray drying to improve its physical properties. Starch characteristics were identified by FTIR, TGA, and XRD analysis. The SEM imaging showed morphological change with reduced surface roughness due to physical modification of the starch. Decreased crystallinity of modified starch (MPS) was confirmed by our XRD results: the peaks of A-type crystalline at 2θ of 13°, 15°, 18°, and 23° were present in PSS, yet mostly absent in MPS. Thermogravimetric analysis showed that MPS behaved differently from PSS and the degradation events occurred at lower temperature. When the starch was spray-dried without prior gelatinization process, the physicochemical characteristics of spray-dried starch resembled untreated starch. Moisture content in PSS (10.66%) decreased after gelatinization to 7.3%. Potential use of MPS was demonstrated by its powder flowability (Student's t test, p < 0.05), swelling capacity (Student's t test, p < 0.05), and compaction profile. In summary, our findings demonstrated that modified pineapple starch showed better physical characteristics and quite promising as a tablet binder and disintegrant.

A Clinical Trial on Biological Half Life of Bioactive Protein from Lumbricus rubellus, DLBS1033 in Healthy Volunteers.

BACKGROUND: DLBS1033 is a bioactive protein fraction extracted from Lumbricus rubellus, with fibrinogenolytic, fibrinolytic and anti-aggregation activities reported in an in vitro study. Plasma half-life is an important parameter to calculate its dose. This study was conducted to evaluate the biological half-life of DLBS1033 by measuring serial plasmin-antiplasmin (PAP) complex. PAP complex is a stable and inactive compound as a result of fibrinolysis process. METHODS: this was an open-label clinical trial in healthy adult subjects. Subjects were divided into two groups to receive single dose drugs (received 3 x 490 mg) or repeated administration until steady state conditions (3 x 490 mg/day for 3 days). Blood samples for PAP complex measurement were collected at time 0 (before drug administration for single dose group), then at 0.5, 1, 1.5, 2, 3, 6, 8, 10, 12, and 24 hours after drug administration. Safety parameters used in this study were creatinine, prothrombin time (PT), activated partial thromboplastin time (aPTT), SGOT, and SGPT. RESULTS: the biological half-life of DLBS1033 was calculated based on the mean of PAP complex concentration on each time sampling. In single dose group, the highest mean of PAP complex concentration was reached before drug administration. Our result showed that the activity of DLBS1033 could not be determined after single dose administration. In steady state condition, the PAP complex concentration increase in 2 hours after last drug administration. The biological half-life of DLBS1033 was 8.6 hours. There were no significant safety findings on all laboratory parameters and no serious adverse events. CONCLUSION: it is concluded that the fibrinolytic effects of DLBS1033 can be measured in steady state condition. The biological half-life of DLBS1033 in steady state condition was 8.6 hours. There were no serious adverse events on two groups of subjects.

Pharmacokinetic equivalence study of nonsteroidal anti-inflammatory drug etoricoxib.

PURPOSE: The current study aimed to evaluate whether a generic product of etoricoxib 120 mg film-coated tablet (the test drug) was bioequivalent to the reference product (Arcoxia® film-coated tablet 120 mg). 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 120 mg 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 120 mg film-coated tablets were bioequivalent to the reference drug.

Protective Effect of Phaleria macrocarpa Water Extract (Proliverenol) against Carbon Tetrachloride-Induced Liver Fibrosis in Rats: Role of TNF-α and TGF-ß1.

Phaleria macrocarpa is one of the Indonesian herbal plants which has been shown to have a hepatoprotective effect. This study was conducted to evaluate the protective effect of water extract of mahkota dewa (Phaleria macrocarpa) in liver fibrosis and to elucidate its mechanism of action. Male Sprague-Dawley rats were treated with carbon tetrachloride (CCl4) for 8 weeks to induce liver fibrosis. Rats were randomly divided into 6 groups (n=5), i.e., control group, CCl4 group, CCl4 + NAC group, CCl4 + various doses of water extract of Phaleria macrocarpa (50, 100, and 150 mg/kg body weight). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), liver histopathology, malondialdehyde (MDA), ratio GSH/GSSG, Tumor Necrosis Factor- (TNF-) α, and Transforming Growth Factor- (TGF-) ß 1 were analyzed. This study demonstrated that water extract of Phaleria macrocarpa and NAC significantly protected CCl4-induced liver injury as demonstrated by reduced AST, ALT, ALP, and fibrosis percentage compared with the CCl4-only group. In addition, water extract of Phaleria macrocarpa and NAC significantly reduced the levels of MDA, TNF-α, and TGF-ß 1 as well as increasing the ratio of GSH/GSSG. Water extract of Phaleria macrocarpa prevents CCl4-induced fibrosis in rats. The prevention of liver fibrosis was at least in part through its antioxidant and anti-inflammatory activities and through its capacity to inhibit hepatic stellate cells (HSC) activation by reducing fibrogenic cytokine TGF-ß 1.