Effects of Peel Extract from Citrus reticulata and Hesperidin, A Citrus Flavonoid, on Macrophage Cell Line

The extract of Citrus reticulata has been studied for its biological activities, due to its citrus flavonoid content. The extract and its flavonoid compounds exhibit growth inhibition properties in several cancer cell lines and in vivo models. Conversely, the extract can also induce cell proliferation and angiogenesis, and shows estrogenic effects, in vitro and in vivo. Because of the contrasting effects that depend on the concentration or dosage, the precise action of the extract and its flavonoids need to be elucidated in various cell types. The objective of this study is to evaluate the effect of Citrus reticulata peel extract (Citrus extract) and hesperidin, a citrus flavonoid, on the modulation of cell proliferation in the RAW 264.7 macrophage cell line. Cell viability under Citrus extract or hesperidin treatment was assessed by using the MTT assay. The expression of interleukin-10 (IL-10), an anti-inflammatory cytokine, modulated by Citrus extract was also examined by immunostaining. Low concentrations of Citrus extract at 1 and 100 μg/mL were able to induce cell proliferation, though not significantly, as shown by cell viability of 138 and 114%, respectively. At higher concentrations of 500, 750, and 1000 μg/mL, Citrus extract decreased cell viability significantly by up to 64, 46, and 36%, respectively. Accordingly, hesperidin at low (3.1 μg/mL−61.1 μg/mL) or high (152.6 μg/mL−305.3 μg/mL) concentrations increased or reduced cell viability significantly by up to 116−136% or 10−61%, respectively. The value of the 50% inhibitory concentration (IC50) of Citrus extract was more than three times higher (756 μg/mL) than that of hesperidin (203 μg/mL = 332 μM). Additionally, 250 μg/mL of Citrus extract was able to induce IL-10 expression compared with the control. These results demonstrate that Citrus extract and hesperidin exert a biphasic effect on macrophage cells. The future development of Citrus extract as a co-chemotherapeutic, anticancer, or immunomodulatory agent should include careful consideration of its biphasic effect on each cell type

Curcumin Analogs Induce Apoptosis and G2/M Arrest In 4T1 Murine Triple-Negative Breast Cancer Cells

Chemotherapy is the first-line treatment for triple-negative breast cancer (TNBC), yet toxicity and resistance effects have been the current problems. Curcumin,a natural compound, has been reported to exert anti-proliferative effects on various cancer cells, including breast carcinoma cells. However, the β-diketone moiety influences the stability of curcumin. Curcumin analogs, pentagamavunon-0 (PGV-0), and pentagamavunon-1 (PGV-1) were synthesized to improve the stability and activity of curcumin by modified the β-diketone moiety into mono-ketone pentanone. In this study, we evaluated the cytotoxicity, inhibition of cell cycle progression, and induction of apoptosis of curcumin and its analogs (PGV-0 and PGV-1) in murine triple-negative breast cancer 4T1 cell line. The cytotoxic evaluation was done by MTT assay, while apoptosis induction and cell cycle evaluation was performed by annexin V staining and detected by flow cytometry. Curcumin and its analogs, PGV-0, and  PGV-1, significantly inhibit the viability of 4T1 breast cancer cells with an IC50 value of 34.34µg/mL, 13.76µg/mL and 38.21μg/mL, respectively. Apoptosis analysis with a dose of 10µg/mL and 15µg/mL in 4T1 breast cancer cells showed that curcumin and its analogs effectively induce apoptotic in a dose-dependent manner. In cell cycle analysis using a dose of 15µg/mL, curcumin inhibited the cell cycle progression in the S phase, whereas PGV-0 and PGV-1 inhibited the cell cycle in the G2/M phase. It could be concluded that curcumin analogs, PGV-0 and PGV-1, have higher potential to be developed as anti-cancer agents by inducing cell cycle arrest and apoptosis in triple-negative breast cancer

The effect of Phyllanthus niruri L extracts on human leukemic cell proliferation and apoptosis induction

Objective: To investigate the effect of Phyllanthus niruri Linn (Euphorbiaceae) in the proliferation of human leukemic cells (MOLT-4 and K562).Methods: Phyllanthus niruri L (P.niruri) was macerated by using various solvents to obtain the crude extracts. Cytotoxicity of the extracts against MOLT-4 and K562 cells was tested using MTT assay to find the IC50 value. To analyse cell cycle progression, cellular DNA was measured using propidium iodide (PI) staining. Apoptosis induction was evaluated using Annexin V-FITC and PI staining and analysed using FACSVerse flow cytometry. Finally, the expression of p53 on MOLT-4 and K562 cell lysate was measured by western blotting, to identify the possible mode of action of the anticancer activity.Results: P. niruri crude extracts demonstrated a potential anti-cancer effect towards MOLT-4 cells (IC50 range was 42.21 ± 4.98 to 97.06 ± 18.29 µg/ml). However, against K562 cells, P.niruri extracts exhibited a lower inhibitory potency (the IC50 was 120.19 ± 8.48 to 256.55 ± 26.22 µg/ml). The results showed the selectivity of the toxic effect of the extracts against MOLT-4 and K562.  To evaluate the possible mechanism of action the anticancer effect, we evaluated P. niruri extract action in apoptosis induction and p53 expression. The results showed that methanol and hexane extract inhibited MOLT-4 cell progression from G1 to S-phase, indicating G1 cell arrest. Moreover, apoptotic cell population following treatment of MOLT-4 and K562 cells with methanol extract was markedly increased, showing morphological signs of apoptosis including membrane degradation and chromatin condensation. Furthermore, we found that there was an increase in p53 expression following MOLT-4 treatment with methanol extract, suggesting that p53 induction may be involved in cell apoptosis.Conclusions: The results indicated the involvement of p53 pathway in the mechanism of anti-cancer activity exerted by P. niruri extract on MOLT-4 cells. However, for cancer cells lacking P53 expression, such as K562 cells, apoptosis might take place via other pathways

Cost Effectiveness Analysis of Rivaroxaban Compared to Warfarin and Aspirin for Stroke Prevention Atrial Fibrillation (SPAF) in the Indonesian healthcare setting

Main drugs used in the prevention of stroke among atrial fibrillation (AF) patients are antiplatelets (aspirin) and oral anticoagulants (OAC). OAC therapy can be difficult to administer due to drug and food interactions, adds the burden of required blood monitoring, narrow therapeutic window, and requirements for dose titration. Rivaroxaban is a single-dose oral anticoagulant which does not require blood monitoring, dose titration or has dietary interactions. Phase III clinical data from the ROCKET trial have recently been reported the non-inferiority of rivaroxaban over warfarin for the prevention of strokes in AF patients. To develop an economic model evaluating the clinical and cost-effectiveness of rivaroxaban for the prevention of stroke in non-valvular AF patients in the Indonesian health care settings. We conducted cost effectiveness analysis from the perspective of payer (national health insurance). Effectiveness data used the international data from previous RCT and network metaanalysis studies. Costs data used local data of Indonesia from national health insurance’s reimbursement tariffs. Markov model was used, comprised of health and treatment states describing the management and consequences of AF. The main analysis was based on data from the phase III trials. Three months was used as cycle length. The time horizon was set at patients’ lifetime (20 years). Costs and outcomes were discounted at a 3% annual rate. Subgroup analysis and extensive sensitivity analysis was conducted. Willingness to pay (WTP) threshold in Indonesia was set as 3 times GDP of Indonesia in 2015, equal about IDR 133,375,000 per quality-adjusted life year (QALY). Base case rivaroxaban vs warfarin has ICER of IDR 141,835,063per QALY at the current cost of rivaroxaban IDR 23,500 and ICER of 130,214,687 per QALY at the proposed cost of rivaroxaban IDR 22,000. One-way sensitivity analysis showed that the key drivers of cost-effectiveness were the utility decrement applied to stable warfarin patients, discontinuation/subsequent discontinuation rates for rivaroxaban, and discontinuation/subsequent discontinuation rates for warfarin. The probabilistic sensitivity analysis suggested that rivaroxaban was cost-effective compared to warfarin in about 45% of cases at the WTP per QALY. Rivaroxaban with the proposed price of IDR 22,000 was considered to be more cost-effective when compared to warfarin

Response Surface Methodology used in the Optimization of RP-HPLC Condition for Quantitative Analysis of Carmine and Rhodamine B

The objective of this study was to optimize reversed-phase high-performance liquid chromatography (RP-HPLC) using an experimental design approach based on the response surface methodology of Central Composite Design (CCD) for separation and analysis of carmine (CAR) and Rhodamine B (RHO) in lipstick products. Some factors (independent variables) responsible for RP-HPLC separation including pH of buffer phosphate (X1), the acetonitrile ratio (X2), flow rate of mobile phase (X3), and column temperature (X4) were investigated. While, the responses (dependent variables) evaluated were resolution between CAR and RHO (Y1), tailing factor of CAR (Y2), tailing factor of RHO (Y3), retention time of CAR (Y4), retention time of RHO (Y5), peak area of CAR (Y6) and peak area of RHO (Y7). CCD showed that separation of CAR and RHO was influenced by these independent variables (factors). The optimum predicted conditions for the separation of CAR and RHO based on statistical results was pH buffer of 3.4, ACN 55%, the flow rate of 1.1 mL/min and column temperature of 35oC with the desirability of 1. Both CAR and RHO were clearly separated using optimum conditions, as suggested by CCD. The developed techniques were effective for optimizing chromatographic separation, therefore, the time consumption and a large number of running could be hindered

Evaluation of Pain Scale Decrease and Adverse Effects of Ketorolac Injections: An Observational Study in Patients with Postoperative Pain

The use of ketorolac injections in Indonesia is restricted with the provision of 2-3 ampoules per day with a maximum of two days even though the literature states that ketorolac could be used for no more than five days. This study aimed to determine the decrease in pain scale as well as gastrointestinal and renal adverse effects of ketorolac injections in two days of use. This study was an observational study with one-group pre-test post-test design conducted prospectively. The group was a group of patients with postoperative pain who received ketorolac injections and were treated during January till April 2018 in an academic hospital in Yogyakarta. The results showed that ketorolac injections did not provide a statistically significant decrease in pain scale in two days of use compared to before surgery (median [range] = 2.0[0.0-9.33] vs 1.33[0.0-8.33]; p=0.32). Ketorolac injections decreased the kidney function of subjects in two days of use compared to before surgery based on creatinine values (0.76mg/dL vs 0.80mg/dL; p=0.024) and GFR (96.13mL/min/m2 vs 87.52mL/min/m2; p=0.023), and as many as 31 subjects (43.06%) experienced complaints that were suspected to be the gastrointestinal adverse effects of ketorolac injections with the three most complaints were bloating (18.06%), nausea (16.67%), and heartburn (15.28%). Those three results support the use of ketorolac injections following what has been regulated in the Indonesian National Formulary

Pharmacokinetics Interaction and Biodistribution of 5 Fluorouracil with Radiopharmaceuticals 99mTc Glutathione for Cancer Diagnostic in Mice Cancer Model

Radiopharmaceutical 99mTc-Glutathione has been developed for cancer diagnostic in nuclear medicine. Interactions between chemotherapy drugs and radiopharmaceuticals can altered radiopharmaceuticals performance.  Drug interaction 5-fluorouracil (5-FU) with a radiopharmaceutical 99mTc-Glutathione in mice cancer model has been proven in pharmacokinetics study. The biological half-life distribution of 99mTc-Glutathione for cancer model mice when administrated with 5-FU become longer to 0.340±0.121h if compared with 99mTc-Glutathione. Biological half-life elimination for cancer model mice given with 99mTc-Glutathione is 72.712±2.427h. Administration of 5-FU makes the biological half-life elimination of 99mTc-Glutathione shorter to 17.030±3.459h. Biodistribution study of 5-FU continued with 99mTc-Glutathione for cancer model mice showed higher physiological uptake in the kidney was observed (39.77±2.70%ID/g) for 99mTc-Glutathione has lower uptake on kidney (29.55.3.73 %ID/g) with p<0.05. Based on calculation on cancer model mice with colon cancer compared with muscle, shown target/non-target (T/NT) ratio 2.93 for 5-FU continued with 99mTc-Glutathione has ratio 0.42. Low ratio T/NT may affect to poor organ visualization for cancer diagnosis.  Acute toxicity study has shown drugs safety for clinical purpose. The knowledge about chemotherapy drug interaction with a radiopharmaceutical is important to have a correct diagnosis of the patient on clinical application

Profile of Biofilm-Producing Staphylococcus epidermidis from Intravenous Catheter Colonisation at Prof. Dr. Margono Soekarjo Hospital Purwokerto

Biofilm- producing Staphylococcus epidermidis has evolved to be a significant human pathogen, particularly in the use of medical devices such as an intravenous catheter. Furthermore, biofilm-producing bacteria 10-1000 fold less susceptible to several antimicrobial agents than free-bacteria. This simple survey aimed to describe the profile of biofilm-producing S. epidermidis from intravenous catheter colonization of some patients in surgical and internal medicine wards at the hospital Margono Soekarjo, Purwokerto, and the antibiotics resistance pattern. A vitek® 2 compact (Enseval Medika Prima) was performed to identify the bacterial species and to examine the 73 antibiotics for understanding the resistance pattern automatically. Microtiter plate biofilm assay with crystal violet staining was performed to measure biofilm optical density (OD) for analyzing the biofilm production capabilities. A scanning electron microscopy  (SEM)  was done to compare the thickness of ultrastructure of biofilm-producing S. epidermidis visually. The present study found that 2 of  8 Gram-positive bacteria (25%) were biofilm-producing  S. epidermidis.  One of  S. epidermidis was moderate whereas the other was high biofilm-producing bacteria. Images of SEM showed that a high biofilm-producing S. epidermidis has a thicker ultrastructure of biofilm than the moderate biofilm-producing, whereas a control, the weak biofilm-producing  S. epidermidis ATCC 12228 has the least biofilm. Both of S. epidermidis strains were sensitive to Gentamicin, Moxifloxacin, Quinupristin/Dalfopristin, Linezolid, Vancomycin, Doxycycline, Minocycline, Tetracycline, Tigecycline, and Nitrofurantoin. Furthermore, both  S. epidermidis strains were resistant to the other (63) antibiotics. In conclusion, two strains of S. epidermidis in this study have different capabilities to form the biofilm which were showed that high biofilm-producing strain was thicker than moderate biofilm-producing strain by scanning electron microscopy. However, both of them were resistant to the same number of antibiotics.

Effect of Platelet-rich Plasma on Caspase-3 and IGF-1 mRNA expression in the diabetic rat testis

Testicular damage is a serious complication of diabetes mellitus resulting in male infertility, which is associated with caspase-3 and IGF-1 mRNA expression. Platelet-rich plasma (PRP), with its rich growth factor composition, has proven beneficial in regenerative therapy. It is believed that PRP has not been studied in testes for diabetes mellitus and there are no studies in the literature concerning the influence of PRP on expressions of growth factors in testes.The aim of this study was to investigate the efficacy of adjunctive PRP in insulin treatment for repair of testicular damage in a diabetic rat model. Diabetes was induced by administering single dose 60 mg/kg streptozotocin. Twenty Wistar male rats were divided into four groups: group 1, control group; group 2, diabetes without treatment; group 3, diabetes with treated insulin; and group 4, diabetes with treated insulin and PRP. Rats were euthanized after two weeks of treatment, and testes were taken for caspase-3 and IGF-1 mRNA expression measurements.Diabetes mellitus induction caused a significant increase in caspase-3 mRNA expression with p=0.049 and significant decrease in IGF-1 mRNA expression with p=0.004. There was no difference in caspase-3 and IGF-1 mRNA expression of the diabetic rat testis given insulin and PRP compared to without PRP

The Effect of Thionamide to TRH, TSH, IL-4, T-REG, and Anti-TPO in Graves’ Disease

The most common cause of hyperthyroidism is Graves' disease. TRH and TSH are hormonal factors that modulate and control thyroid function in Graves' disease. In the immunological aspect, Graves' disease is played by the role of T-reg, IL-4, and anti-TPO. Graves' disease treatment goal is to inhibit thyroid hormone secretion by administering thionamide. The evaluation of this treatment is its hormonal and immunological aspects. To describe the effect of thionamide on serum TRH, TSH, IL-4, T-reg, and anti-TPO levels in Graves' disease. This study is a clinical trial study in 25 study participants. All study participants were given thionamide, namely PTU 300mg for three months and blood samples were taken for laboratory tests. Serum TRH, TSH, IL-4, T-reg FOXP3, and anti-TPO levels were examined by ELISA. The mean levels at the beginning and after three months of therapy are: serum TRH 92.589pg/mL and 115.944pg/mL; serum TSH 0.041mU/L and 0.223mU/L; serum IL-4 19.759pg/mL and 23.040pg/mL; T-reg FOXP3 gene polymorphism 0.621ng/mL and 0.518 ng/mL; serum anti-TPO 2697.539pg/mL and 2604.710pg/mL. Increased levels of serum TRH and TSH levels were statistically significant. The change in serum IL-4, T-reg FOXP3 gene polymorphism, and anti-TPO levels were not statistically significant. The administration of thionamide in Graves' disease for three months will significantly decrease Wayne index and serum FT4 levels, increase serum TRH and TSH levels