Ml Of Phosphate Buffer Solution Research Articles

ANTIMICROBIAL EFFECT OF OZONIZED PHYSIOLOGICAL SOLUTION ON BACTERIA CAUSING CYSTITIS IN DOGS

Bacterial cystitis is a common clinical problem among dogs and is one of the reasons for the empirically administration of antimicrobials. This practice facilitates the selection of bacteria that are multidrug-resistant to antibiotics. In this context, it is urgent to understand and validate therapeutic modalities that complement antimicrobial treatment in cystitis cases. Ozone therapy has been proposed by scientists both in human and animal medicine. Thus, the objective of this study was evaluated the in vitro antimicrobial activity of ozonized saline solution over standard strains and resistant or multi-drug resistant isolates commonly associated with cystitis in dogs. The plating method was used to evaluate the antimicrobial activity of the exposure of 1 mL of phosphate buffer solution containing 108 colony forming units (CFU) mL-1 for 60 s to 4 mL of ozonized saline solution at 78 µg mL-1 over the standard strains (Staphylococcus aureus and Escherichia coli) and resistant or multi-resistant isolates (Proteus mirabillis, Klebsiella sp. e Enterococcus sp.), and the broth microdilution test to determine the minimum inhibitory and bactericidal concentration for S. aureus, E. coli and Pseudomonas aeruginosa strains from the same solution. The plating was the only assay in which treatment with ozonized saline resulted in bactericidal activity, promoting a reduction of more than 99% of colony-forming units for most of the tested strains, except for P. mirabillis. These results are promising, as these disease is the most common lower urinary tract disorder in dogs, with limited allopathic therapy due to increased bacterial resistance.

PSI-4 Evaluation of chemical mitigants when soybean meal was inoculated with porcine epidemic diarrhea virus, porcine reproductive and respiratory virus, and Seneca Valley virus 1

Abstract Chemical mitigants have been found to decrease virus concentrations in feed and ingredient matrices. Continued research is needed to identify the appropriate inclusion levels and application time for different viruses in these matrices. Therefore, the objective was to evaluate different inclusion levels of two different chemical mitigants when applied either before or after inoculation of porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV) and Seneca Valley virus 1 (SVV1) to soybean meal. The experiment was designed in a 2×2×2 factorial with two chemical mitigants, a formaldehyde-based product (Termin-8, Anitox Corp. Lawrenceville, GA) and a product containing Synergistic blend of phytochemicals and carboxylic acids (Finio, Anitox Corp. Lawrenceville, GA), two applications times, before virus inoculation (prevention) and after virus inoculation (mitigation), and either a low or high inclusion level (Termin-8: 2 or 3 kg/MT; Finio: 1.75 or 2.75 kg/MT). On d 1, samples of the respective matrices were weighed in 50 g aliquots and added to 500 mL bottles. Chemical mitigants were applied to the prevention samples at their respective inclusion levels and 50 µL each of 1×107 TCID50/mL PEDV, 1×108 TCID50/mL PRRSV, and 1×108 TCID50/mL SVV1 were added to the mitigation samples. All bottles were shaken and allowed to sit at room temperature for 24 h. On d 2, virus was added to the prevention samples and chemical mitigants were added to the mitigation bottles, samples incubated at room temperature for 1 h before 200 mL of phosphate buffer solution was added, and bottles incubated at 4°C for 18 h. After incubation, liquid from the samples was centrifuged and the supernatant analyzed via triplex PCR at Kansas State University Veterinary Diagnostic Laboratory. Cycle threshold and proportion PCR positive were analyzed using SAS GLIMMIX v 9.4 (SAS, Inc., Cary, NC) with each virus and matrix combination analyzed individually. A chemical mitigant×application time×inclusion level interaction was not observed (P > 0.05) for any virus. A chemical mitigant×application time interaction was observed (P < 0.05) in all three viruses, where less viral RNA was detected (P < 0.05) when either Termin-8 or Finio were applied after inoculation (mitigation) than when applied prior to inoculation or to the positive controls. An application time×inclusion level interaction was noticed (P < 0.05) for SVV1 and PEDV in soybean meal. Chemical mitigants applied at either the low or high inclusion level after inoculation (mitigation) had less detectable RNA (P < 0.05) than their prevention counterparts. Overall, Termin-8 and Finio can reduce detectable RNA immediately in contaminated soybean meal, with ongoing research evaluating the effect of extended contact times between the mitigants and soybean meal.

The immune system contributes to the effectiveness of vaccine therapy in patients with metastatic melanoma

The aim of the study was to identify differences in the immune system parameters between metastatic melanoma patients who responded and did not respond to dendritic cell vaccination.Material and Methods. The study group included 20 patients with stage III–IV metastatic melanoma, who received vaccine therapy with dendritic cells (DC) in a prophylactic mode. The control groups included 13 patients who had symptoms of disease progression at the time of starting vaccine therapy, and 5 healthy donors. The DC-vaccine was prepared in the form of a suspension of the patient’s autologous dendritic cells loaded with tumor antigens in vitro. A single dose had 2 million dendritic cells in 1 ml of phosphate buffer solution, which was administered intradermally in the nearest site to the regional lymphatic collectors. The immune system status was assessed before starting vaccination. The immune system status was evaluated according to the indices of 25 peripheral blood cell populations using multicolor flow cytometry and integral characteristic in the form of the visual image generated by the visualization method of multidimensional data (NovoSpark, Canada).Results. The immune status in patients with metastatic melanoma at the start of DC-vaccination differed and was associated with the effectiveness of subsequent vaccine therapy. The response to vaccination was observed in patients whose immune system status was similar to that of healthy individuals. Low efficacy of DC-vaccine therapy was shown in patients whose immune system status corresponded to that of patients with disease progression. Alterations of the immune system in patients with metastatic melanoma were registered both at the level of individual immunological parameters and at the level of visualized integral characteristics. The integral characteristics of the immune system associated with the patient’s immunocompromised status can be considered as a criterion for stratification of patients with metastatic melanoma for the effective DC-vaccine therapy.Conclusion. The effectiveness of vaccine therapy with dendritic cells in patients with metastatic melanoma is associated with the immune system state before starting this therapy.

Immunopathological Assessment of Hydatid Cyst and Cysticercus Tenuicollis Sonicated Protoscoilces Antigens in Mice

The present study was designed to investigate the cross-protection (protective immunity) between Hydatid cyst and Bladder worm and evaluate the immunologic response of both humerol and cellular immunity in mice. To achieve these goals, 120 mice were used and equally divided into four groups immunized subcutaneously with 2 doses of antigen at the first and 14 days of the experiment. Mice in the first group (n=30) were immunized with 0.3 ml of hydatid cyst sonicated protoscolex antigen. Those in the second group (n=30) were immunized s/c with 0.3 ml of Cysticercus tenuicollis sonicated protscolex antigen. The third group (n=30) was immunized with 0.3 ml of both antigens (0.15 + 0.15), and the fourth group was a control group in which the mice were intraperitoneally injected with 0.2 ml of phosphate buffer solution. At the end of the experiment (30 days), blood samples were taken from the hearts of mice in all groups after being anesthetized by intramuscular injection of Ketamine 60 mg/kg, and Xylazine 12 mg/kg for the assessment of mouse Interleukin-12, IgG, and tumor necrosis alpha levels. The skin test results 24 hours (day 28) post-immunization showed an increase in the skin thickness against both antigens in the treatments, compared to the control. However, there was a decrease at 48 hours (day 29) post-examination in all groups. The results of TNFα titer showed higher titer in the third group, compared to the first, second, and fourth groups. Interleukin 12 concentration showed a higher titer in the third group than in the first, second, and fourth groups. The IgG concentration showed higher titer in the third group compared to the first, second, and fourth groups. In conclusion, immunopathological studies have shown that Ags used in the study, induce humoral and cellular immunity, compared to each Ag alone, and the mixed antigens were much more immunogenic. This cross-reactivity and synergistic interactions between the two parasites may be the cause of their antigenic activities.

Highly sensitive determination of three kinds of amanitins in urine and plasma by ultra performance liquid chromatography-triple quadrupole mass spectrometry coupled with immunoaffinity column clean-up

建立了超高效液相色谱-三重四极杆质谱高灵敏测定尿液和血浆中α-鹅膏毒肽、β-鹅膏毒肽和γ-鹅膏毒肽的方法。经过免疫亲和柱净化,尿液样品浓缩20倍、血浆样品浓缩10倍,以Kinetex Biphenyl色谱柱(100 mm×2.1 mm, 1.7 μm)作为分析柱,甲醇-0.005%(v/v)甲酸水溶液作为流动相进行梯度洗脱分离,电喷雾电离、负离子、多反应监测模式下检测,外标法定量。3种鹅膏毒肽的线性范围为0.1~200 ng/mL,相关系数(r)>0.999。尿液和血浆中3种鹅膏毒肽的基质效应和提取回收率分别为92%~108%和90%~103%,变异系数均小于13%。尿液中3种鹅膏毒肽的准确度为-9.4%~8.0%,重复性和中间精度分别为3.0%~14%和3.5%~18%,当取样量为2.00 mL时,方法的检出限均为0.002 ng/mL;血浆中3种鹅膏毒肽的准确度为-13%~8.0%,重复性和中间精度分别为3.9%~9.7%和5.5%~12%,当取样量为1.00 mL时,方法的检出限均为0.004 ng/mL。该法操作简单、灵敏、准确,已在中毒患者摄入野生蘑菇后138 h的尿液中检出0.0067 ng/mL α-鹅膏毒肽和0.0059 ng/mL β-鹅膏毒肽。该法已成功解决中毒患者尿液和血浆中超痕量鹅膏毒肽的检测难题,对于疑似中毒病人的早诊断、早治疗、降低死亡率都具有非常重要意义,也为今后开展此类毒素毒理作用及机体代谢规律的研究提供了可靠的技术支撑。

IMMUNOLOGICAL INDICATORS IN ANIMAL ORGANISMS UNDER THE INFLUENCE OF ALLOGENEIC ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS

The studies were conducted on 2-3-months-old males of mice weighing 20-24 g. О ur work was to study the functional state of the organs of the immune system of C57Bl/6 mice after introduction of allogeneic MSCs of adipose tissue origin . Obtaining and cultivating of MSCs were carried out in a sterile laminar box with compliance of conditions of asepsis and antiseptics. C57B l /6 mice adipose tissue cultured in a CO 2 incubator at 37 о C and 5% CO 2 in DMEM with 10-15% of fetal bovine serum, 1% of antibiotic-antimycotic solution (Sigma-Aldrich, USA). The following groups of animals were formed: 1 group – intact (control group); 2 group - animals, to whom 0.5 ml of 0.9% NaCl solution (placebo) were injected into the caudal vein; 3 group – animals, to whom were injected 10 4 of allogen e ic MSCs from adipose tissue in 0.5 ml of phosphate buffer solution into the caudal vein. The weight index , content of lymphoid cells of thymus and spleen in C57Bl/6 mice investigated after the introduction of MSC s on 7, 18 and 25 days . To assess the content of lymphocytes in lymphoid organs, the latter were weighed. Whole thymus and 50 mg of spleen were triturated and filtered through the kapron tissue. After that, the tissue homogenate was applied to the gradient of ficoll-urografin (density 1.077) in a ratio of 3:2. The test tubes were centrifuged at a rate of 1500 rpm for 30-40 minutes. After centrifugation the layer of lymphocytes which was above the gradient was collected by a Pasteur pipette and washed twice with an arbitrary amount of Hanks' solution by centrifugation at a rate of 1500 for 10 minutes. 1 ml of Hanks's solution was added to lymphocytes after washing. Lymphocytes were counted in the Goryaev chamber. Calculation of the cells of lymphoid organs was performed on 1 mg of tissue. The administration of allogenic adipose derived mesenchymal stem cells affects on the central and peripheral organs of the immune system. Administration of allogenic adipose derived mesenchymal stem cells cause a significant increase in the content of lymphoid cells in the thymus at 7, 18 and 25 days by 71, 57 and 53 % respectively ( *- p <0,05 ) compared to the control. Quantity lymphoid cells in the spleen significant increase at the 7 and 18 days of the immune response by 33 and 19 %, respectively (p<0.01, p <0.05) compared to the control under administration of allogenic adipose derived mesenchymal stem cells. On the 25th day, lymphoid cell and spleen index values return to normal. Weight index of the thymus and spleen directly correlates with the content of lymphoid cells in its. Key words: thymus , spleen , lymphoid cells , mice , weight index , allogenic mesenchymal stem cells , adipose tissue

Effects of Chemical Mutagen (Sodium Azide) on Onion Grown in Organic and Inorganic Fertilized Soil

The effects of chemical agent (Sodium Azide) on Onion growing in organic and inorganic fertile soil was to be examined during this study. The analysis work was carried out within the green house of the research laboratory technology of the Oke Ogun Science Laboratory Technology, Saki, Oyo State. Onion seeds were soaked inside different beakers containing the mixture of Sodium Azide and water mixed with 10 ml of Phosphate solution for 4 hours. Also, the control was soaked with normal water and 10 ml of Phosphate buffer solution. The treated seeds of onions was planted in plastic containers containing 4.2 g of weighed humus soil within the green house at the Department of research lab Technology of The Oke Ogun polytechnic school, Saki Oyo State. The samples parameter were taken daily for six consecutive months. The result obtained was additionally subjected to statistical analysis by using DMRT techniques. The results showed that the stem length was ranged from 11.39±0.62 and 9.98±0.52 with sample of onion without sodium Azide and inorganic had the highest stem length values and samples of onion with Sodium Azide and inorganic had very cheap stem length. However, the leave length ranged from 29.63±0.12 and 22.45±0.10 with the Onion samples with inorganic and Organic fertilizers which had the highest leave length and sample of onion without Sodium Azide was very low leave length. The results of this study showed that each one the parameters studied within the plant were low with Sodium Azide treatment. The decrease in plant growth, plant heights, root lengths, and Phaonerogam survival, fruit yield per plant and height at maturity with agent concentration. It is hereof suggested that Sodium Azide (NaN3) was expected to produce mutation in onion that area unit extremely liable to harmful pathogens and making them cheap to be useful for farmers.

Safety and immunogenicity of a novel quadrivalent subunit influenza vaccine in animal models

ABSTRACT Background: Compared with trivalent influenza vaccines, quadrivalent influenza vaccines are expected to provide wider protection against influenza B virus infections. We developed a novel quadrivalent subunit influenza vaccine which was distinct from the influenza vaccines available on the market in production process. In this research, we evaluated the safety and immunogenicity of the quadrivalent subunit influenza vaccine in animal models. Methods: In toxicity assessment, 40 SD rats were randomly assigned to be intramuscularly injected with 1.0 ml of the tested vaccine (33 μg/ml) or 0.9% sodium chloride solution. In irritation assessment, eight rabbits were randomly assigned to receive 0.5 ml of tested vaccine or phosphate buffer solution intramuscularly. Thirty-two guinea pigs were randomly assigned to be intramuscularly injected with high-dose tested vaccine (0.5 ml), low-dose tested vaccine (0.05 ml), ovalbumin, or 0.9% sodium chloride solution, respectively, for sensitization assessment. In immunogenicity assessment, 50 BALB/c mice were equally randomized to receive one dose of tested vaccine, two doses of tested vaccine with an interval of 14 days, 0.5 ml of trivalent subunit influenza vaccine, 0.5 ml of monovalent subunit influenza vaccine, or 0.5 ml of phosphate buffer solution. Orbital blood was collected before and 28 and 42 days after administration of the injections for detecting influenza antibody titers. Results: No abnormal toxicity and irritation in rats and rabbits showed in the gross autopsy and histopathological examinations. The results of sensitization in guinea pigs indicated that no obvious allergic symptoms observed in the high-dose and low-dose vaccine groups within 30 min after twice provocations, and the result of sensitization evaluation was negative. Vaccine induced significant immune responses in mice with 100% seroconversion rates at 28 and 42 days after the first dose. The geometric mean titers (GMTs) of hemagglutination inhibition (HI) antibodies at day 28 in one-dose quadri–vaccine and two-dose quadri–vaccine groups were comparable to those in the tri–vaccine or mono-vaccine groups for shared influenza strains. However, the GMTs of HI antibodies against H1N1 (P = 0.025) and BV (P = 0.049) at day 42 in one-dose quadri–vaccine group were significantly lower than those in the tri–vaccine or mono-vaccine groups. The GMTs of HI antibodies against H1N1, H3N1, BY, and BV at day 28 and day 42 were comparable between one-dose quadri–vaccine and two-dose quadri–vaccine groups. Conclusions: The quadrivalent subunit influenza vaccine was safe and immunogenic in animal models. One dose of the vaccine could elicit a satisfactory antibody response in mice.

АКТИВНІСТЬ СУКЦИНАТДЕГІДРОГЕНАЗИ МІТОХОНДРІЙ ПЕЧІНКИ ТВАРИН-РЕЦИПІЄНТІВ ЗА ВПЛИВУ АЛОГЕННИХ МЕЗЕНХІМАЛЬНИХ СТОВБУРОВИХ КЛІТИН

The studies were conducted on 2-3-months-old males of C57BL/6 mice weighing 20-24 g. О btaining of allogenic bone marrow and adipose derived MSCs were carried out in a sterile laminar box. Cells were cultivated in a CO 2 -incubator at 37 о C and 5 % CO2 in DMEM with 10-15 % of fetal bovine serum, 1% of antibiotic-antimycotic solution (Sigma-Aldrich, USA). The following groups of animals were formed: 1 group – intact (control) animals; 2 group – animals, to whom 0.5 ml of 0.89% NaCl solution (placebo) were injected into the caudal vein; 3 group – animals, to whom 10 4 of allogenic AD MSCs in 0.5 ml of phosphate buffer solution were injected into the caudal vein, 4 group – animals, to whom 10 4 of allogenic BM MSCs in 0.5 ml of phosphate buffer solution were injected into the caudal vein. On 12th day after transplantation of MSCs activity of succinate dehydrogenase in animals were determined. The determination of the activity of the enzyme was carried out in accordance with the method, the principle of which is the restoration of potassium ferricyanide (K 3 [Fe(CN) 6 ]) to potassium ferrocyanide (K 4 [Fe(CN) 6 ]) by succinate with involvement of succinate dehydrogenase . Activity was determined by the volume of recovered ferricyanide. Statistical processing of the results was performed with using of Origin 6.1 software and Student's t-criterion. All data are presented in the form of arithmetic average and standard deviations. It was found that at the 12 day of the study, the activity of the succinate dehydrogenase in the third experimental group was 57.7 ± 1.6 mmol / l K 3 [Fe (CN) 6 ] / mg * min (p <0.001), which is significantly higher than in the animals of the first and second group (45.9 ± 0.7 and 43.3 ± 1.2 mmol / l K 3 [Fe (CN) 6 ] / mg * min, respectively). The activity of the enzyme in animal of the fourth experimental groups was also reliable higher compared to the first two groups and was 53.3 ± 1.4 mmol/l K 3 [Fe(CN) 6 ] / mg * min (p < 0.01). It should be noted that mitochondrial succinate dehydrogenase activity in the liver of recipient animals after transplantation of MSCs from adipose tissue is significantly higher than after transplantation of MSCs, obtained from bone marrow (p < 0.05). Thus, reliable raising of mitochondrial succinate dehydrogenase activity in the liver of recipient animals after transplantation of allogenic mesenchymal stem cells from adipose tissue and bone marrow has been determined. Keywords : mesenchymal stem cells, mitochondria, succinate dehydrogenase, mice, bone marrow, fatty tissue.

Analysis of Anthocyanins Extracted from the Acai Fruit (Euterpe oleracea): A Potential Novel Vital Dye for Chromovitrectomy.

Purpose To classify and quantify anthocyanins in a vital dye extracted from the acai fruit (Euterpe oleracea), adjust pH and osmolarity, and perform lyophilization to develop a new chromovitrectomy dye. Methods Three dye concentrations 10%, 25%, and 35% (equivalent to 100, 250, and 350 mg of lyophilized acai fruit pulp extract samples) were evaluated when diluted in 1 ml of phosphate-buffered solution (pH 7 and 300 mOsm). The dye was analyzed by mass spectrometry and high-performance liquid chromatography (HPLC) to identify and quantify anthocyanins molecules. Results The pH and osmolarity correction and lyophilization were performed without damaging the anthocyanin molecular structure. Mass spectrometry confirmed the presence of five anthocyanins in the three concentrations of the dye. Cyanidin-3-O-glucoside was the major anthocyanin found. HPLC showed that the concentration of anthocyanin was similar, independent of the dye concentration tested. Conclusions Lyophilization and the correction of pH and osmolarity (7.00 and 300 mOsm, resp.) were performed successfully. Five anthocyanins are present in the dye from the acai fruit. The major anthocyanin is cyanidin-3-O-glucoside. Independent of the dye concentration tested, the anthocyanin concentration was similar. Standardized chemical characteristics of this new dye may allow its use during chromovitrectomy in humans.

The system influence of allogeneic adipose tissue derived mesenchymal stem cells on the functional state of immune organs

The studies were conducted on 2–3-months-old males of mice weighing 20–24 g. Оur work was to study the functional state of the organs of the immune system of C57Bl/6 mice after introduction of allogeneic MSCs of adipose tissue origin. Obtaining and cultivating of MSCs were carried out in a sterile laminar box with compliance of conditions of asepsis and antiseptics. C57Bl/6 mice adipose tissue cultured in a CO2 incubator at 37 °C and 5% CO2 in DMEM with 10–15% of fetal bovine serum, 1% of antibiotic-antimycotic solution (Sigma-Aldrich, USA). The following groups of animals were formed: 1 group – intact (control group); 2 group – animals, to whom 0.5 ml of 0.9% NaCl solution (placebo) were injected into the caudal vein; 3 group – animals, to whom were injected 104 of allogeneic MSCs from adipose tissue in 0.5 ml of phosphate buffer solution into the caudal vein. The weight index, content of lymphoid cells of thymus and spleen in C57Bl/6 mice investigated after the introduction of MSCs on 7, 18 and 25 days. To assess the content of lymphocytes in lymphoid organs, the latter were weighed. Whole thymus and 50 mg of spleen were triturated and filtered through the kapron tissue. After that, the tissue homogenate was applied to the gradient of ficoll-urografin (density 1.077) in a ratio of 3:2. The test tubes were centrifuged at a rate of 1500 rpm for 30-40 minutes. After centrifugation the layer of lymphocytes which was above the gradient was collected by a Pasteur pipette and washed twice with an arbitrary amount of Hanks' solution by centrifugation at a rate of 1500 for 10 minutes. 1 ml of Hanks's solution was added to lymphocytes after washing. Lymphocytes were counted in the Goryaev chamber. Calculation of the cells of lymphoid organs was performed on 1 mg of tissue. The administration of allogenic adipose derived mesenchymal stem cells affects on the central and peripheral organs of the immune system. Administration of allogenic adipose derived mesenchymal stem cells cause a significant increase in the content of lymphoid cells in the thymus at 7, 18 and 25 days by 71, 57 and 53% respectively (P &lt; 0.05) compared to the control. Weight index of the thymus directly correlates with the content of lymphoid cells and its value was r = 0.57, P &lt; 0.01 on 7 day and 18 day r = 0.50, P &lt; 0.05. Quantity lymphoid cells in the spleen significant increase at the 7 and 18 days of the immune response by 33 and 19%, respectively (P &lt; 0.01, P &lt; 0.05) compared to the control under administration of allogenic adipose derived mesenchymal stem cells. On the 25th day of experience, the content of lymphoid cells in spleen and spleen index values return to normal. Weight index of the spleen directly correlates with the content of lymphoid cells – r = 0.91–0.94 (P &lt; 0.001).

Antitumor effect of Ferula assa foetida oleo gum resin against breast cancer induced by 4T1 cells in BALB/c mice

BackgroundFerula assa foetida commonly consumed as a healthy beverage has been demonstrated to have various biological activities, including antioxidation, anti-obesity and anti-cancer. ObjectiveOur study aims to investigate the antitumor effect of asafoetida in vivo using mouse mammary carcinoma 4T1 cells. Materials and methodsIn the study, female BALB/c mice were divided into two groups (n = 6), which were control (untreated) and other group of mice with breast cancer treated with 100 mg/kg of asafoetida, respectively, by oral gavage. All mice were injected into the mammary fat pad with 4T1 cells (1 × 105 4T1 cells/0.1 ml of phosphate buffer solution). Asafoetida was administered on day 15 after the tumor had developed for 3 weeks. At end of experiment, tumor weight, tumor volume and tumor burden were measured and lung, liver, kidney and tumor were harvested and sections were prepared for histopathological analysis. Lipoxygenase inhibitory and antioxidant activity of asafoetida was also determined. ResultsOur results showed that treatment with asafoetida was effective in decreasing the tumor weight and tumor volume in treated mice. Body weight significantly increased in female BALB/c mice against control. Apart from the antitumor effect, asafoetida decreased lung, liver and kidney metastasis and also increased areas of necrosis in the tumor tissue respectively. ConclusionsThe present study demonstrated that asafoetida has potent antitumor and antimetastasis effects on breast cancer and is a potential source of natural antitumor products.

In vivo Antimicrobial Activity of Ozonized Theobroma Oil Ovules against Candida albicans

Title: Effects of ozonated theobroma oil and ketoconazole ovules on rats infected with Candida albicans. Background: Candidiasis is an infection caused by a yeast shaped fungus which includes Candida gender. Candida albicans is an opportunistic microorganism, which causes more than 80% of vaginal infections. The aim of this study was to determine the effect of ozonized theobroma oil vaginal ovules in the treatment of induced vaginal candidiasis compared to Ketoconazole ovules in Sprague Dawley female rats. Methods and Findings: Animals were ovariectomized and injected with a hormonal treatment after 14 days in order to know the oestrus cycle. After 48 hours rats with keratinous cellules were vaginally infected with an inoculum of 106-107 Candida albicans in 0.1 mL of phosphate buffer solution. Five animals groups were studied: group I (without treatment), group II (treated with unozonized theobroma oil ovules), group III (treated with ketoconazole ovules), group IV (treated with 10% ozonized theobroma oil ovules), and group V (treated with 20% ozonized theobroma oil ovules). Exudates were made before beginning the treatment, 5 and 10 days during the treatment and 48 hours after the end of treatment. Results demonstrated a decrease of 0.7 log of the number of rats with infection after 5 days of treatment with 20% ozonized theobroma oil ovules; however, it was not observed infection in rats after 10 days. A similar result was obtained with ketoconazole ovules. Conclusions: Due to antimicrobial activity of 20% ozonized theobroma oil ovules, it can be recommended their use for treatment of Candidiasis

Anti-breast cancer properties and toxicity of Dillenia suffruticosa root aqueous extract in BALB/c mice

ObjectiveTo determine the anti-breast cancer activities and the safety oral consumption of Dillenia suffruticosa root aqueous extract (DRAE) in BALB/c mice. MethodsIn the anti-breast cancer study, female BALB/c mice were divided into five groups (n = 12), which were (1) positive control (with breast cancer, untreated), (2) negative control (without breast cancer, untreated) and other three groups of mice with breast cancer treated with 1000, 500 and 250 mg/kg of DRAE, respectively, by oral gavage for 28 days. All mice except from the negative control group were injected into the mammary fat pad with 4T1 cells (1 × 105 4T1 cells/0.1 mL of phosphate buffer solution). DRAE was administered orally on Day 11 after the tumor has developed. ResultsThe tumor volume of the 1000 mg/kg of DRAE group reduced significantly compared to the positive control while treatment with 500 mg/kg of DRAE had significantly inhibited metastasis to the heart. In the acute toxicity study, treatment with up to 5000 mg/kg of DRAE was not toxic to the animals, indicating its safety when a large amount of this plant extract was ingested. Based on the sub-acute toxicity study, treatment of the highest dose of DRAE (1000 mg/kg) had mild liver toxicity indicated by mild focal hemorrhage. ConclusionsDRAE possesses anti-breast cancer properties but at the same time it shows mild toxicity to the liver. The non observable adverse effect dose for DRAE is 500 mg/kg.

Bioequivalence of Telmi plus V tablet 80/12.5 mg to Micardis plus tablet 80/12.5 mg (telmisartan/hydrochlorothiazide 80/12.5 mg)

The aim of this study is to assess the quality of Telmi plus V® tablet (test formulation) by comparing its pharmacokinetic parameters with Micardis plus® tablet (reference formulation) in forty healthy Korean male volunteers. Seven subjects were withdrawn during the periods. This study was performed under fasted condition with a randomized, single-dose, 2-period crossover design. The dissolution studies of both formulations were conducted using USP apparatus 2 at 75 rpm with 1000 mL of phosphate buffer solution (pH 7.5) at 37 ± 0.5 °C. Forty healthy Korean male volunteers were enrolled in this study with 19–34 years (24.7 ± 3.9), height 164–184 cm (174.9 ± 5.3) and weight 52–85 kg (71.2 ± 9.0). The results of telmisartan were as follows: the mean AUC0−∞ of reference tablet and test tablet was 3290 ± 2270 and 3080 ± 2010 ng h/mL, respectively; the mean Cmax was 674 ± 366 and 612 ± 252 ng/mL, respectively; the mean Tmax was 0.92 ± 0.44 and 0.99 ± 0.46 h, respectively. The results of HCTZ were as follows: the mean AUC0−∞ of reference tablet and test tablet was 525 ± 94.3 and 536 ± 96.8 ng h/mL, respectively; the mean Cmax was 90.8 ± 25.4 and 86.2 ± 21.4 ng/mL, respectively; the mean Tmax was 1.58 ± 0.60 and 1.78 ± 0.66 h, respectively. The 90 % confidence intervals for geometric mean ratios of test to reference formulation of AUC0−t and Cmax were 0.9132–1.0441 and 0.8579–1.1048 for telmisartan, and 0.9737–1.0713 and 0.8801–1.0326 for HCTZ, respectively. The results of this study in healthy Korean male volunteers showed that the test and reference formulations of 80/12.5 mg telmisartan/hydrochlorothiazide met the MFDA regulatory criteria for bioequivalence. Both formulations were well tolerated, with no serious adverse events reported.

Pharmacokinetic Properties and Bioequivalence of 2 Formulations of Valsartan 160-mg Tablets: A Randomized, Single-Dose, 2-Period Crossover Study in Healthy Korean Male Volunteers

BackgroundThe solubility of valsartan is dependent on pH and thus may cause patient variability in drug absorption and failure in bioequivalence studies; thus, increasing the solubility and release of valsartan at low pH has been suggested for a more favorable pharmacokinetic profile. However, due to this pH dependence, the change in the formulation process could alter the disintegration and/or dissolution profile of the drug, possibly making the results of bioequivalence studies misleading. ObjectiveThe aim of this study was to assess the bioavailability and tolerability of a newly developed oral formulation of valsartan 160 mg (wet-granulation tablet) in healthy Korean male volunteers. MethodThis study was performed with the subjects under fasted conditions, using a randomized, single-dose, 2-period crossover design. Subjects were assigned to receive, in randomized order, a single dose of the test formulation and a reference formulation (valsartan 160-mg dry-granulation tablet), with a washout period of 7 days between the administrations. Blood samples were collected up to 24 hours after dosing, and pharmacokinetic parameters were determined after the plasma valsartan concentration was analyzed using UPLC-MS/MS. The dissolution studies of both formulations were conducted using USP apparatus 2 at 50 rpm with 1000 mL of phosphate buffer solution (pH, 6.8) at 37°C ± 0.5°C. Bioequivalence was defined per Korean Food and Drug Administration’s regulatory criteria as 90% CIs of the geometric mean test/reference ratios of AUC0–t and Cmax within the range of 0.8 to 1.25. Tolerability was assessed using physical examination and subject interviews. ResultsSixty subjects were enrolled (mean [SD] age [range], 23.6 [2.4] years [21–31]; height, 173.7 [6.6] cm [161–190]; and weight, 68.0 [8.7] kg [54–85]). The mean AUC0–∞ values with the test and reference tablets were 31,784 (13,844) and 32,714 (14,512) ng·h/mL, respectively; Cmax, 5094 (2061) and 5064 (1864) ng/mL; Tmax, 2.92 (1.04) and 3.08 (1.01) hours. The 90% CIs for the geometric mean test/reference ratios of AUC0–t and Cmax were 0.9295 to 1.0546 and 0.9190 to 1.0848, respectively, which met the criteria for bioequivalence. The most frequently reported adverse event was dizziness after blank blood sampling, recorded in 4 subjects, 2 cases each with the test and reference formulations. ConclusionsIn this study in healthy Korean male volunteers, the test and reference formulations of 160-mg valsartan met the Korean Food and Drug Administration’s regulatory criteria for bioequivalence despite the difference in formulation (wet granulation vs dry granulation). Both formulations were well tolerated, with no serious adverse events reported.

Pharmaceutical Microencapsulation Technology for Development of Controlled Release Drug Delivery systems

This article demonstrated development of controlled release system of an NSAID drug, Diclofenac sodium employing different ratios of Ethyl cellulose. Diclofenac sodium and ethyl cellulose in different proportions were processed by microencapsulation based on phase separation technique to formulate microcapsules. The prepared microcapsules were then compressed into tablets to obtain controlled release oral formulations. In-vitro evaluation was performed by dissolution test of each preparation was conducted in 900 ml of phosphate buffer solution of pH 7.2 maintained at 37 ± 0.5 °C and stirred at 50 rpm. At pre- determined time intervals (0, 0.5, 1.0, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 hrs). The drug concentration in the collected samples was determined by UV spectrophotometer at 276 nm. The physical characteristics of diclofenac sodium microcapsules were according to accepted range. These were off-white, free flowing and spherical in shape. The release profile of diclofenac sodium from microcapsules was found to be directly proportional to the proportion of ethylcellulose and coat thickness. The in-vitro release pattern showed that with ratio of 1:1 and 1:2 (drug: polymer), the percentage release of drug at first hour was 16.91 and 11.52 %, respectively as compared to 1:3 which is only 6.87 % with in this time. The release mechanism followed higuchi model for its release pattern. Tablet Formulation (F2) of present study was found comparable in release profile the marketed brand Phlogin-SR, microcapsules showed an extended release beyond 24 h. Further, a good correlation was found between drug release and proportion of ethylcellulose in the microcapsules. Microencapsulation based on coacervation found as good technique to control release of diclofenac sodium for making the controlled release formulations. Keywords—Diclofenac sodium, Microencapsulation technology, Ethylcellulose, In-Vitro Release Profile

In vitro degradation behavior of chitosan based hybrid microparticles.

The degradation properties of the MPs is important to the long-term benefits of the use of the chitosan (CS) based hybrid MPs in bone tissue-engineering, because the degradation kinetics could affect a multitude of processes within the cell, such as cell growth, tissue regeneration, and host response. The aim of this study was to investigate the degradation of solid, hybrid CS microparticles (MPs), CS-10% calcium phosphate (CaHPO4, w/w), and CS-10% calcium carbonate (CaCO3, w/w) MPs in phosphate buffered solution (PBS) over a 30-week period. The hybrid MPs were synthesized by emulsification technique, cross-linked with 64% sodium tripolyphosphate (TPP), purified and air dried overnight. Each sample had 30 mg of MPs was placed in a glass vial with 9 ml of PBS added and then the vial was closed to prevent evaporation. Every week 4 ml of the incubated solution was removed for sample measurement and all samples were replaced with an equivalent amount of fresh medium. The samples were maintained at 37°C under continuous shaking. The hybrid MPs were measured for pH and calcium release, every week in triplicate. At 0, 5, 10, 15, 20, 25, and 30 weeks, surface and bulk morphology were analyzed with a scanning electron microscope (SEM). The degradation data suggested that the hybrid MPs were stable at least up to 25 week and maintain the physiologically relevant pH. Therefore, we can use these hybrid MPs to apply in the bone tissue engineering applications since they do not degrade within a short period.

Evaluation of dispersive liquid–liquid microextraction for the simultaneous determination of chlorophenols and haloanisoles in wines and cork stoppers using gas chromatography–mass spectrometry

Dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography–mass spectrometry (GC–MS) was evaluated for the simultaneous determination of five chlorophenols and seven haloanisoles in wines and cork stoppers. Parameters, such as the nature and volume of the extracting and disperser solvents, extraction time, salt addition, centrifugation time and sample volume or mass, affecting the DLLME were carefully optimized to extract and preconcentrate chlorophenols, in the form of their acetylated derivatives, and haloanisoles. In this extraction method, 1 mL of acetone (disperser solvent) containing 30 μL of carbon tetrachloride (extraction solvent) was rapidly injected by a syringe into 5 mL of sample solution containing 200 μL of acetic anhydride (derivatizing reagent) and 0.5 mL of phosphate buffer solution, thereby forming a cloudy solution. After extraction, phase separation was performed by centrifugation, and a volume of 4 μL of the sedimented phase was analyzed by GC–MS. The wine samples were directly used for the DLLME extraction (red wines required a 1:1 dilution with water). For cork samples, the target analytes were first extracted with pentane, the solvent was evaporated and the residue reconstituted with acetone before DLLME. The use of an internal standard (2,4-dibromoanisole) notably improved the repeatability of the procedure. Under the optimized conditions, detection limits ranged from 0.004 to 0.108 ng mL −1 in wine samples (24–220 pg g −1 in corks), depending on the compound and the sample analyzed. The enrichment factors for haloanisoles were in the 380–700-fold range.

Immobilization of lipase on methyl-modified silica aerogels by physical adsorption

In this work, methyl-modified silica aerogels, a new kind of macro-porous material with high porosity, were used as carriers to immobilize lipase by adsorption. SEM, TEM, nitrogen adsorption device, and thermogravimetric analysis were used to characterize the properties of modified aerogels. The surface area was 395.6m2/g, and the average pore diameter was 68.72nm. The contact angle of aerogel particles increased from 20.9° to 99.2° after methyl modification. Reaction characteristics of the material after enzyme loading were also discussed. The results showed that adsorption capacity could reach 67.42mg/g; and the maximal enzyme activity was 19.87μmolmin−1mg−1 (protein), and activity retention could reach 56.44%. It is worth mentioning that the amount of modified aerogels added had significant effects on the diameter of droplets and the mass transfer behavior of substrates in the reaction emulsion. Online microscope was used to visualize the droplets in the emulsion, where the aerogel particles were observed locating at the interface of oil and water. The average diameter of droplets reached the minimum when 0.06g of modified aerogels was added into the reaction emulsion which contained 10ml of oil and 10ml of phosphate buffer solution. The phenomenon was resulted from the wettability of methyl-modified silica aerogels.