Asian Pacific Journal of Tropical Biomedicine

702–706

Authors: Rosario Galarza-Seeber, Juan David Latorre, Xochitl Hernandez-Velasco, Amanda Drake Wolfenden, Lisa Renee Bielke, Anita Menconi, Billy Marshall Hargis, Guillermo Tellez

Objective: To evaluate the ability of Bacillus spp. as direct-fed microbials (DFM) to biodegrade aflatoxin B1 (AFB1) by using an in vitro digestive model simulating in vivo conditions. Methods: Sixty-nine Bacillus isolates were obtained from intestines, and soil samples were screened by using a selective media method against 0.25 and 1.00 mg/mL of AFB1 in modified Czapek-Dox medium. Plates were incubated at 37
Cand observed every two days for two weeks. Physiological properties of the three Bacillus spp. candidates were characterized biochemically and by 16S rRNA sequence analyzes for identification. Tolerance to acidic pH, osmotic concentrations of NaCl, bile salts were tested, and antimicrobial sensitivity profiles were also determined. Bacillus candidates were individually sporulated by using a solid fermentation method and combined. Spores were incorporated into 1 of 3 experimental feed groups: 1) Negative control group, with unmedicated starter broiler feed without AFB1; 2) Positive control group, with negative control feed contaminated with 0.01% AFB1; 3) DFM treated group, with positive control feed supplemented with 109 spores/g. After digestion time (3:15 h), supernatants and digesta were collected for highperformance liquid chromatography fluorescence detection analysis by triplicate. Results: Three out of those sixty-nine DFM candidates showed ability to biodegrade AFB1 in vitro based on growth as well as reduction of fluorescence and area of clearance around each colony in modified Czapek-Dox medium which was clearly visible under day light after 48 h of evaluation. Analysis of 16S-DNA identified the strains as Bacillus amyloliquefaciens, Bacillus megaterium and Bacillus subtilis. The three Bacillus strains were tolerant to acidic conditions (pH 2.0), tolerant to a high osmotic pressure (NaCl at 6.5%), and were able to tolerate 0.037% bile salts after 24 h of incubation. No significant differences (P > 0.05) were observed in the concentrations of AFB1 in neither the supernatants nor digesta samples evaluated by high-performance liquid chromatography with fluorescence detection between positive control or DFM treated groups. Conclusions: In vitro digestion time was not enough to confirm biodegradation of AFB1. Further studies to evaluate the possible biodegradation effects of the Bacillus-DFM when continuously administered in experimentally contaminated feed with AFB1, are in progress.

714–719

Authors: Haytham Ahmed Zakai, Wajihullah Khan

Objective: To analyse the efficacy of diethylcarbamazine (DEC), tetramisole and chlorpromazine on the longevity and activity of glucose-6-phosphatase and succinate dehydrogenase in the microfilariae recovered from the peripheral circulation of the rats before and after the treatment. Methods: Setaria cervi worms were implanted in white rats via laparotomy and microfilaraemic rats were divided into 4 groups. Groups 1, 2 and 3 were treated with DEC, tetramisole and chlorpromazine respectively, while Group 4 served as infected control. Longevity of microfilariae and differential leucocyte counts were recorded till the disappearance of microfilariae from peripheral blood. Glucose-6-phosphatase and succinate dehydrogenase enzymes were localized in the microfilariae recovered from normal and treated rats. Results: The microfilariae survived for 48 days in untreated rats while survival was reduced to 15, 21 and 27 days after treatment with DEC, tetramisole and chlorpromazine, respectively. Eosinophils and neutrophils increased during 2nd and 3rd weeks, whereas the lymphocytes increased during 4–7 weeks. DEC treatment resulted in slight decrease in the localization of succinate dehydrogenase but not in glucose-6-phosphatase. Tetramisole and chlorpromazine treatment did not show any appreciable change in the localization of both the above enzymes. Conclusions: DEC proved the most effective drug which cleared the microfilaraemia within 15 days and reduced the activity of succinate dehydrogenase to some extent followed by tetramisole and chlorpromazine which took more time for the clearance of microfilariae and had no effect on the localization of both glucose-6-phosphatase and succinate dehydrogenase.

728–732

Authors: Farida Hussan, Rina Haryani Osman Basah, Mohd Rafizul Mohd Yusof, Nur Aqilah Kamaruddin, Faizah Othman

Objective: To determine the effect of Plantago major (P. major) extract on the liver injury following acetaminophen (APAP) toxicity. Methods: The male Sprague Dawley rats (n = 38) were randomly divided into normal control (n = 6) and experiment (n = 32) groups. The latter was subdivided into four groups and induced with APAP (1000 mg/kg) per oral, followed by P. major extract and N-acetylcysteine orally to the respective groups for six days. Results: On the seventh day, the serum bilirubin, liver enzymes and tissue malondialdehyde were increased in APAP groups whereas the total protein in serum, tissue superoxide dismutase and glutathione levels were reduced. The plant extract treatment reduced the histological deteriorations such as aggregation of hepatocellular cords, formation of binucleated cells and vacuolisation of the cells with scanty cytoplasm. It also revealed significant reduction of malondialdehyde and increased level of superoxide dismutase and glutathione. The findings in the extract treated groups were comparable to the group treated with N-acetylcysteine. Conclusions: In conclusion, P. major can enhance innate antioxidant activity and ameliorate the APAP-induced liver injury.

738–743

Authors: Hataipan Phienwej, Ih-si Swasdichira, Surattana Amnuoypol, Prasit Pavasant, Piyamas Sumrejkanchanakij

Objective: To investigate the effect of Tinospora crispa (T. crispa) extract on matrix metalloproteinase-13 (MMP-13) expression and cell migration. Methods: The cytotoxicity of T. crispa extract was examined by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay on head and neck squamous cell carcinoma (HNSCC) cell lines. The effect on expression of MMP-13 was analysed by RT-PCR and ELISA. The migration was assessed by wound healing assay. Results: MMP-13 mRNA was highly expressed in the metastatic human HNSCC cell lines, HN22 and HSC-3. T. crispa extract at a concentration of 100.0 mg/mL caused about 50% reduction of cell survival. T. crispa extract at a non-toxic concentration of 12.5, 25.0 and 50.0 mg/mL significantly suppressed MMP-13 mRNA expression and secreted MMP- 13 in both HN22 and HSC-3. The expression of tissue inhibitors of metalloproteinase-2 (TIMP-2) by HSC-3 cells was attenuated by 25.0 and 50.0 mg/mL of T. crispa extract. Addition of the extract to cells in a wound healing assay showed inhibition of cell migration by HN22 cells. Conclusions: These data suggest that T. crispa could be considered as a potential therapeutic drug to prevent metastasis of HNSCC.

748–755

Authors: Anastasia Wheni Indrianingsih, Sanro Tachibana, Rizna Triana Dewi, Kazutaka Itoh

Objective: To isolate and investigate antioxidant and a-glucosidase inhibitor compounds in the leaves of Quercus gilva Blume (Q. gilva). Methods: Dry leaves of Q. gilva were extracted with methanol and the methanolic extract was further separated by silica gel column chromatography using several solvents with increasing polarity. The antioxidant activities of the isolated compounds were evaluated using various in vitro assays: 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, hydrogen peroxide radical scavenging activity, b-carotene bleaching assay, and reducing power assay. The a-glucosidase inhibitory assay was conducted against aglucosidase from Saccharomyces cerevisiae. Results: Three compounds were isolated and their structures were identified as catechin (1), epicatechin (2), and tiliroside (3) using an instrumental analysis. Compound 2 had higher antioxidant activity with inhibitory concentrations (IC50) of (22.55 ± 2.23) mmol/L than that of quercetin, which was used as the standard, with an IC50 of (28.08 ± 2.39) mmol/L, followed by compound 1 with IC50 of (40.86 ± 3.45) mmol/L. On the other hand, compound 3 had the lowest antioxidant activity with an IC50 of (160.24 ± 8.15) mmol/L. However, compound 3 had the highest a-glucosidase inhibitory activity with an IC50 of (28.36 ± 0.11) mmol/L, followed by compounds 1 and 2 with (168.60 ± 5.15) and (920.60 ± 10.10) mmol/L, respectively. Conclusions: The results obtained for the antioxidant activities and a-glucosidase inhibitory activities in a methanolic extract from the leaves of Q. gilva confirmed the potential of this plant as a source of natural antioxidants and antidiabetic medicine.

761–767

Authors: Hai Nguyen Thanh, Hue Pham Thi Minh, Tuan Anh Le, Huong Duong Thi Ly, Tung Nguyen Huu, Loi Vu Duc, Thu Dang Kim, Tung Bui Thanh

Objective: To investigated the protective potential of ethanol extracts of Scutellaria baicalensis (S. baicalensis) against lipopolysaccharide (LPS)-induced liver injury. Methods: Dried roots of S. baicalensis were extracted with ethanol and concentrated to yield a dry residue. Mice were administered 200 mg/kg of the ethanol extracts orally once daily for one week. Animals were subsequently administered a single dose of LPS (5 mg/ kg of body weight, intraperitoneal injection). Both protein and mRNA levels of cytokines, such as tumor necrosis factor alpha, interleukin-1b, and interleukin-6 in liver tissues were evaluated by ELISA assay and quantitative PCR. Cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor-kB protein levels in liver tissues were analyzed by western blotting. Results: Liver injury induced by LPS significantly increased necrosis factor alpha, interleukin-1b, interleukin-6, cyclooxygenase-2, inducible nitric oxide synthase, and nuclear factor-kB in liver tissues. Treatment with ethanol extracts of S. baicalensis prevented all of these observed changes associated with LPS-induced injury in liver mice. Conclusions: Our study showed that S. baicalensis is potentially protective against LPSinduced liver injury in mice.

773–777

Authors: Mustafa Kemal Çelen, Murat Sayan, Tuba Dal, Celal Ayaz, Alicem Tekin, Tuncer O¨ zekinci, Suda Tekin Koruk, Tunga Barcin, Recep Tekin, Mehmet Sinan Dal, Sevgi Kalkanlı

Objective: To detect the subtype characterization and drug-resistant mutations in HIV-1 strains after the refugee movement from Syria to Turkey between 2011 and 2014 in south east border lines. Methods: A total of 65 patients were included in this study, of which 57 (88%) patients were antiretroviral therapy-naive patients. HIV-1 RNA was detected and quantified by real-time PCR assay. HIV-1 subtypes and circulating recombinant forms (CRFs) were identified by phylogenetic analysis (neighbor-joining method), and drug-resistant mutations were analyzed. Results: Three major HIV groups were indicated. Two of these groups were located in subtype B. The other group showed heterogeneity. Subtype B (48/65, 73.8%), followed by CRFs (12/65, 18.5%) was the most common strain. Subtype of CRFs consisted of CRF01_AE (9/65, 13.8%) and CRF02_AG (3/65, 4.6%). Subtype C (1/65, 1.5%), sub-subtypes A1 (2/65, 3.1%) and F1 (2/65, 3.1%) were also detected with low prevalence. The rate of overall primary antiretroviral resistance was 4.9% (3/61). Drug-resistant rate for non-nucleoside reverse transcriptase inhibitors was 4.9%. The thymidine analogue mutation rate was 13.1% (8/61). Conclusions: HIV molecular epidemiology studies are necessary to determine transmission patterns and spread. Subtype B and CRF01_AE, CRF02_AG are the most prevalent strains in the south-east of Turkey. However, subtype C, sub-subtypes A1 and F1 are of low prevalence but persist in the south-east of Turkey. In the near future, changing of HIV epidemiology will be possible in Turkey due to migration movement in border lines and resistance testing will play an important role in HIV management.

781–784

Authors: D´ebora Oro, Andreia Heissler, Eliandra Mirlei Rossi, Diane Scapin, Patrıcia da Silva Malheiros, Everton Boff

Objective: To evaluate the antifungal effect of Cinnamomum zeylanicum (C. zeylanicum) and Melaleuca alternifolia essential oils and honey against strains of Candida sp. from HIV-positive patients in order to subsidize new therapeutic strategies for candidiasis. Methods: The study evaluated the antifungal effect of natural antimicrobials against 30 strains of Candida sp. isolated from oral cavities in HIV-infected patients. Then, they were compared to the action of fluconazole and amphotericin B. Antifungal susceptibility was evaluated by the broth macrodilution technique and the minimum inhibitory concentration and the minimum fungicidal concentration were determined. Results: Among all antifungals evaluated in this study, amphotericin B was the one showing the best results; however, all compounds studied here showed inhibitory activities against isolates of Candida sp. Honey (0.0313–64 mg/mL) demonstrated fungistatic activity inhibiting 70% of the isolates. C. zeylanicum essential oil (0.0313–64 mg/ mL) inhibited 93.3% of the Candida strains and Melaleuca alternifolia essential oil (0.0313–64 mg/mL) was able to inhibit 73.3% of them. Conclusions: Therefore, all natural compounds evaluated in this study, especially C. zeylanicum essential oil, may become promising agents for oral candidiasis therapy including in HIV-positive patients.