Asian Pacific Journal of Tropical Biomedicine

11-17

Authors: Jarinyaporn Naowaboot, Nuntiya Somparn, Suphaket Saenthaweesuk

Objective: To evaluate the renoprotective effect of umbelliferone in high-fat diet/streptozotocin-induced type 2 diabetic rats. Methods: We established a streptozotocin-induced type 2 diabetic model in male Wistar rats. The rats were fed with high-fat diet (45 kcal% lard fat) and injected with 35 mg/kg streptozotocin. Diabetic rats were treated with umbelliferone for 8 weeks. At the end of the experimental period, the serum and kidney were used for measuring biochemical parameters, protein expression and histological analysis. Results: After 8-week treatment, umbelliferone decreased fasting plasma glucose, concentrations of malondialdehyde and monocyte chemoattractant protein-1 in the plasma and tissues. It also significantly reduced serum creatinine, blood urea nitrogen, serum advanced glycation end products, as well as kidney weight in type 2 diabetic rats (P<0.05). Moreover, umbelliferone reduced the 24-h urine albumin, but increased 24-h urine creatinine excretion (P<0.05). In renal protein expression, umbelliferone decreased the levels of transforming growth factor-ß1 and fibronectin while increasing the levels of superoxide dismutase and catalase (P<0.05). Renal histological examination revealed an enlarged glomerular size in diabetic rats, which was smaller in umbelliferone-treated diabetic rats. Conclusions: Umbelliferone alleviates renal dysfunction in diabetes via decreasing hyperglycemia, oxidative stress, inflammation and glycation.

23-32

Authors: Nik Abdul Aziz Nik Kamarudin, Judy Nur Aisha Sat, Nur Fatihah Mohd Zaidi, Khairul Mohd Fadzli Mustaffa

Objective: To isolate and characterize RNA aptamers that are specific to human CD36 protein using systematic evolution of ligands by exponential enrichment (SELEX) technology to identify candidates for adjunct therapy to reverse the binding of Plasmodium-infected erythrocytes. Methods: RNA aptamers were isolated using nitrocellulose membrane-based SELEX and binding analysis was screened using an electrophoretic mobility shift assay and enzyme-linked oligonucleotide assay. Results: Thirteen cycles of nitrocellulose membrane-based SELEX yielded three aptamers (RC60, RC25, RC04) exhibiting high binding against CD36 protein as shown on electrophoretic mobility shift assay. The sequence analysis revealed a G-quadruplex sequence within all the isolated aptamers that might contribute to aptamer binding and thermodynamic stability. The specificity assay further showed that RC60 and RC25 were highly specific to CD36. The competitive inhibition assay demonstrated that RC60 and RC25 shared a similar binding epitope recognized by mAb FA6-152, a specific monoclonal antibody against CD36. Conclusions: RC60 and RC25 are promising candidates as anti-cytoadherence for severe malaria adjunct therapy.

42-46

Authors: Ulfet Cetinkaya, Gülay Sezer, Arzuv Charyyeva

Objective: To evaluate the anti-microsporidial effects of the active component of Nigella sativa seeds, thymoquinone, against Encephalitozoon intestinalis using an in vitro model. Methods: Anti-microsporidial effect of thymoquinone against Encephalitozoon intestinalis was evaluated by using various concentrations of thymoquinone (0, 1, 5, 10, 15, 20, 30, 35, and 40 μΜ) and sterile dimethyl sulfoxide. Real time PCR was used to evaluate the inhibitory effects of thymoquinone on the life cycle of Encephalitozoon intestinalis. Results: The cytotoxic effect of thymoquinone on HEK293 cell line was observed with 30, 35, and 40 μM concentrations of thymoquinone after 24, 48, and 72 hours of incubation. It was observed that 10, 15, 20, and 30 μM concentrations of thymoquinone decreased the spore density compared with the control; however, it was significant only at 30 μM. Conclusions: Thymoquinone shows potent anti-microsporidial effects against Encephalitozoon intestinalis in the in vitro model; however, the toxic concentrations of thymoquinone are also toxic to the host cells.

365-372

Authors: Mariano Nicolás Alemán, Sara Serafina Sánchez, Stella Maris Honoré

Objective: To evaluate the potential of Smallanthus sonchifolius (S. sonchifolius) roots in ameliorating hepatic damage of rats fed with a high fructose diet. Methods: The effect of S. sonchifolius roots on energy intake, body weight, fat and liver mass was determined in male rats fed with a high-fructose diet. Plasma glucose, triglycerides, total cholesterol, lipoproteins and aspartate aminotransferase and alanine aminotransferase were analyzed. Histological changes of the livers were evaluated by electronic microscopy and apoptosis was examined using the TUNEL method. The levels of malondialdehyde, reduced-glutathione and antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase) activities were also determined. Results: S. sonchifolius roots significantly decreased energy intake, body weight, fat and liver mass (P < 0.05). S. sonchifolius roots ameliorated liver steatosis and mitochondrial morphology, avoiding cellular apoptosis and normalizing transaminase activity in the liver of rats fed with high fructose. Enzymatic assays revealed that S. sonchifolius roots had a modulatory effect on the oxidative stress induced by fructose-feeding by reducing lipid peroxidation (P < 0.05) and antioxidant enzyme activities (P < 0.05) in liver. Conclusions: S. sonchifolius roots can ameliorate non-alcoholic fatty liver disease by improving oxidative stress and liver injury.

381-388

Authors: Mohamed Bouhrim, Hayat Ouassou, El Hassania Loukili, Mohammed Ramdani, Hassane Mekhfi, Abderrahim Ziyyat, Abdelkhaleq Legssyer, Mohammed Aziz, Mohamed Bnouham

Objective: To assess the antidiabetic effect of Opuntia dillenii seed oil on rats with diabetes mellitus. Methods: A rat diabetes model was established by intraperitoneal injection of rats with 50 mg/kg streptozotocin. Thirty albino Wistar rats were divided into five groups: the diabetic control group and normal control group were treated only with distilled water, two diabetic groups received 1 and 2 mL/kg of oil per day, respectively, for 30 days and one diabetic group received 2 mg/kg of glibenclamide. In addition, blood glucose was determined weekly. Body weight, average daily food, water intake and urinary volume of each animal were determined before and after the treatment period. After the treatment period, hepatic glycogen was determined using the anthrone reagent, and glycosuria, total cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, urea, creatinine and uric acid were estimated using common clinical diagnostic kits. Results: Oral intake of the oil at 1 and 2 mL/kg for the diabetic animals significantly diminished blood glucose, glycosuria, total cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, urea, creatinine and uric acid, accompanied by a noticeable elevation in the amount of hepatic glycogen in comparison with the diabetic control group. Similarly, Opuntia dillenii seed oil significantly increased the food intake and decreased the urinary volume per day in treated rats of the same groups in comparison with the period before the treatment intervention and attenuated body weight loss in the diabetic rats. Moreover, this effect of the oil was dose dependent. On the other hand, the oil did not affect their need for water. Conclusions: The results show that Opuntia dillenii seed oil has a very important antidiabetic effect on streptozotocin-induced diabetic rats. Hence, we suggest it as a preventive control of diabetes mellitus.

397-404

Authors: Waode Munaeni, Widanarni , Munti Yuhana, Mia Setiawati, Aris T Wahyudi

Objective: To analyze compounds in Eleutherine bulbosa (E. bulbosa) (Mill.) Urb. extract and to determine its antibacterial capability against Vibrio parahaemolyticus (V. parahaemolyticus). Methods: E. bulbosa bulb extract was preprared using 96% ethanol by the maceration method. Phytochemical investigation of E. bulbosa extract was analyzed using GC-MS, spectrophotometry and titrimetry methods. The zone of inhibition was identified by the diffusion agar method. The minimum inhibitory concentration and minimum bactericidal concentration were determined using the plate count method. The inhibitory rate against V. parahaemolyticus was determined by the microdilution method. Cellular leakage was evaluated by spectrophotometry and cellular damage was observed by scanning electron microscopy. Results: GC-MS analysis showed the high compound of the E. bulbosa extract was securixanthone E (7-hydroxy-1,2-dimethoxyxanthone). The compound groups also included fatty acid esters, isoquinolines, naphthalenes, and phenolics. The total phenolic content was (2.50 ± 0.00) mg/g, flavonoid (6.61 ± 0.00) mg/g, and tannin (0.03 ± 0.00)%. The greatest zone of inhibition and inhibitory rate were (11.83 ± 0.06) mm and (91.32 ± 2.76)%, respectively, at 10 mg/mL. The minimum inhibitory concentration was 0.156 mg/mL, while the minimum bactericidal concentration was 10 mg/mL. The E. bulbosa extract caused leakage and cellular damage to V. parahaemolyticus. Conclusions: The E. bulbosa extract possesses inhibitory activities against V. parahaemolyticus and causes cellular leakage and damage.

323-332

Authors: Nagaraju Jalli, KV Santhi Sri, Sairengpuii Hnamte, Subhaswaraj Pattnaik, Parasuraman Paramanantham, Busi Siddhardha

Objective: To investigate the antioxidant and anti-infective potential of Phrynium capitatum and Dryptes indica extract. Methods: The antioxidant potentials were determined by DPPH radical scavenging, reducing power, hydroxyl radical scavenging and total antioxidant assays. We further examined anti-quorum sensing activity and inhibition of synthesis of pathogenic factor of Chromobacterium violaceum and Pseudomonas aeruginosa PAO1. Bioactive compounds were determined using gas chromatography–mass spectrometry analysis. In silico analysis was conducted to determine the binding affinity of bioactive compounds of plant extracts for the quorum sensing regulatory receptor LasR. Results: DPPH assay showed that the ethanolic extract of Phrynium capitatum and Dryptes indica at 500 μg/mL showed (86.96 ± 4.07)% and (74.83 ± 3.47)% inhibition, respectively. Hydroxyl radical scavenging assay showed (73.17 ± 3.03)% and (62.63 ± 4.59)% activity, respectively. The ethanolic extract of Phrynium capitatum and Dryptes indica showed high level of attenuation of quorum sensing regulated pyocyanin production. Confocal laser scanning microscopic analysis revealed that the extracts had the potential to effectively inhibit biofilm formation of Pseudomonas aeruginosa. Molecular docking analysis showed a better binding affinity of bioactive compounds from the extracts for the structure of LasR protein of Pseudomonas aeruginosa. Conclusions: The ethanolic extracts of Phrynium capitatum and Dryptes indica possess antioxidant activity and the potential to inhibit the quorum sensing system and its regulatory irulence traits in Pseudomonas aeruginosa PAO1.

339-345

Authors: Nurul Hidayah Wahab, Nur Afina Mohd Din, Yee Ying Lim, Noor Izani Noor Jamil, Nor Fazila Che Mat

Objective: To examine the proapoptotic properties of Oroxylum indicum methanol extract on cervical cancer cells. Methods: Methylene blue assay was used to determine the IC50 value of the extract. Western blotting assays were done to analyze the expression of HPV oncoproteins (HPV18 E6 and E7) and apoptotic molecules (caspase-3 and caspase-8). Reverse transcriptase PCR assays were performed to determine genetic alteration of tumor suppressors p53 and pRb and apoptosis markers Fas and FasL. Enzyme-linked immunosorbent assay (ELISA) was done to determine the expression of cytokine levels (IL-6 and IL-12). Results: The determination of IC50 value indicated a higher anti-proliferative activity of the extract compared to cisplatin. After 24 hours of treatment, Western blot analysis showed that treated HeLa cells exhibited a significant down-regulation of HPV18 oncoproteins E6 and E7, and a significant induction of caspase-8 and caspase-3 activation level. Meanwhile, the mRNA expressions of p53, pRb, Fas and FasL were significantly upregulated in treated cells. Moreover, ELISA showed an increased IL-12 and decreased IL-6 production after Oroxylum indicum treatment. Conclusions: The methanol extract of Oroxylum indicum has an anti-proliferative activity and proapoptotic potential. It induces localized-immunity improvements by altering cytokine production in HPV-positive cervical cancer cells.

353-358

Authors: Mostafa Kardan, Zahra Yazdani, Zaher Morsaljahan, Mohammad Ali Ebrahimzadeh, Alireza Rafiei

Objective: To evaluate antitumor activities of Fritillaria imperialis and Eryngium caucasicum methanolic extracts on human hepatoma (HepG2) and colon cancer (HCT116) cell lines in comparison to human foreskin fibroblasts as the normal cells. Methods: Methanolic extracts of Fritillaria imperialis and Eryngium caucasicum were prepared by the maceration method. The effect of the extracts at various concentrations (100, 200, 400, 600, and 800 μg/mL) on cell survival was evaluated using the MTT method. Besides, fluorescence staining was used to evaluate death patterns of the cells. Results: MTT assay showed that Fritillaria imperialis significantly decreased the viability of all cell lines after 24 and 48 hours of treatments. However, Eryngium caucasicum extract did not show any significant cytotoxicity effect on the cell lines. Fluorescence staining revealed that Fritillaria imperialis induced apoptosis of HCT116 cells at 550 μg/mL. Conclusions: Fritillaria imperialis extract has antiproliferative and cytotoxic effects on HCT116 and HepG2 cancer cells and therefore, may serve as an anticancer agent.

278-283

Authors: Roopesh Poojary, Nayanatara Arun Kumar, Reshma Kumarchandra, NA Vinodini, K Bhagyalakshmi, Ganesh Sanjeev

Objective: To explore the effect of hydroalcoholic extract of Cynodon dactylon on the whole body radiation-induced oxidative status of the cerebellum and cognitive impairments in mice. Methods: Swiss albino mice were randomly divided into the control group, radiation control group, low dose and high dose Cynodon dactylon extract treated groups and pre-treated with Cynodon dactylon extract before irradiation. Cynodon dactylon extract was administered for 7 d daily in low dose (0.25 g/kg) and high dose (1 g/kg). On day 7, mice were irradiated with a sublethal dose of 5 Gy gamma rays. Motor coordination was assessed by elevated rotarod test and spatial memory was studied by water maze test. Subsequently, biochemical markers (glutathione, lipid peroxidation and nitric oxide levels) in the cerebellum were evaluated. Results: The gamma irradiated group showed significant impairment in motor coordination and spatial memory compared to normal mice. Mice treated by Cynodon dactylon extract prior to gamma radiation showed good improvement in both paradigms compared to the radiation control group. Moreover, glutathione level was increased, while lipid peroxidation and nitric oxide levels were significantly reduced in mice receiving low dose and high dose of Cynodon dactylon extract compared to the radiation control group. Conclusions: The present study suggests the neuroprotective role of Cynodon dactylon against radiation-induced cognitive impairment and oxidative stress on the cerebellum of mice.

291-298

Authors: Kedsara Junmarkho, Pintusorn Hansakul

Objective: To study the inhibitory effect of rice bran extracts of Thai black Kam Muang and red Hawm Dawk Mali Deang on oxidative stress factors including superoxide (O2•-), nitric oxide (NO•), and inducible nitric oxide synthase (iNOS). Methods: Bran extracts (40% ethanol) of Kam Muang and Hawm Dawk Mali Deang were obtained and evaluated for in vitro 2-2’-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and NO• scavenging activity. Their inhibitory effects on cellular O2•- and NO• were measured in phorbol 12-myristate 13-acetate-stimulated neutrophil-like HL-60 cells and lipopolysaccharide- stimulated RAW264.7 macrophages, respectively, and their viability was monitored using the MTT assay. The effect on iNOS expression was also assessed by the Western blotting assay. Total contents of phenolics, flavonoids, and subtypes were also determined. Results: Hawm Dawk Mali Deang exhibited about 3.5-fold greater cellular O2•- inhibitory activity than Kam Muang [EC50 values of (23.57±4.54) and (81.98±1.45) μg/mL, respectively] in phorbol 12-myristate 13-acetate-stimulated HL-60 cells. Hawm Dawk Mali Deang exhibited about 2-fold higher in vitro ABTS•+ and NO• scavenging activity than Kam Muang, but it exerted cellular NO• inhibitory activity of only about 26% (undetermined EC50 value) in lipopolysaccharide-stimulated RAW264.7 cells. Conversely, Kam Muang exerted potent cellular NO• inhibitory activity [EC50 value: (281.13±59.18) μg/mL] and dose-dependently decreased iNOS levels. No cytotoxicity of both extracts was detected in both cell types. As for corresponding contents, Hawm Dawk Mali Deang contained higher contents of phenolics and flavonoids than Kam Muang. Moreover, Kam Muang and Hawm Dawk Mali Deang had a high content of total anthocyanins [(14.73±0.52) mg C3GE/g of extract] and total proanthocyanidins [(115.13±1.47) mg CE/g of extract], respectively. Conclusions: Based on these data, bran extracts of Thai black Kam Muang and red rice Hawm Dawk Mali Deang can help lower oxidative stress and inflammation attributed partly to O2•- and NO•.

307-314

Authors: NI Mongalo, OS Soyingbe, TJ Makhafola

Objective: To evaluate the antimicrobial, antioxidant, cytotoxicity and anticancer activity of fractions from Jatropha zeyheri roots and to explore the phytochemical profile of the most biologically active fraction. Methods: Fractions from Jatropha zeyheri ethyl acetate extract were investigated for antimicrobial activity against a plethora of pathogenic microorganisms of different origins. The cytotoxicity studies of fractions were evaluated in vitro using tetrazolium-based calorimetric assay against human dermal fibroblast, colon adenocarcinoma (Caco-2), breast cancer (MCF-7) and lung cancer (A547) cell lines. The anti-oxidant activity of fractions was determined in vitro against 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis-(3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt (ABTS) and chelation of iron (Fe2+). Gas chromatography mass spectrometry analysis was performed to detect phytochemical constituents in fraction with potent biological activity. Results: Fraction 2 of Jatropha zeyheri roots exhibited the lowest minimum inhibitory concentration of 40 μg/mL against Klebsiella pneumoniae and 80 μg/mL against Candida albicans, Staphylococcus aureus and Mycoplasma hominis. The fractions revealed some varying degrees of cytotoxicity against human dermal fibroblasts yielding LC50 values ranging from 28.96 to 166.52 μg/mL. Fraction 3 exhibited the highest selectivity index value of 2.08 against Klebsiella pneumoniae. Moreover, fraction 2 selectively inhibited the growth of Caco-2 with LC50 of 8.83 μg/mL, compared to other cancerous cell lines. Fraction 2 of Jatropha zeyheri further exhibited IC50 of 19.66, 22.63 and 1.82 μg/mL against DPPH, ABTS and Fe2+, respectively. Gas chromatography mass spectrometry analysis revealed the presence of cyclotetracosane (10.08%), 9-hexacosene (9.40%), hexadecanoic acid (3.90%), (Z)-9-octadecenamide (3.63%), octacosane (2.27%), 11-n-decylheneicosane (2.23%), ethyl vallesiachotamate (2.17%), heneicosanoic acid (2.10%), and octadecanoic acid (2.08%) in fraction 2 of Jatropha zeyheri. Conclusions: These identified compounds, particularly cyclotetracosane (hydrocarbon), may well explain the biological activity of fraction 2 of Jatropha zeyheri, which possesses higher biological activity than other fractions. These compounds can be further investigated for use in treating various bacterial and fungal opportunistic infections associated with HIV-AIDS and related cancers.

232-239

Authors: Marjan Zare, Abbas Rezaianzadeh, Hamidreza Tabatabaee, Hossain Faramarzi, Mohsen Aliakbarpour, Mostafa Ebrahimi

Objective: To establish an early warning system for cutaneous leishmaniasis in Fars province, Iran in 2016. Methods: Time-series data were recorded from 29 201 cutaneous leishmaniasis cases in 25 cities of Fars province from 2010 to 2015 and were used to fit and predict the cases using time-series models. Different models were compared via Akaike information criterion/ Bayesian information criterion statistics, residual analysis, autocorrelation function, and partial autocorrelation function sample/model. To decide on an outbreak, four endemic scores were evaluated including mean, median, mean + 2 standard deviations, and median + interquartile range of the past five years. Patients whose symptoms of cutaneous leishmaniasis began from 1 January 2010 to 31 December 2015 were included, and there were no exclusion criteria. Results: Regarding four statistically significant endemic values, four different cutaneous leishmaniasis space-time outbreaks were detected in 2016. The accuracy of all four endemic values was statistically significant (P<0.05). Conclusions: This study presents a protocol to set early warning systems regarding time and space features of cutaneous leishmaniasis in four steps: (i) to define endemic values based on which we could verify if there is an outbreak, (ii) to set different time-series models to forecast cutaneous leishmaniasis in future, (iii) to compare the forecasts with endemic values and decide on space-time outbreaks, and (iv) to set an alarm to health managers.

249-256

Authors: Wan Suriyani Wan-Ibrahim, Norzila Ismail, Siti Farhanah Mohd-Salleh, Aidy Irman Yajid, Michael Pak-Kai Wong, Mohd Nizam Md Hashim

Objective: To determine the anti-proliferative activity of Abrus precatorius (A. precatorius) leaf extracts and their effect on cell death. Methods: A. precatorius leaves were extracted successively with hexane, ethyl acetate and methanol by Soxhlet extraction. Aqueous extract was prepared by decoction at 50 C. Extracts of A. precatorius leaves were used to treat selected cancer and normal cell lines for 72 h. Furthermore, 3-(4,5-dimethyl thiazol-2-yl) 2,5-diphenyl tetrazolium bromide assay was performed to determine cell viability. Analysis of cell cycle arrest, apoptosis assay and apoptosis protein expressions were determined by flow cytometry. Results: Methanolic extract of A. precatorius leaves showed the lowest IC50 on MDA-MB-231 cells at (26.40±5.40) μg/mL. Flow cytometry analysis revealed that cell arrest occurred at G0/ G1 phase and the apoptosis assay showed the occurrence of early apoptosis at 48 h in MDA-MB-231 cells treated with methanolic extract of A. precatorius leaves. Methanolic extract of A. precatorius leaves induced apoptosis by upregulation of Bax, p53 and caspase-3 and downregulation of Bcl-2. Conclusions: Methanolic extract of A. precatorius leaves promotes MDA-MB-231 cell death by inducing cell cycle arrest and apoptosis possibly via the mitochondrial-related pathway.

263-270

Authors: Pukar Khanal, BM Patil

Objective: To identify alpha-glucosidase inhibitors from Ficus benghalensis and analyze gene set enrichment of regulated protein molecules. Methods: The phytoconstituents of Ficus benghalensis were queried for inhibitors of alpha-glucosidase, also identified as aldose reductase inhibitors. Druglikeness score, absorption, distribution, metabolism, excretion and toxicity profile, biological spectrum, and gene expression were predicated for each compound. Docking study was performed to predict the binding affinity with alpha-glucosidase and aldose reductase and compared with clinically proven molecules. Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed for the regulated genes to identify the modulated pathways. Results: Apigenin, 3,4’,5,7-tetrahydroxy-3’-methoxyflavone, and kaempferol were identified as inhibitors of alpha-glucosidase and aldose reductase. Kaempferol was predicted to possess the highest binding affinity with both targets. The p53 signaling pathway was predicted to modulate the majority of protein molecules in diabetes mellitus. All the alpha-glucosidase inhibitors were also predicted as membrane integrity agonist and anti-mutagenic compounds. Conclusions: The current study indicates alpha-glucosidase inhibitors from Ficus benghalensis can act as aldose reductase inhibitors after absorption from the intestinal tract. Furthermore, these phytoconstituents are involved in the regulation of numerous protein molecules and pathways. Hence, the anti-diabetic efficacies of these compounds are due to their action on multiple protein molecules and synergistic effects which should be confirmed by future investigations.