Asian Pacific Journal of Tropical Biomedicine

431-435

Authors: Chanisala Sereewong, Monticha Chaiyasaeng, Naiyana Senasri, Jukkrid Chaiyos, Smarn Tesana

Objective: To investigate the maturity development of miracidia in uterine eggs from four portions of the Opisthorchis viverrini uterus and environmental factors possibly affected in maturation and infectivity of distal part uterine eggs. Methods: Uteri of adult worms were divided into 4 equal parts. Development of eggs was determined under light microscope. Only embryonated eggs were used to evaluate the effects of physicochemical factors: temperature, salinity, acidity, ultraviolet A, B, C. Infection success was evaluated by feeding treated eggs to intermediate host snails and determining by using a PCR approach. Results: Eggs obtained from the uterus closest to the ovary (regions 1 and 2) failed to develop in vitro. Eggs from region 4 of the uterus (close to the genital pore) were used to study effects of physicochemical factors. The highest survival and infection success was in groups of eggs kept at 30 °C (95.20%). The calculated period of loss infection success (LI50 and LI95) on miracidia in distal uterine eggs by exposure to UV-A, UV-B and UV-C were 73 and 1 523 d; 8 and 20 d; 1 and 2 d, respectively. Lethal concentrations (LC50 and LC95) of salinity, HCl and NaOH on miracidia in distal uterine eggs were 45.43 and 120.09 ppt, 0.01 and 0.25 M; 0.01 and 0.11 M, respectively, after 24 h exposure. Conclusions: Opisthorchis viverrini eggs display a high tolerance to environmental conditions, especially after snail host eating for infection.

446-455

Authors: Amit Kandhare, Uday Phadke, Abhay Mane, Prasad Thakurdesai, Sunil Bhaskaran

Objective: To assess the safety and efficacy of herbal formulation rich in standardized fenugreek seed extract (IND-2) add-on therapy in type 2 diabetes mellitus (T2DM) patients who were on insulin treatment in prospective, single arm, open-label, uncontrolled, multicentre trial. Methods: T2DM patients (n=30) with aged 18-80 years who were stabilized on insulin treatment with fasting blood sugar (FBS) level between 100-140 mg/dL received IND-2 capsules (700 mg, thrice a day) for 16 weeks. The primary endpoints were an assessment of FBS at week 2, 4, 6, 8, 12 and 16. Secondary end-points include post-prandial blood sugar level, glycosylated Hb (HbA1c), reduction in the dose of insulin and number of hypoglycemic attacks, and improvement in lipid profile at various weeks. Safety and adverse events (AEs) were also assessed during the study. Results: Study was completed in twenty T2DM patients, and there was no significant reduction in FBS and post-prandial blood sugar level after addon therapy of IND-2. However, add-on therapy of IND-2 significantly reduced (P<0.01) the HbA1c values, requirements of insulin and hypoglycemic events as compared with baseline. Total cholesterol, high-density lipoproteins-cholesterol, and low-density lipoprotein-cholesterol levels were significantly increased (P<0.01) after IND-2 add-on therapy. Body weight and safety outcomes did not differ significantly in IND-2 add-on therapy group at week 16. Additionally, add-on therapy of IND-2 did not produce any serious adverse events. Conclusions: The results of present investigation suggest that add-on therapy of IND-2 with insulin in T2DM patients improves glycaemic control through a decrease in levels of HbA1c and number of insulin doses needed per day without an increase in body weight and risk of hypoglycemia. Thus, IND-2 may provide a safe and well-tolerated add-on therapy option for the management of T2DM.

463-470

Authors: Dayane Kelly Dias do Nascimento Santos, Weslley Henrique de Oliveira Melo, Anastássia Mariáh Nunes de Oliveira Lima, Iranildo José da Cruz Filho, Gláucia Manoella de Souza Lima, Túlio Diego da Silva, Maiara Celine de Moura, Márcia Silva do Nascimento, Ana Maria Souto Maior, Thiago Henrique Napoleão, Cristiane Moutinho Lagos de Melo

Objective: To evaluate the structural and chemical composition of plant and the antioxidant and antimicrobial activities promoted by hexanic, ethanolic and ethyl acetate fractions obtained from leaves of Conocarpus erectus. Methods: Organic fractions were characterized through UPLC-MS and GC-MS. Antioxidant potential was performed through DPPH and molybdenum phosphate techniques. Antibacterial and antifungal assays were performed in accordance with Clinical and Laboratory Standards Institute protocols. Results: The obtained biomass of Conocarpus erectus leaves showed the high presence of glucose (0.45 g/L), cellulose (28.69%), Na (55.126 μg/L) and K (31.163 μg/L). We identified seven compounds in the hexanic and ethyl acetate fractions, and eight compounds in ethanolic fraction. Moreover, phenolic compounds are prevalent in all organic fractions with values of (10.04 ± 0.24), (221.26 ± 1.84), (340.53 ± 0.84) mg/g GAE to hexanic, ethyl acetate and ethanolic fraction, respectively. Antioxidant results showed a high potential in ethyl acetate fraction (71.82 ± 6.87)% and (10.89 ± 0.05)% in DPPH and molybdenum phosphate techniques, respectively. The ethanolic fraction showed moderate bacteriostatic and bactericidal activity against Staphylococcus aureus and presented a high fungistatic potential for all Candida species tested. Conclusions: Organic fractions obtained from leaves of Conocarpus erectus present antimicrobial and antioxidant properties, and these findings contribute to scientific information for the effectiveness on use of this plant in the development of a phytotherapic compound.

387-393

Authors: Marzieh Rezai, Mehdi Mahmoodi, Ayat Kaeidi, Mojgan Noroozi Karimabad, Alireza Khoshdel, Mohammad Reza Hajizadeh

Objective: To investigate the effect of crocin carotenoid on BNDF and CREB gene expression in the brain ventral tegmental area (VTA) and the serum level of BDNF in morphine-treated rats compared to control. Methods: In this study, 40 male Wistar rats (200-250 g) were used in 5 experimental groups: 1) non morphine treat rats (control); 2) non morphine-treated rats with 25 mg/kg crocin carotenoid (i.p., for 21 d); 3) morphine treated rats (10 mg/kg twice a day, s.c., 21 d); 4 and 5) morphine-treated rats with 12.5 and 25 mg/kg crocin carotenoid, respectively. By the end of research, BDNF and CREB expression was determined by real-time-PCR method. ELISA analysis was also applied for assessing the serum BDNF level. Results: The data indicated that morphine treatment could cause a significant decrease in BDNF and CREB gene expression (P<0.01 and P<0.001, respectively) in brain VTA as well as serum level of BDNF (P<0.01) in comparison to control group. Treatment with 25 mg/kg crocin carotenoid caused a significant enhancement in BDNF and CREF gene expression (P<0.01 and P<0.05, respectively) and serum level of BDNF (P<0.01) in morphine-treated rats in comparison to morphine-treated group. Conclusions: Regarding to obtained results, crocin carotenoid can inhibit unfavorable effects of morphine on the neural system to some extent through enhancing BDNF and CREB gene expression in brain VTA and serum level of BDNF.

403-410

Authors: Farouk K El-Baz, Gehad A Abdel Jaleel, Dalia O Saleh, Rehab A Hussein

Objective: To investigate the possible protective and/or therapeutic potentials of Dunaliella salina (D. salina) biomass, its carotenoid and polar fractions on cardiac dysfunction associated with D-galactose (D-GAL) induced aging in rats. Methods: Aging associated cardiac dysfunction was induced in rats by injection of D-GAL (200 mg/kg; i.p) for 8 weeks. D-GAL injected rats were treated with two regimens; protective regimen where D. salina biomass (250 mg/kg), its carotenoid (250 μg/kg) and polar (250 μg/kg) fractions were given orally for two weeks concurrently with D-GAL injection as well as treatment regimen where the three treatments were given orally for 28 consecutive days after D-GAL injection. Results: D-GAL injection for 8 weeks was accompanied with dramatic electrocardiographic changes as well as profound elevation in serum levels of homocysteine, creatinine kinase isoenzyme and lactate dehydrogenase in addition to the reduction of the cardiac content of glucose trasporter 4. D-GAL also induced reduction in cardiac superoxide dismutase activity and elevation of inducible nitric oxide synthetase and interleukin-6. On the other hand, oral administration of D. salina carotenoid fraction as well as the total biomass significantly attenuated the D-GAL-induced disturbances in the above mentioned parameters where the protective regimen appeared more successful in controlling the manifestations of cardiac dysfunction. The histopathological examination further emphasized the promising results. Besides, the HPLC analysis of the carotenoid fraction of D. salina revealed the presence of 2.31% β -carotene. Conclusions: D. salina carotenoid fraction as well as the total biomass ameliorate D-GAL-induced aging associated cardiac dysfunction which is attributed to the potent antioxidant activity of β -carotene.

418-424

Authors: Sateesh Chandra Gupta, Tusha Tripathi, Shravan Kumar Paswan, Annie G Agarwal, Chandana V Rao, Om P Sidhu

Objective: To undertake metabolite profiling of various plant parts of Citrullus colocynthis, and assess antioxidant and wound healing activities of fractions for therapeutical applications. Methods: Extracts from leaves, stem, root, fruit pulp and seeds were analyzed using gas chromatography-mass spectrometry and high performance liquid chromatography. Variation in antioxidant potential was assayed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay. The extract with highest antioxidant potential was subjected on in-vivo wound healing activity using excision wound model. Results: Metabolite profiling of Citrullus colocynthis identified 70 chemically diverse metabolites from different plant parts by using a combination of GC-MS and HPLC. Concentration of colocynthin, a principal active secondary metabolite, ranged from 3.15 mg/g dry weight to 242.00 mg/g dry weight, the lowest being in leaves and highest in fruit pulp. DPPH radical scavenging activity of free radical (IC50) ranged from 196.44 μg/mL in fruit pulp to 413.33 μg/mL in leaves tissues. Significant wound contraction and increase in hydroxyproline content of granulation tissue were observed with ointment formulated from methanolic extract of fruit pulp. Conclusions: The study indicates that the methanol extract of Citrullus colocynthis fruit pulp when applied topically may promote wound contraction in rat model attributable to the accumulation of colocynthin. The high quantity of colocynthin (242.00 mg/g dry weight) and substantial concentration of 2,4-di-tert butyl phenol (3.2%), squalene (4.2%) and δ -tocopherol (2.5%) make this plant to provide new opportunities for development of medicinal, nutraceutical and dietary supplements with optimized functionality.

340-344

Authors: M Choi, JY Baek, I Park, SY Lim

Objective: To evaluate effects of docosahexaenoic acid-enriched phosphatidylcholine (DHA-PC) on cytokine production induced by lipopolysaccharide (LPS). Methods: The culture supernatants of splenocytes exposed to DHA-PC along with LPS were harvested to determine the production of Th 1 (IFN- γ and IL-2) and Th2 [IL-4, IL-5, IL-6 and IL-12/IL-23(p40)] cytokines. Cytokines were measured using ELISA. Results: Co-administration of DHA-PC with LPS resulted in significantly lower IL-2 expression compared to that observed with administration of only LPS (P<0.01). Treatment with DHA-PC and LPS significantly increased IL-5 expression (P<0.01). Moreover, co-administration of DHA-PC with LPS significantly decreased IL-6 and IL-12/IL-23(p40) expressions compared to that observed with administration of only LPS (P<0.01). Conclusions: Our results suggest that DHA-PC inhibits pro-inflammatory cytokines [IL-2, IL-6 and IL-12/IL-23(p40)] expression on induction of inflammation.

352-359

Authors: Amit D Kandhare, Anwesha A Mukherjee, Subhash L Bodhankar

Objective: To evaluate the protective effect of morin against pentylenetetrazol (PTZ)-induced tonic-clonic convulsions in mice. Methods: Swiss albino mice (18-22 g) was used to induce convulsions by intraperitoneal (i.p.) administration of PTZ (90 mg/kg). Mice were either pretreated with morin (10, 20 and 40 mg/kg) or vehicle (distilled water, 10 mg/kg) 45 min before PTZ administration. Various behavioral and biochemical parameters were assessed. Results: PTZ administration resulted in significant production (P<0.001) of tonic-clonic conclusion and mortality in mice. PTZ-induced increase in the duration of convulsion, onset of convulsion and mortality was inhibited significantly by morin (20 and 40 mg/kg) administration. The PTZ-induced decrease in brain GABA, dopamine and Na+K+ATPase levels and increase in xanthine oxidase activity were inhibited significantly by morin (20 and 40 mg/kg) treatment. The increased levels of malondialdehyde and nitric oxide level were significantly decreased by morin (20 and 40 mg/kg) treatment. Also, reduced levels of superoxide dismutase and glutathione were increased significantly by morin treatment. Conclusions: Results of the present study indicate that morin showed its anti-convulsant effect via modulating the levels of brain GABA, Na+K+ATPase, and oxido-nitrosative stress. Thus, morin can be a potential candidate for further clinical evaluations as an anti-epileptic agent.

365-370

Authors: Sunjukta Ahsan, David Summers

Objective:To identify the region conferring stability to pBSSB2 (a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods:The open reading frame (ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results:The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB(plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions:This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.

292-301

Authors: Patricia Carara dos Santos, Jean Carlos Vencioneck Dutra, Juliana Macedo Delarmelina, Lilian Valadares Tose, Wanderson Romão, Claudia Masrouah Jamal, Hildegardo Seibert França, Maria do Carmo Pimentel Batitucci

Objective: To evaluate the influence of fertilization and phenological stages on secondary metabolites production and chemoprotective effects of Coriandrum sativum (C. sativum) L. Methods: The plants were grown under organic or chemical fertilizer, collected at vegetative and flowering development stages and their hydroalcoholic extracts were analyzed by phytochemicals methods, mass spectrometry, antioxidant and antimutagenic assays. Results: All extracts exhibited metabolites such as coumarins, flavonoids and steroids, and mass spectrometry showed similar molecular peaks among the extracts evaluated, suggesting the presence of palmitic and 毩-linolenic acids. Vegetative C. sativum extract grown under chemical fertilizer showed better antioxidant activity, according to the DPPH assay. Vegetative C. sativum extracts grown under organic and chemical fertilizer were able to effectively reduce micronucleous frequency in the simultaneous and pre-treatment protocols, especially reaching 55.90% of damage reduction in the pre-treatment protocol. Conclusions: These findings suggest that chemical fertilization promotes an increase in the content of flavonoids in C. sativum and, consequently, leads to better antioxidant and antimutagenic activities, as well as reinforces the potential uses of this culinary plant in health promotion and disease prevention. Asian Pacific Journal of Tropical Biomedicine 2018; 8(6): 292-301 Asian Pacific Journal of Tropical Biomedicine Journal homepage: www.apjtb.org This is

313-319

Authors: Cheong Wei Ong, Yik Sin Chan, Kong Soo Khoo, Hean Chooi Ong, Nam Weng Sit

Objective: To evaluate antifungal and cytotoxic activities of four underutilised fruit species, i.e. Artocarpus altilis (breadfruit), Cynometra cauliflora (nam-nam), Mangifera pajang (M. pajang) (Bambangan) and Physalis minima (wild gooseberry). Methods: Extracts from the fresh flesh of Artocarpus altilis and Cynometra cauliflora, the flesh and kernel of M. pajang, and the whole fruit of Physalis minima were obtained by sequential extraction using hexane, chloroform, ethyl acetate, ethanol, methanol and distilled water. Each extract was assessed against six species of human fungal pathogens using a colourimetric broth microdilution method. The cytotoxicity was evaluated using African monkey kidney epithelial (Vero) cells. Results: All 30 extracts showed inhibitory activity against Cryptococcus neoformans. However, none of the extracts were active against Aspergillus fumigatus. The ethanol, methanol and water extracts from the kernel of M. pajang fruit showed the strongest activity against three species of Candida and Trichophyton interdigitale, with a minimum inhibitory concentration range of 0.001 – 0.630 mg/mL. The corresponding mean 50% cytotoxic concentrations for these three extracts were 358.7, 158.4 and 261.3 μg/mL, respectively against Vero cells. In contrast, the flesh of M. pajang fruit (hexane, chloroform and ethyl acetate extracts) showed statistically significant (P<0.001; ANOVA) strong toxicity against the cells, with 30.6, 13.5 and 22.2 μg/mL of mean values of 50% cytotoxic concentrations, respectively. Conclusions: The results suggest that the bioactivity of the kernel of M. pajang fruit is more selective towards fungi and thus is a potential source of new antifungal agents.

328-332

Authors: Bhagavathi Sundaram Sivamaruthi, Periyanaina Kesika, Chaiyavat Chaiyasut

Atopic dermatitis (AD) is one of the chronic inflammatory skin diseases characterized by conflicts in epidermal barrier and wired immune response. About 10%-20% of the population is affected by AD, especially infants. Topical application of corticosteroids, antihistamines, and antibiotics are used to treat severe AD condition. Recent studies suggest that disturbance in skin and intestinal microbiota is majorly linked to skin diseases. Probiotics are known for the positive alteration of an individual’s microbiome and associated with several health benefits. Clinical studies suggest that probiotic and synbiotic supplementation protect infants from a stringent AD to some extent. Reduction in the risk of AD development upon probiotic supplementation was not observed in all studied populations. Further studies are needed to regularize microbiome of skin and intestine in AD patients that may reduce AD severity. Present review summarizes the outcomes from clinical studies on AD using probiotic as an alternative treatment candidate.

245-253

Authors: Bruno Rafael Barboza, Bárbara Rafaela da Silva Barros, Bárbara de Azevedo Ramos, Maiara Celine de Moura, Thiago Henrique Napoleão, Maria Tereza dos Santos Correia, Luana Cassandra Breitenbach Barroso Coelho, Iranildo José da Cruz Filho, Ana Maria Souto Maior, Túlio Diego da Silva, Leylianne de Cássia Rodrigues Nerys, Edson Renan Barros de Santana, Cláudia Sampaio de Andrade Lima, Virgínia Maria Barros de Lorena, Cristiane Moutinho Lagos de Melo

Objective: To evaluate antimicrobial and antioxidant properties of saline extract from Tithonia diversifolia leaves by phytochemical bioprospecting, and investigate its safety against animal cells. Methods: The saline extract was prepared, with NaCl (0.15 M), by constant stirring of the dried and pulverized leaves, followed by volume reduction by lyophilization. The extract was phytochemical characterized using ultra-performance liquid chromatography, and total phenol and flavonoid analysis also was performed. The antioxidant capacity was determined through DPPH· radical, the antimicrobial property was evaluated against standard bacteria and fungi, and the viability assays were performed against mice splenocytes. Results: Fifteen compounds were identified belonging to two main classes terpenoids and phenolics. The extract showed 22.185 mg GAE/g of total phenolic compounds and 3.220 mg QE/g of flavonoid. Moreover, extract showed higher antioxidant ability similar to butylated hydroxytoluene a standard molecule [(3.042±0.019) mg AAE/g and (4.12±0.10) mg AAE/g to saline extract and butylated hydroxytoluene, respectively]. The antimicrobial assays demonstrated that the extract had a significant antifungal potential against Candida species and could be used with safety against mice splenocytes, in concentrations lower than 50 μg/mL, promoting higher proliferation in these cells. Conclusions: Saline extract from Tithonia diversifolia leaves presents potential antioxidant, antifungal properties and induces immunostimulation in mice splenocytes.

261-267

Authors: Abdullah Amira Raudhah, Hapidin Hermizi, Abdullah Hasmah

Objective: To understand the effects of combination treatment of pamidronate with isolated Quercus infectoria semi-purified fraction (QIsm-F) on human foetal osteoblast cell model (hFOB 1.19 cell line) through assessment of Runt related transcription fraction-2 (Runx2) and Osterix (Osx). Methods: The isolation and purification of QIsm-F were conducted by chromatographic technique. In order to assess relative efficacy of QIsm-F to the osteoblast model, the determination of half maximal effective concentration (EC50) was performed by MTT assay. hFOB 1.19 cells were cultured in DMEM F-12 and supplemented with 10% fetal bovine serum along with 1% penicillin-streptomycin incubated in 5% CO2 at 37 °C. Expression of Runx2 and Osx was assessed through western blotting and confirmed with immunofluorescence staining. Results: Results of western blot analysis and immunofluorescence staining demonstrated that compared to hFOB 1.19 cells treated with single individual treatment of QIsm-F and control groups, levels of Runx2 and Osx were elevated with higher fluorescence intensity and more rapid proliferation in hFOB 1.19 cells treated with combined treatment of QIsm-F and pamidronate. Conclusions: The finding demonstrates the synergistic effect between osteoporotic drug pamidronate and established QIsm-F. The combination treatment helps increase the efficiency of pamidronate acting on osteoblast cells by stimulating osteoblast proliferation and differentiation via expression of Runx2 and Osx.

273-278

Authors: Damrongpan Thongwat, Ratchanaporn Chokchaisiri, Lucksagoon Ganranoo, Nophawan Bunchu

Objective: To evaluate the larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri endocarp against Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus mosquitos. Methods: Larvicidal activity was tested according to World Health Organization standard protocol. The third-stage larvae of each mosquito species were exposed to various concentrations of Dracaena loureiri crude extract and six groups of Dracaena loureiri fractionated extracts (RC-DT 009–014). Larval mortality rates were observed after 24 h and 48 h of exposure. Then, a computerized probit analysis of the mortality data was performed to determine lethal concentration 50 (LC50) and lethal concentration 90 values. Results: Anopheles minimus larvae (24-h LC50 77.88 mg/L) had the highest susceptibility to crude extract, whereas others (Aedes aegypti, 24-h LC50 224.73 mg/L; Aedes albopictus, 24-h LC50 261.75 mg/L; and Culex quinquefasciatus, 24-h LC50 282.86 mg/L) were significantly less susceptible. The most effective groups of fractionated extracts were RC-DT 012 and RC-DT 013. The mosquito species most susceptible to fractionated extracts was Culex quinquefasciatus, with 24-h LC50 values of 0.66 and 0.94 mg/L for RC-DT 012 and RC-DT 013, respectively. Conclusions: The larvicidal activity of fractionated extracts is more effective than that of crude extract against all tested mosquito species. For the most effective alternative larvicide, purification and a phytochemical constituent analysis must be performed.