Asian Pacific Journal of Tropical Biomedicine

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.

188-195

Authors: Ramakrishnan Sabitha, Kumari Nishi, Vinoth Prasanna Gunasekaran, Govindhan Annamalai, Balupillai Agilan, Mathan Ganeshan

Objective: To examine the effects of p-coumaric acid on ethanol-induced kidney injury in Swiss Wistar rats. Methods: Ethanol (25% v/v) was used to induce nephrotoxicity in rats. p-Coumaric acid was orally administered at 50, 100, or 200 mg/kg body weight. The levels of oxidative parameters were determined; pro-inflammatory biomarkers were analyzed by Western blotting and apoptotic protein was analyzed by immunohistochemistry. Results: Ethanol treated rats showed decreased levels of antioxidants and aberrant production of pro-inflammatory cytokines (IL-6, IL1β, TNF-α), NF-κB activation and imbalance of proand anti-apoptotic proteins (Bcl-2, Bax, caspase 3). Meanwhile, p-coumaric acid restored antioxidant levels and decreased the levels of inflammatory cytokines, NF-κB, and proapoptotic proteins and increased Bcl-2 expression. Conclusions: p-Coumaric acid ameliorates ethanol-induced kidney injury by restoring antioxidant production and suppressing cellular apoptosis and inhibiting NF-κB expression. p-Coumaric acid should be further investigated as a promising candidate for ethanol-induced kidney toxicity.

204-208

Authors: Ratree Tavichakorntrakool, Aroonlug Lulitanond, Arunnee Sangka, Seksit Sungkeeree, Natthida Weerapreeyakul

Objective: To evaluate antibacterial activity and the bioactive compounds of 50% hydroethanolic extract of Alpinia zerumbet (A. zerumbet) rhizomes. Methods: Eight reference microbial strains including two Gram-positive bacteria [Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212)] and six Gram-negative bacteria [Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATTC 700603), Proteus mirabilis (DMST 8212), Salmonella enterica subsp. enterica serovar Vellore. (ATCC 15611), Shigella flexneri (ATCC 12022) and Pseudomonas aeruginosa (ATCC 27853)], were used to test antimicrobial susceptibility by the broth microdilution method. Bioactive compounds were analyzed by using HPLC. Results: The minimum inhibitory concentration values of A. zerumbet extract were 8 mg/mL for Staphylococcus aureus, Escherichia coli and Shigella flexneri and 16 mg/mL for Enterococcus faecalis and the other four Gram-negative bacilli. HPLC chromatograms revealed that the A. zerumbet extract contained hydroxybenzoic acids, hydroxycinnamic acids and flavonoids. Conclusions: The constituents of A. zerumbet rhizomes could be a potential source of antibacterial compounds, warranting further study of A. zerumbet extract.

217-221

Authors: Umay Chen, Md. Saddam Hussain, Tanoy Mazumder, SM Naim Uddin, Sujan Banik

Objective: To evaluate the neuropharmacological properties of Costus speciosus (C. speciosus) rhizome using different experimental mouse models. Methods: The anxiolytic effect was investigated by hole-board test, elevated plus maze and light/dark test, while central nervous system (CNS) depressant effect was evaluated by thiopental sodium-induced sleep test. Finally, antidepressant effect was evaluated by forced swimming test and tail suspension test. Results: In both elevated plus maze and hole board test, 400 mg/kg C. speciosus showed more significant CNS depressant effect than 1 mg/kg diazepam. Both 200 mg/kg and 400 mg/kg C. speciosus extract produced a significant dose-dependent decrease in onset of sleep. In forced swimming test, C. speciosus rhizome showed a decrease in duration of immobility in a dose-dependent manner. Imipramine (10 mg/kg) and C. speciosus extract at 400 mg/kg dose exhibited a significant reduction in duration of immobility in tail suspension test which provided additional evidence of antidepressant effect of C. speciosus rhizome. Conclusions: Our study indicates that C. speciosus rhizome possesses CNS depressant, anxiolytic and antidepressant-like activities. Further studies are warranted determine the exact phytoconstituents and mechanism of action responsible for the neuropharmacological effect.

140-149

Authors: Nurul Hazirah Che Hamzah, Arifullah Mohammed, KNS Sirajudeen, Mohd Asnizam Asari, Zulhazman Hamzah, Ibrahim Khalivulla Shaik

Objective: To investigate the wound-healing effect of Alocasia longiloba (A. longiloba) petiole extract on wounds in rats. Methods: Twenty-two male Sprague-dawley rats were randomly assigned to receive 10% solcoseryl gel, phosphate buffer saline, 50% ethanol, 95% ethanol and hexane extracts of A. longiloba at 1.5%, 3% and 6% doses, respectively. A full thicknesses wound (6 mm) was created on the dorsal of the rat; and all rats were applied with the extract solutions, 10% solcoseryl gel and phosphate buffer saline once a day topically until day 12. The wound was photographed on day 1, 6 and 12, and the percentage of wound contraction was calculated. On day 12, rats were sacrificed and histological examination of granulation tissue was carried out using haematoxylin & eosin and Masson’s Trichrome stain to determine the wound healing effect. Results: In this study, 6% of 50% and 95% ethanol extracts of A. longiloba showed 82.50% and 82.32% wound contraction, respectively, and were comparable with 10% solcoseryl gel (82.30%). Meanwhile, phosphate buffer saline treated group showed the lowest wound contraction (69.86%). Histological assessment of wound treated with 6% of 95% ethanol extract of A. longiloba showed distinct epidermal and dermal layer, higher proliferation of fibroblast and more angiogenesis with collagen compared to other wound treated groups. Conclusions: A. longiloba petiole extracts have a wound healing potential and 6% of 95% ethanol extract of A. longiloba is more effective. Further studies are required to understand the wound healing mechanism of action of the extract.

158-167

Authors: Ali Abdulqader, Faisal Ali, Amin Ismail, Norhaizan Mohd Esa

Objective: To identify and determine the composition of antioxidant compounds, and to evaluate the antioxidant abilities of Gac fruit parts (peel, pulp, seed and aril) grown in Malaysia. Methods: LC-MS/MS was used for identification of antioxidant compounds and UV-Vis for estimation of the contents of phenolics, flavonoids, and carotenoids. Lycopene and β -carotene were quantified using high-performance liquid chromatography. DPPH (2, 2-diphenyl-1- picrylhydrazyl) and ferric reducing antioxidant power assays were employed to evaluate antioxidant capacities. Results: Phytochemicals were found amongst all the fruit parts. Notably, significant amounts of carotenoids [(107.4 ± 4.5), (85.7 ± 4.4), (110.6 ± 2.1) mg/100 g dry weight (DW)], and relatively high levels of both phenolics [(27.3 ± 1.7), (28.9 ± 2.4), (30.8 ± 2.7) mg/100 g DW] and flavonoids [(38.1 ± 2.2), (8.8 ± 1.3), (24.5 ± 3.3) mg/100 g DW] were found in the fruit’s peel, pulp and aril, respectively. Seed part also showed a relatively high level of flavonoids [(18.1 ± 2.3) mg/100 g DW]. Lycopene and β -carotene were found to be significantly high (P < 0.05) in aril [(579.3 ± 22.7) and (621.0 ± 35.0) μg/g DW], followed by peel [(51.0 ± 7.5) and (210.0 ± 12.5) μg/g DW] and pulp [(37.6 ± 10.9) and (205.6 ± 22.1) μg/g DW)]. Antioxidant assays revealed that aril possessed the highest scavenging activity (IC50 = 865 μg/mL), while the peel possessed the highest ferric reducing power of 140 pmol FeSO4/μg. Conclusions: The current results demonstrate that Gac fruit grown in Malaysia is a rich source of phytochemicals, especially carotenoids, and possesses antioxidant activities. Thus, such findings suggest Gac fruit as a source of an antioxidant plant.

176-180

Authors: Enegide Chinedu, Peter A Akah, Dabum L Jacob, Ifeoma A Onah, Chimere Y Ukegbu, Chukwuma K Chukwuemeka

Objective: To evaluate the antimalarial activity of the ethylacetate and butanol fractions of Combretum nigricans (C. nigricans) leaf extract in mice. Methods: C. nigricans solvent (butanol and ethylacetate) fractions were screened for their phytochemical constituents using standard procedures illustrated by Harborne and Evans. The Peters’ 4-day suppressive test against early malaria infection, Rane’s curative test against established malaria and prophylactic test for residual activity were employed for evaluating the antimalarial potential in mice. Results: The phytochemical screening revealed the presence of alkaloids, terpenoids, saponins, and flavonoids in both fractions at different intensity. Both fractions exhibited significant antimalarial activity in all test models (P<0.05). The ethylacetate fraction of C. nigricans had better chemosuppressive and curative effects compared to the butanol fraction, which however, elicited a better chemoprophylactic effect. The chemosuppressive effect of C. nigricans ethylacetate fraction (200-800 mg/kg) was 77.6%, 69.1% and 86.1%; curative effect was 62.3%, 71.3% and 72.4%; while the chemoprophylactic activity was 32.1%, 48.6% and 61.2% respectively. C. nigricans butanol fraction (200-800 mg/kg) had 40.3%, 54.1% and 69.1% chemosuppression; 26.2%, 36.9% and 34.5% curative effect; and 48.4%, 70.0% and 87.4% chemoprophylaxis. Conclusions: Both solvent fractions of C. nigricans possess antimalarial activity, and may be useful at different stages of malaria therapy.