Asian Pacific Journal of Tropical Biomedicine

343-352

Authors: Nguyen Bao, Jean-Pière LE CAER, Phan Thi Khanh Vinh

Objective: To determine the new M-superfamily conotoxins from molluscivorous snail Conus bandanus in Vietnam. Methods: Conus bandanus venom was fractionated and purified on HPLC system with an analytical reversed-phase C18 column in order to screen small conotoxins. The primary structure of peptide was analyzed by matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry using collision-induced dissociation and confirmed by Edman’s degradation method. Results: Five new conotoxins were biochemically characterized from the crude venom of the mollusk-hunting cone snail Conus bandanus, which were collected at Ke Ga reef of the Nha Trang Bay (Vietnam). Each conotoxin had 15 or 16 amino acid residues and shared the same characteristic cysteine framework V as –CC–C–C–CC–. They were termed as Bn3b, Bn3c, Bn3d, Bn3e and Bn3f following the conotoxins nomenclature. Conclusions: The conotoxins Bn3b, Bn3e, and Bn3f are categorized in the mini-M conotoxins of the M1 branch, while conotoxins Bn3c and Bn3d are categorized in the mini-M conotoxins of the M2 branch. The homological analysis reveals that these conotoxins could serve as promising probe compounds for voltage-gated sodium channels

361-368

Authors: Abdi Wira Septama, Ibrahim Jantan, Pharkphoom Panichayupakaranant, Mohd Fadhlizil Fasihi Mohd Aluwi, Eldiza Puji Rahmi

Objective: To evaluate the immunosuppressive effect on human phagocytes and antibacterial activity of dihydromorin and norartocarpetin isolated from Artocarpus heterophyllus heartwoods. Methods: Dihydromorin and norartocarpetin were isolated from Artocarpus heterophyllus heartwoods. A modified Boyden chamber was used to determine the chemotactic activity of human phagocyte. The respiratory burst was evaluated by chemiluminescence assay. Myeloperoxidase (MPO) activity was quantified using a colorimetric assay. The broth microdilution method was performed to assess their antibacterial activity. Results: Dihydromorin exhibited potent inhibitory effect on the chemotactic activity of polymorphonuclear neutrophils (PMNs) with an IC50 value of 5.03 μg/mL. Dihydromorin also inhibited reactive oxygen species production of whole blood cells, PMNs, and monocytes with IC50 values of 7.88, 7.59 and 7.24 μg/mL, respectively. Interestingly, dihydromorin also strongly inhibited the MPO activity of PMNs with an IC50 value of 5.24 μg/mL, which was lower than indomethacin (24.6 μg/mL). Molecular docking of dihydromorin and crystal structure of MPO showed that dihydromorin had close interaction with key amino acid residues such as Arg239 and Gln91. Antibacterial activity assay showed that only dihydromorin had a strong effect against Streptococcus pyogenes with MIC and MBC values of 15.62 and 31.25 μg/mL, respectively. Conclusions: The results suggest that dihydromorin could be developed as an anti-inflammatory and antibacterial agent.

379-386

Authors: Dornadula Sireesh, Natarajan Suganya, Suvro Chatterjee, Kunka Mohanram Ramkumar

Objective: To investigate the effects of Gymnema montanum leaf extract against endoplasmic reticulum (ER) stress-induced toxicity in endothelial cells. Methods: The immortalized endothelial hybrid cell, EA.hy926 was treated with different concentrations of Gymnema montanum leaf extract (0-100 μg/mL) and the ER stress inducer, tunicamycin. The cytotoxicity was assessed by MTT as well as lactate dehydrogenase and malondialdehyde levels were determined. The levels of ER stress markers, GRP78 and CHOP were analysed by Western blot assay. The Gymnema montanum leaf extract-mediated activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was assessed by cell-based luciferase enzyme fragment complementation assay and antioxidant responsive element driven luciferase reporter assay. The levels of phosphoproteins of the MAPK pathway were analyzed using the Bioplex system. Results: A dose-dependent cytoprotective effect of Gymnema montanum leaf extract was observed in tunicamycin-induced toxicity. Gymnema montanum leaf extract significantly reduced lactate dehydrogenase activity and malondialdehyde levels in ER stress-induced endothelial cells. It also suppressed ER stress markers dose dependently and inhibited the phosphorylation of JNK, ERK, MEK and p38 MAPK in tunicamycin-induced endothelial cells. Moreover, Gymnema montanum leaf extract increased the expression of Nrf2 and its downstream targets in endothelial cells. Conclusions: Gymnema montanum leaf extract attenuates ER stress by increasing the expression of Nrf2 and its downstream genes.

308-315

Authors: Xiao-Yan Zhu, Chun-Ling Zhang, Yukiat Lin, Min-Yan Dang

Objective: To assess the anti-inflammatory efficacy of ferruginol on dextran sulfate sodium (DSS) stimulated ulcerative colitis mice. Methods: Ulcerative colitis was induced in C57BL/6J mice by administering 2% of DSS through drinking water for 7 d. The mice in the treatment group were treated with DAA+50 mg/kg/day ferruginol orally. In the positive control group, sulfasalazine (50 mg/kg/day) was used alongside with DSS. After induction, the bodyweight, character of stool and feces occult blood were recorded daily, the disease activity index was calculated, and the colon length, colon weight, and spleen weight were recorded. The myeloperoxidase activity was assayed by spectrophotometry. Interleukin (IL)-6, IL-1β, and tumor necrosis factor-a were determined by ELISA method, and nuclear factor-κB, cyclooxygenase-2, matrix metalloproteinases-9, and inducible nitric oxide synthase by Western blotting assays. Results: Ferruginol significantly increased the bodyweight, colon weight, colon length, and decreased disease activity index and spleen weight. It exhibited anti-inflammatory activity against DSS induced ulcerative colitis in mice by reducing the activities of myeloperoxidase, tumor necrosis factor-α, nuclear factor-κβ, IL-1β, cyclooxygenase-2, matrix metalloproteinases-9, IL-6, and inducible nitric oxide synthase. Conclusions: Ferruginol could be used to treat ulcerative colitis by attenuating the inflammation in colon cells and maintaining colonic mucosal barrier integrity.

325-332

Authors: Phuong T.M. Nguyen, Minh T.H. Nguyen, Lien T Quach, Phuong T.M. Nguyen, Lam L Nguyen, Dong V Quyen

Objective: To investigate the antibiofilm activity of alpha- mangostin (AMG) loaded nanoparticle (nanoAMG) against dental caries pathogen Streptococcus mutans. Methods: AMG was isolated from the peels of Garcinia mangostana L. using silica gel columns and chemically analysed by high performance liquid chromatography and nuclear magnetic resonance. NanoAMG was prepared using the solvent evaporation method combined with high-speed homogenization. The nanoparticles were characterized using dynamic light scattering, field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared spectroscopy (FTIR). The toxicity of nanoAMG in fibroblast NIH/3T3 cell line was determined using MTT method. The antibiofilm effect of nanoAMG was determined through the evaluation of biofilm formation by Streptococcus mutans using a 96-well plate. Biofilm biomass was quantified using crystal violet. Cell viability was observed under confocal microscopy using LIVE/DEAD BacLight staining. Moreover, gene expression was determined by quantitative real-time PCR and membrane permeabilization activity by measuring the uptake of o-nitrophenol- β-D-galactoside. Results: NanoAMG size was in a range of 10-50 nm with a polydispersity index of < 0.3 and zeta potential value of -35.2 mV The size and the incorporation of AMG in the nanoparticles were confirmed by FE-SEM and FTIR analyses. The IC50 values of the test agents on NIH/3T3 cells were (9.80 ± 0.63) μg/mL for AMG and (8.70 ± 0.81) μg/mL for nanoAMG, while no toxicity was generated from excipients used to prepare nanoparticles. In the early stage of biofilm formation, treatment with 6.25 μmol/L nanoAMG caused a reduction in biofilm biomass up to 49.1%, compared to 33.4% for AMG. In contrast, biofilms at the late stage were more resistant to the test agents. At 96 μmol/L (= 10 × MIC), nanoAMG reduced only 20.7% of biofilm biomass while AMG did not show any effect. Expressions of gtfB and gtfC genes involved in biofilm formation were down-regulated 3.3 and 12.5 folds, respectively, compared to AMG (2.4 and 7.6 folds, respectively). LIVE/DEAD BacLight fluorescence staining and microscopy observation indicated that biofilm cells were killed by both nanoAMG and AMG at 48 μmol/L (= 5 × MIC). In addition, membrane permeabilization activity was increased in a time dependent manner and higher in nanoAMG treated cells compared to AMG. Conclusions: AMG coated nanoparticle can enhance AMG bioactivity and can be used as a new and promising antibiofilm agent.

248-253

Authors: Fatemeh Nemati Zargaran, Mosayeb Rostamian, Alisha Akya, Hamid M Niknam

Objective: To compare limiting dilution assay and real-time PCR methods in Leishmania tropica parasite load measurement in vaccinated mice. Methods: BALB/c mice were vaccinated by Leishmania tropica soluble Leishmania antigen or recombinant Leishmania tropica stress- inducibleprotein-1 with/without adjuvant. After three vaccinations, mice were challenged by Leishmania tropica promastigotes. Two months after challenge, the draining lymph nodes of mice footpad were removed and parasite load was assayed by limiting dilution assay and real-time PCR methods. Limiting dilution assay was done by diluting tissue samples to extinction in a biphasic medium. For real-time PCR, DNA of the lymph nodes was extracted, equal dilutions of each sample were prepared and hot-start real-time PCR was done using appropriate primers. The data of the two methods were compared by appropriate statistical methods. Results: Both methods were able to measure different levels of parasite load in vaccinated/unvaccinated mice. In addition, wherever parasite load of a group was estimated high (or low) by one method, the estimated parasite load by another method was the same, although statistically significant differences were found between some groups. Conclusions: Both methods lead to approximately similar results in terms of differentiating parasite load of the experimental groups. However, due to the lower errors and faster process, the real-time PCR method is preferred.

263-272

Authors: Nancy B Mohamed, Amira H Mohamed, Nashwa A Abu-Aita, Soad M Nasr, Somia A Nassar, Kawkab A Ahmed

Objective: To evaluate the effect of Moringa oleifera leaf ethanol extract as an adjunct treatment on lead acetate induced hepato- nephrotoxicity in rabbits. Methods: Thirty-six male New Zealand White rabbits were assigned into two main groups. The first group (14 rabbits) served as normal control. The secondgroup (22 rabbits) was administered orally with lead acetate at a dose of 40 mg/kg/day, 5 days/week for 8 weeks. At the 4th and the 8th week of treatment, 6 animals (3 animals at each period) of the second group were sacrificed while the remaining animals (16 rabbits) were assigned randomly into 2 subgroups (8 rabbits each): treated and non-treated. The first subgroup was orally given 1 mL phosphate-buffered saline for further 4 weeks while the second subgroup was administered orally with Moringa oleifera leaf ethanol extract at a dose of 400 mg/kg/day for the same period. Blood samples were collected to determine hematological and serum biochemical indices. Tissue specimens were collected from the liver and kidney for evaluation of the oxidant/antioxidant markers and for histopathological examinations. Results: Lead acetate exposure decreased the mean body weight gain, hematocrit, hemoglobin, mean corpuscular volume, and lymphocytes count. Moreover, it markedly increased counts of monocytes and platelets, serum enzyme activity, levels of creatinine, total cholesterol, triglycerides, and low-density lipoprotein cholesterol. Malondialdehyde level was markedly increased while the reduced glutathione content was significantly decreased in liver tissue of lead intoxicated-rabbits. Histopathological alterations were also noticed in the liver and kidney of lead intoxicated rabbits. Moringa oleifera leaf ethanol extract significantly improved hematological and serum biochemical parameters and histopathological structure of the liver and kidney. Conclusions: Moringa oleifera leaf ethanol extract ameliorates hemato-biochemical and histopathological alterations caused by lead acetate and improveshepatic and renal functions.

281-292

Authors: Perwez Alam, Nasir A Siddiqui, Ali S Alqahtani, Anzarul Haque, Omer A Basudan, Saleh I Alqasoumi, Abdullah A AL-Mishari, MU Khan

Objective: To optimize the ultrasonication method for efficient extraction of P-sitosterol and lupeol from the roots of Astragalus atropilosus using Box-Behnken design of response surface methodology (RSM), and its validation by high performance thin layer chromatography (HPTLC) method. Methods: Ultrasonication method was used to extract β-sitosterol and lupeol from Astragalus atropilosus (roots). RSM was used to optimize the different extraction parameters viz. liquid to solid ratio (10-14 mL/g), temperature (60-80 °C) and time (40-60 min) to maximize the yield of β-sitosterol and lupeol. The quantitative estimation of β-sitosterol and lupeol was done in chloroform extract of Astragalus atropilosus by validated HPTLC method on 10 cm × 20 cm glass-backed silica gel 60F254 plate using hexane and ethyl acetate (8:2, v/v) as mobile phase. Results: A quadratic polynomial model was found to be most appropriate with regard to R1 (yield of total extraction; R2/% CV = 0.994 8/0.28), R2 (β-sitosterol yield; R2/% CV = 0.992 3/0.39) and R3 (lupeol yield; R2/% CV = 0.994 2/0.97). The values of adjusted R2/predicted R2/signal to noise ratio for R1, R2, and R 3 were 0.978 2/0.955 1/48.77, 0.990 4/0.911 0/31.33, and 0.992 7/0.940 1/36.08, respectively, indicating a high degree of correlation and adequate signal. The linear correlation plot between the predicted and experimental values for R1, R2, and R3 showed high values of R2 ranging from 0.990 5-0.997 3. β-sitosterol and lupeol in chloroform extract of Astragalus atropilosus were detected at Rf values of 0.22 and 0.34, respectively, at X max = 518 nm. The optimized ultrasonic extraction produced 8.462% w/w of Rl, 0.451% w/w of R2 and 0.172% w/w of R3 at 13.5 mL/g liquid to solid ratio, 78 C of temperature and 60 min of time. Conclusions: The experimental findings of RSM optimized extraction and HPTLC analysis can be further applied for the efficient extraction of β-sitosterol and lupeol in other species of Astragalus.

201-207

Authors: Saideh Yousefi, Ali Reza Zahraei-Ramazani, Yavar Rassi, Mohammad Reza Aflatoonian, Mohammad Reza Yaghoobi-Ershadi, Abbas Aghaei-Afshar, Amir Ahmad Akhavan, Masoumeh Amin, Azim Paksa

Objective: To determine the diversity of sand flies in different biotopes of mountainous and plain areas of Bam County as the most infected focus of anthroponotic cutaneous leishmaniasis in southeast Iran, and synanthropic index of Phlebotomus sergenti Parrot, and Phlebotomus papatasi Scopoli as the main vectors of cutaneous leishmaniasis in Iran. Methods: Sand flies were captured once a month using sticky traps in domestic, peri-domestic, agricultural, and sylvatic biotopes in the plain and mountainous areas. Alpha diversity indices, including richness, evenness, Shannon-Wiener; beta diversity indices (Jaccard’s and Sorensen’s similarity indices) and synanthropic index were calculated. Results: A total of 2 664 specimens of 9 sand fly species were collected from mountainous (47%) and plain (53%) areas. Species richness, species evenness, and Shannon-Wiener indices were obtained as 9, 0.637, and 1.399, respectively in the mountainous area. Phlebotomus sergenti and Phlebotomus papatasi were constant species with the synanthropic index of -18.463 and -29.412, respectively. In addition, species richness, species evenness, and Shannon-Wiener indices were 4, 0.690, and 0.956, respectively in the plain area. Phlebotomus sergenti and Phlebotomus papatasi were dominant species with the synanthropic index of +9.695 and +36.207, respectively. Similarity indices were low among different biotopes of plain and mountainous areas. Conclusions: A basic knowledge about the diversity of sand flies in various biotopes is essential to design sound control programs. Biodiversity and synanthropic indices of sand flies are different in plain and mountainous areas due to the difference in biotic and abiotic factors between the two areas.

216-223

Authors: Gorkem Kismali, Ahmet Ceylan, Ogunc Meral, Merve Alpay, Funda Kosova, Dilek Ulker Cakir, Begum Yurdakok-Dikmen, Neslihan Tascene, Tevhide Sel

Objective: To investigate whether piperlongumine can sensitize prostate cancer cells to tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) and trigger apoptosis in prostate cells. Methods: Human prostate cancer cell lines PC3, LNCaP, and VCaP were cultured with piperlongumine and TRAIL. Then, cell proliferation, migration, caspase activation, apoptotic protein expressions, and death receptor expressions were measured. Results: Piperlongumine inhibited cell proliferation at low doses (<10 μM) alone and in combination with TRAIL (25 ng/mL), induced apoptosis, and suppressed cyclooxygenase activation. Additionally, piperlongumine induced expression of death receptors which potentiated TRAIL-induced apoptosis in cancer cells but did not affect decoy receptors. Piperlongumine also downregulated tumor cell-survival pathways, inhibited colony formation and migration of cancer cells alone or in combination with TRAIL. The combination of piperlongumine with TRAIL was found to be synergistic. Conclusions: Our findings indicate that piperlongumine can sensitize cancer cells to TRAIL through the upregulation of death receptors and can trigger apoptosis with the downregulation of anti- apoptotic proteins.

232-238

Authors: Shaymaa Fadhel Abbas Albaayit, Abdullah Rasedee, Noorlidah Abdullah, Yusuf Abba

Objective: To investigate the anti-inflammatory properties of methanolic extract of Clausena excavata in lipopolysaccharide (LPS)-activated macrophages (J774A.1) and the effect on skin wound in a rat model through determining cytokine levels and gene expressions. Methods: The effects of methanolic extract of Clausena excavata on in vitro viability and TNF-α, IL-6, IL-10, and nitric oxide release by LPS-activated J774A.1 cells were determined. In addition, relative expressions of BAX, BCL-2 and COX-2 genes were examined in healed wounds of rats. Results: The methanolic extract of Clausena excavata was not toxic to J774A.1 cells at the highest dose of 400 μg/mL. It decreased levels of TNF-α and IL-6, while increasing IL-10 level in LPS- activated J774A.1 cells and in the healed wounds of rats. The methanolic extract of Clausena excavata also inhibited nitric oxide production in LPS-activated J774A.1 cells. The BAX and COX-2 genes were downregulated while the BCL-2 gene was upregulated in the healed wound of rats. Conclusions: The methanolic extract of Clausena excavata promotes wound healing via its anti-inflammatory and anti-apoptotic activities.

156-163

Authors: Ahmet Altan, Hatice Balci Yuce, Őzkan Karataş, Mehmet Murat Taşkan, Fikret Gevrek, Sefa Çolak, Nihat Akbulut

Objective: To evaluate the effect of free and liposome form of gallic acid on bone regeneration in critical defects in Wistar rats. Methods: Thirty-two female Wistar rats were divided into four study groups: group 1, negative control; group 2, positive control; group 3, gallic acid powder; group 4, gallic acid liposome. A critical-sized defect was created in all rats. Groups 2 to 4 had xenograft, autograft and membrane placement while negative control rats did not receive any treatment. The defect area was sutured and rats were kept alive for 30 d. At the end of the study, a bone specimen including the defect area was removed from calvaría. All specimens were evaluated under the stereomicroscope, then underwent histological analysis. Inflammatory cell counts, osteoblast, osteoclast counts, receptor activator of nuclear factor κ-B (RANKL), osteoprotegerin (OPG), runt-related transcription factor 2 (Runx2), bone morphogenetic protein-2 (BMP-2), and alkaline phosphatase were determined. Results: The biggest unhealed defect area was observed in the negative control group and the smallest was observed in the gallic acid liposome group. There were no differences between the positive control group vs. the gallic acid powder group and the gallic acid powder group vs. the gallic acid liposome group. The severity of inflammation was the highest in the negative control group and the lowest in the gallic acid liposome group with significant differences between the groups. All groups had similar osteoblast counts while osteoclast counts were the highest in the positive control group. Gallic acid groups had a lower number of osteoclasts compared with the positive control group. Runx2 and alkaline phosphatase levels were similar in the groups while OPG and BMP-2 levels exhibited a significant increase compared with the negative control group and the positive control group. RANKL was similar in the negative control group, the positive control group, and the gallic acid powder groups but decreased in the gallic acid liposome group. Conclusions: Gallic acid powder and liposome significantly improve bone regeneration in Wistar rats with calvarial defects. The improvement in healing is evident with decreased inflammation and RANKL expressions and increased OPG and BMP-2 expressions.

172-182

Authors: Verica Aleksic Sabo, Emilija Svircev, Neda Mimica-Dukic, Dejan Orcic, Jelena Narancic, Petar Knezevic

Objective: To examine the effect of Rumex crispus (R. crispus) and Rumex sanguineus (R. sanguineus) plant extracts against isolates of Acinetobacter baumannii (A. baumannii) from wounds, including multidrug-resistant strains. Methods: Six prepared Rumex extracts were subjected to liquid chromatography-tandem mass spectrometry. Antimicrobial activity of extracts and pure compounds (catechin, quercetin, isoquercitrin, emodin, and gallic acid) was examined by a microtiter plate method, while for determination of compound binary combinations activity a checkerboard method was applied. Active fractions of extracts were detected by agar-overlay high-performance thin- layer chromatography-bioautography assay followed by liquid chromatography - diode array detection - mass spectrometry analysis. Results: A total of 28 compounds were detected in two extracts of R. crispus and 26 compounds in four different R. sanguineus extracts, with catechin as a dominant component. Anti-A. baumannii activity was confirmed for all six R. sanguineus and R. crispus extracts at the concentration range from 1 to 4 mg/mL. Neither examined single compounds nor their binary combinations exhibited an anti-A. baumannii activity (MIC>256 μg/mL). The bioautography showed that fractions with the most prominent anti-A. baumannii activity tended to contain more polar compounds, predominantly flavonol (quercetin and kaempherol) glycosides; but also fractions containing flavanone (eriodictyol) glycosides and anthraquinone (emodin) glycosides; and less polar eriodictyol aglycone. Conclusions: The results justify and elucidate the traditional application of R. sanguineus and R. crispus extracts for wound healing, indicating the necessity for their further examination in combat against multidrug-resistant A. baumannii isolates from wounds.

189-192

Authors: Shui-Mao Zhou, Xian-Ling Jin, Hao Wang, Hua-Tang Luo, Xi-Shuai Jia

Objective: To investigate the prevalence of Calodium hepaticum (C. hepaticum) in rodents and insectivores from Wuhan section of the Yangtze River in China, and to provide evidence for the prevention and treatment of hepatic Calodium infection. Methods: Rodents and insectivores were captured from three selected Yangtze River beaches using mousetraps. The three survey sites were divided into six areas according to natural conditions, with 60 mousetraps placed in each area. The liver lesions in the captured rodents were observed by the naked eye and the eggs in the liver tissue were observed by microscopic examination. Results: A total of 1 080 mousetraps were placed, and 1 075 mousetraps were retrieved, with the retrieve rate as 99.5%. A total of 101 Apodemus agrarius, 12 Rattus norvegicus, and 9 Crocidura attenuata were caught. The average density of rodents and insectivores was 10.5% and 0.8%, respectively. DNA of egg nodules from infected rodents showed 98% similarity with that of C. hepaticum 18S rRNA (LC425008.1). One Rattus norvegicus was infected with C. hepaticum, with an infection rate of 3.23% in the Erqi river beach; the other two beaches did not show the incidence of C. hepaticum. Conclusions: The monitoring of C. hepaticum in the Yangtze River beaches should be strengthened to reduce the risk of human C. hepaticum infection.