Asian Pacific Journal of Tropical Biomedicine

101-110

Authors: Narisara Paradee, Duangta Kanjanapothi, Tawat Taesotikul, Sarawut Kongkarnka, Adchara Prommaban, Pimpisid Koonyosying, Somdet Srichairatanakool

Objective: To study the effect of perilla fruit oil against carbon tetrachloride (CCl4)-induced liver damage in rats. Methods: Perilla fruit oil was analyzed in terms of fatty acids, tocopherols and tocotrienols using chromatography. Sub-chronic toxicity of perilla fruit oil was investigated in rats for 90 d followed by a 28 d recovery period. Hematological, biochemical and pathological parameters were determined. To evaluate hepatoprotection, rats were divided into five groups and orally administered with Tween 80 for 10 d; Tween 80, silymarin, perilla fruit oil (0.1 mL/200 g) and perilla fruit oil (1 mL/200 g) for 10 d together with subcutaneous injection of CCl4 (2 mL/200 g) on days 9 and 10. Liver enzymes and pathological parameters were determined. Results: Perilla fruit oil contained α-linolenic acid (56.55% of total fatty acid), β-tocopherol (49.50 mg/kg) and γ-tocotrienol (43.65 mg/ kg). Rats showed significant changes in the percentage of monocytes and platelet indices following perilla fruit oil consumption for 90 d; in the percentage of neutrophils and lymphocytes, and RBC indices in the recovery period when compared with the deionized water group. Total protein and creatinine levels were increased while alkaline phosphatase and aspartate aminotransferase levels were decreased (P < 0.05). Organ weight index and pathological indicators did not change significantly. The liver of CCl4-induced rats showed remarkable centrilobular fatty changes, which was ameliorated by perilla fruit oil pretreatment. Aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were decreased (P < 0.05) in rats given perilla fruit oil. Conclusions: Perilla fruit oil is rich in α-linolenic acid, β-tocopherol and γ-tocotrienol and improves blood biomarker levels and protects against CCl4-induced hepatotoxicity. Further studies are required before supporting its use for the treatment of hepatitis.

120-135

Authors: Chim-Kei Chan, Kind-Leng Tong, Pooi-Fong Wong, Habsah Abdul Kadir

Objective: To systematically map the stepwise events leading to deoxyelephantopin-induced cell death of HCT116 human colorectal cancer cells and evaluate the effectiveness of deoxyelephantopin in vivo. Methods: HCT116 cells were treated with deoxyelephantopin at various concentrations and time points. Autophagy was confirmed by the detection of autophagosomes and autophagosomal proteins by electron microscopy and Western blotting assays, respectively, and then validated by siRNA knockdown. In addition, apoptosis was confirmed by the detection of apoptosis-related proteins. The intracellular reactive oxygen species (ROS) level was measured using flow cytometry. The growth inhibitory effect of deoxyelephantopin was further evaluated in vivo using a mouse xenograft model. Results: Deoxyelephantopin firstly elevated ROS production, which then triggered autophagic flux with the accumulation of autophagosomal proteins including LC3A/B, ATG5, and ATG7, followed by the induction of apoptosis via the intrinsic and extrinsic pathways. Pre-treatment with N-acetyl-L-cysteine, a ROS inhibitor, reversed both apoptosis and autophagy. The knockdown of LC3 prevented apoptosis induction which confirmed that deoxyelephantopin induced autophagy-dependent apoptosis in HCT116 cells. Accumulation of ROS also activated apoptosis via the mitogen-activated protein kinases signaling pathway. Furthermore, deoxyelephantopin also inhibited the PI3K/AKT/mTOR pathway, which then released the inhibition of autophagy. In vivo study further showed that deoxyelephantopin significantly suppressed the growth of HCT116 subcutaneous xenograft in nude mice. Conclusions: Our findings revealed that deoxyelephantopin elevates oxidative stress and induces ROS-dependent autophagy followed by apoptosis in HCT116 cells via the concerted modulation of multiple signaling pathways. These findings further support the development of deoxyelephantopin as a therapeutic agent for colorectal cancer.

54-64

Authors: Aarti Sharma, Pushplata Tripathi, Sarita Kumar

Objective: To formulate silver nanocomposites from Achyranthes aspera leaf extracts and evaluate its larvicidal activity against Aedes aegypti. Methods: The silver nanocomposites were synthesized from Achyranthes aspera leaf extracts. The process was optimized and traced through UV-visible and photon correlation spectroscopy. The larvicidal potential of silver nanocomposites of Achyranthes aspera leaf extracts was assessed against the early fourth instars of Aedes aegypti and three non-target organisms. Furthermore, the most effective and eco-safe nanocomposite was characterized by different biophysical techniques including scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy, transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform-infrared spectroscopy (FT-IR). Results: The formulated silver nanocomposites exhibited efficient larvicidal efficacy against Aedes aegypti. Bioassay with silver nanocomposites formulated using different AgNO 3 concentrations (3, 4, and 5 mM) revealed respective LC50 values of 37.570, 6.262 and 1.041 μg/mL; 5.819, 1.412 and 0.489 μg/mL; and 5.519, 1.302 and 0.267 μg/mL after 24, 48 and 72 h. The silver nanocomposites with 4 mM AgNO3 were selected for characterization. SEM and TEM analysis revealed spherical, poly-dispersed structure with varied diameters of 1-25 nm. The XRD analysis established the crystalline and face-centred-cubic structure of silver nanocomposites with the maximum peak at a 2θ value of 37.42°. The EDX pattern showed the presence of Ag, O and C in the nanocomposites in their order of weight%. The FT-IR displayed visibly distinct peaks in different ranges demonstrating the intricacy of silver nanocomposites. In addition, the lethal concentrations of silver nanocomposites of Achyranthes aspera leaf extracts against Aedes aegypti larvae were non-toxic to non-target organisms including Gambusia affinis, Daphnia magna and Moina macrocopa. Conclusions: Silver nanocomposites synthesized with leaf extract of Achyranthes aspera provide a cost-effective and eco-safe alternative to conventional insecticides, and can be utilized as a potent mosquito nano-larvicide.

78-86

Authors: Eun Ok Choi, Hyesook Lee, Cheol Park, Gi-Young Kim, Hee-Jae Cha, Suhkmann Kim, Heui-Soo Kim, You-Jin Jeon, Hye Jin Hwang, Yung Hyun Choi

Objective: To investigate whether ethanol extracts of Hizikia fusiforme could induce apoptosis in human prostate cancer PC3 cells. Methods: Cell viability was evaluated using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis and mitochondrial membrane potential (MMP) were measured using flow cytometry in PC3 cells. DNA damage was assessed by nuclear staining and DNA fragmentation assay. Expressions of apoptosis-associated proteins were determined by Western blotting assays. Activities of caspase-3, -8, and -9 were determined by colorimetric assay. Moreover, intracellular reactive oxygen species (ROS) generation was detected using a flow cytometer and fluorescence microscope. Results: Treatment of PC3 cells with ethanol extracts of Hizikia fusiforme inhibited proliferation, which was associated with induction of apoptosis, and accompanied by increased expression of Fas, Fas-ligand (FasL), Bax and tBid, and decreased expression of Bcl-2. In addition, ethanol extracts of Hizikia fusiforme reduced c-Flip expression and activated caspase-8, -9 and -3, resulting in an increase in poly (ADP-ribose) polymerase (PARP)cleavage. However, in the presence of a pan-caspase inhibitor, ethanol extracts of Hizikia fusiforme-mediated growth inhibition and apoptosis were significantly attenuated. Ethanol extracts of Hizikia fusiforme also destroyed the integrity of mitochondria due to the loss of MMP, leading to cytosolic release of cytochrome c. Moreover, the levels of ROS were markedly increased by treatment with ethanol extracts of Hizikia fusiforme, which was significantly suppressed by the ROS scavenger N-acetyl-L-cysteine. Further investigation of whether ethanol extracts of Hizikia fusiforme-induced apoptosis was related to the generation of ROS was conducted and the results showed that N-acetyl-L-cysteine fully blocked ethanol extracts of Hizikia fusiforme-induced apoptotic events including loss of MMP, activation of caspase-3, the cytosolic release of cytochrome c and cytotoxicity. Conclusions: Ethanol extracts of Hizikia fusiforme have chemopreventive potential via induction of ROS-dependent apoptosis. Therefore, ethanol extracts of Hizikia fusiforme may be useful for developing effective and selective natural sources to inhibit cancer cell proliferation.

532-539

Authors: Mosayeb Rostamian, Hamid M Niknam

Objective: To study the role of antibodies in protection against Leishmania tropica (L. tropica) infection in the experimental model of BALB/c mice. Methods: BALB/c mice were vaccinated against L. tropica by soluble Leishmania antigen or recombinant L. tropica stress-inducible protein-1 (LtSTI1) of L. tropica, and against Leishmania major (L. major) by soluble Leishmania antigen. Monophosphoryl lipid A was used as an adjuvant. The L. tropica- or L. major-vaccinated mice were challenged by L. tropica or L. major, respectively. The levels of anti-Leishmania antibodies (IgG1 and IgG2a) were determined after vaccination and after challenge. Results: All vaccinated groups caused a higher antibody response in comparison with the control group. The L. major-vaccinated group showed lower IgG1 response than the control group after the challenge. Conversely, in L. tropica-vaccinated mice, the levels of antibodies were higher than the control group. Moreover, the group receiving rLtSTI1 and monophosphoryl lipid A showed higher levels of antibodies than those of the rLtSTI1 group. In vaccinated mice, antibody responses against L. tropica remained high until 16 weeks after the challenge. Conclusions: The higher levels of post-challenge antibodies are associated with protective vaccination against L. tropica infection of BALB/c mice. Our findings provide new insight into the association of antibody with vaccine-induced protective immunity against L. tropica infection. More studies are needed to clarify the role of antibody in protection against L. tropica.

547-554

Authors: A Vysakh, Sebastian Jose Midhun, Ninan Jisha, Kuriakose Jayesh, V Vijeesh, Mathew Jyothis, MS Latha

Objective: To evaluate the anti-bacteria! and anti-biofilm activity of ethyl acetate fraction of Rotula aquatica Lour. (EFRA) against clinically isolated uropathogenic Escherichia coli. Methods: In vitro antibacterial and anti-biofilm studies were employed. The antimicrobial activity of EFRA was assayed by the well diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the active fraction were determined by Resazurin method. The time-kill kinetic assay, acridine orange-ethidium bromide staining, propidium iodide uptake assay, and scanning electron microscopic (SEM) analysis were done to evaluate the efficacy of EFRA in killing uropathogenic Escherichia coli. The anti-biofilm activity was determined by 3-[4,5- dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium-bromide (MTT) assay and specific biofilm formation assay. Results: The well diffusion assay of EFRA showed a very clear zone of inhibition against Escherichia coli BRL-17. The MIC and MBC of EFRA were 2.5 mg/mL and 5 mg/mL, respectively. The time-kill kinetic assay, fluorescence microscopic analysis, propidium iodide uptake assay, and SEM analysis displayed the effect of EFRA in killing the bacteria. The MTT assay and specific biofilm formation assay showed that EFRA prevented the formation of biofilms. Conclusions: The results of the present study confirm that EFRA could prevent bacterial growth and inhibit its biofilm formation.

563-568

Authors: Aatika Sadia, Muhammad Azam, Muhammad Asim Raza Basra

Objective: To explore potential inhibitors of viral enzymes of SARS CoV-2. Methods: The in-silico docked potential of anti-viral, antibiotic, and analgesic drugs were studied for inhibition of the nonstructural protein (NSP) 9, NSP3, and NSP15 of SARS CoV-2 using recent structural peculiarities of these enzymes, 3D optimized structures of drugs and algorithm-based ligand inhibitory potential. Results: Teicoplanin, azithromycin, and remdesivir potentially inhibited NSP9 (Dock-score 9 620, 5 472 and 6 252, respectively), NSP3 (Dock-score 9 846, 5 604 and 5 548, respectively) and NSP15 (Dock-score 10 960, 6414 and 6 002, respectively). Conclusions: Teicoplanin acts as a significant receptor antagonist and potentially inhibits the SARS CoV-2 enzymes.

490-495

Authors: Huyen Do Thi Thanh, Phuong Ngo Thi, Hien Nguyen Thi Thu, Nga Nguyen Thi, Hung Le Ngoc, Thao Do Thi, Ha Le Minh

Objective: To evaluate the anti-arthritic effects of Polygonatum kingianum rhizome extract using both in vitro and in vivo models. Methods: Lipopolysaccharide-induced RAW 264.7 macrophages were treated with an ethanol extract of Polygonatum kingianum rhizomes at different concentrations to determine nitric oxide and prostaglandin E2 (PGE2) production. For in vivo study, Polygonatum kingianum ethanol extract was further investigated for its antiinflammatory effect in a mouse model with collagen antibody-induced arthritis. Phytochemical study of Polygonatum kingianum ethanol extract was also performed. Results: Saponins (142 mg/g total yield) was the main component in the Polygonatum kingianum ethanol extract. 5α,8α-ergosterol peroxide, (E,E)-9-oxooctadeca-10,12-dienoic acid and 3-(2’-hydroxy-4’-methoxy-benzyl)-5,7-dihydroxy-8-methyl-chroman-4-one were isolated from the extract. Polygonatum kingianum ethanol extract exhibited potential anti-inflammatory effects by inhibiting nitric oxide and PGE2 production in RAW 264.7 cells in a dose-dependent manner. The level of arthritis in mice with collagen antibody-induced arthritis was significantly reduced (P<0.01) after treatment with Polygonatum kingianum ethanol extract, particularly at a dose of 1 000 mg/kg body weight. Besides, the extract demonstrated the regulatory effects on serum tumor necrosis factor- alpha, interleukin-6, and interleukin-10 in treated mice. Conclusions: Polygonatum kingianum ethanol extract has beneficial effects on inflammatory cytokine regulation and PGE2 inhibition in an experimental mouse model with collagen antibody-induced arthritis. The phytochemical screening reveals that the saponin, as the main component, and sterols (daucosterol and 5α,8α-ergosterol peroxide) from Polygonatum kingianum ethanol extract may contribute to its promising in vitro and in vivo anti-inflammatory activities.

505-515

Authors: Mehmet Kadir Erdogan, Can Ali Ağca, Hakan Aşkın

Objective: To investigate the antiproliferative, anti-angiogenic, and apoptotic effects of extracts of Achillea biebersteinii (ABE) and combined treatments of ABE with 5-fluorouracil (5-FU) on HT-29 cells. Methods: The effects of ABE, 5-FU, and combined treatments on the viability of HT-29 cells were determined by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Isobologram analysis was used to determine synergism between ABE and 5-FU. The apoptotic and anti-angiogenic effects were determined by cell death detection and human vascular endothelial growth factor ELISA method, respectively. Transcriptional and translational expressions of p53, Bax, Bcl-2, p38 MAPK, Akt, PTEN, and mTOR were also evaluated by real-time PCR and Western blotting analysis. Results: ABE decreased the viability of HT-29 cells in a dose-dependent manner. Combined treatment of hexane, chloroform, and methanol extracts of Achillea biebersteinii with 5-FU at IC50 doses decreased the cell viability to 26.0%, 19.1%, and 14.9%, respectively (P<0.001). Furthermore, ABE treatment alone and combination with 5-FU, induced apoptosis, significantly downregulated mTOR, Akt, Bcl-2 expression, upregulated p53, Bax, PTEN, p38 MAPK expression, and exhibited anti-angiogenetic effects. Conclusions: Our findings indicate that ABE shows synergism with 5-FU and inhibits the proliferation of HT-29 cells by inducing apoptosis and suppressing angiogenesis, which may provide biological evidence for further use of ABE in the treatment of colorectal cancer.

442-451

Authors: Uzma Saleem, Shingraf Zaib, Sana Khalid, Fareeha Anwar, Muhammad Furqan Akhtar, Bashir Ahmad

Objective: To evaluate acute oral toxicity and anti-arthritic activity of the methanolic extract of Convolvulus arvensis L. leaves. Methods: Safety was assessed by acute oral toxicity (OECD 425) study. Anti-arthritic activity was explored by in vitro (inhibition of protein denaturation) and in vivo (Complete Freund’s adjuvant-induced arthritis and carrageenan-induced inflammation) methods. Antioxidant potential was determined by assessing ferric reducing power, DPPH inhibition, and H2O2 scavenging assays. Furthermore, molecular docking was done to check interactions between the plant constituents and cyclooxygenases (COX-1 and COX-2). Quercetin, gallic acid, caffeic acid, syringic acid, sinapic acid, and vanillic acid were quantified by HPLC and eight compounds were identified by GC-MS analysis. Results: No mortality and abnormality in biochemical parameters were observed in the toxicity study. Histological analysis of vital organs also supported these biochemical results. The in vitro and in vivo studies showed that the methanolic extract of leaves of Convolvulus arvensis exhibited dose-dependent anti-arthritic and anti-oxidant potential. Molecular docking showed better interactions of plant compounds with cyclooxygenases as compared to standard ibuprofen. Conclusions: Convolvulus arvensis exhibits strong anti-arthritic activity, justifying the traditional use of the herbal drug.

460-469

Authors: Jung-Ha Kil, Fatih Karadeniz, Ga Hyun Yu, Hojun Kim, Junse Kim, Jung Hwan Oh, Jung Im Lee, Chang-Suk Kong, Youngwan Seo

Objective: To investigate matrix metalloproteinases (MMP)-2 and MMP-9 inhibitory effect of Salsola komarovii Iljin, an edible halophyte with health beneficial effects. Methods: Salsola komarovii crude extracts (SKI), and solvent (n-hexane, 85% aq. MeOH, n-BuOH, and H2O) fractionated extracts of SKI were prepared. Gelatin zymography was carried out to observe MMP enzymatic activity. The release of the MMP enzymes was measured by enzyme-linked immunosorbent assay. Expression of MMPs in mRNA and protein level were investigated by polymerase chain reaction analysis and immunoblotting, respectively. Results: SKI and SKI fractions inhibited active MMP-2 and MMP-9 amount in the treated cell culture medium. Also, SKI suppressed the release of MMP-2 and MMP-9 from stimulated HT1080 human fibrosarcoma cells. Furthermore, SKI suppressed the mRNA and protein expression of MMP-2 and MMP-9. SKI fractions showed parallel effects except for H2O fraction which did not yield any significant MMP inhibitory effect. Among fractions, 85% aq. MeOH was the most active fraction to inhibit both the enzymatic effect and expression of MMP-2 and MMP-9. Conclusions: SKI may contain potential MMP release inhibitory compounds. Salsola komarovii is a promising source of compounds against MMP and could be utilized in the development of antitumor agents.