POSTER SESSIONS (5 DECEMBER 2018)

TRACK 6: CANCER II

Poster 6.1

Validation of PASD1 as an immunotherapeutic target in colorectal cancer (CRC)

Joanne E. Soh1, Nadiah Abu1, Hui-min Neoh1, Luqman Mazlan2, Azyani Yahaya3 and Rahman Jamal1*

1UKM Medical Molecular Biology Institute (UMBI), Malaysia
2Department of Surgery, Faculty of medicine, University of Malaya, Malaysia
3Department of Pathology, Faculty of Medicine, National University of Malaysia, Malaysia

Corresponding author: Prof. Rahman Jamal, rahmanj@ppukm.ukm.edu.my

Background

Colorectal cancer (CRC) is the third commonest malignancy in Asia including Malaysia. Despite the advances in screening, surgery and conventional therapies, a significant proportion of CRC patients failed to achieve long term remission or experienced toxic side effects of treatment. The identification of PAS (Per ARNT Sim) domain containing 1 (PASD1), a promising cancer testis antigen (CTA) as an immunotherapeutic target, represents one approach to improve treatment options for CRC. The aim of the present study was to investigate the expression of PASD1 and CD8 T-cells pulsed with PASD1 immune responses in CRC patients.

Methods

PASD1 mRNA expression was determined via RT-PCR on paired tumors and normal tissue samples from 25 CRC patients. Tissue expression of PASD1 was assessed via immunochemistry (IHC) in another 18 CRC samples. Four immunogenic PASD1 peptides predicted to bind to Major Histocompatibility Complex (MHC) Class 1 allele were identified using web-based algorithms. Peripheral blood mononuclear cells from CRC patients were used to investigate the immunogenicity of these HLA-A*2402 restricted peptides in vitro using IFN-y release ELISpot assay. The effects of effector CD8+ T-cells pulsed with PASD1 peptides against different cancer cells were detected in cytolytic and granzyme-B release ELISpot cytotoxicity assays. PASD1 mRNA expression was determined via RT-PCR on paired tumors and normal tissue samples from 25 CRC patients. Tissue expression of PASD1 was assessed via immunochemistry (IHC) in another 18 CRC samples. Four immunogenic PASD1 peptides predicted to bind to Major Histocompatibility Complex (MHC) Class 1 allele were identified using web-based algorithms. Peripheral blood mononuclear cells from CRC patients were used to investigate the immunogenicity of these HLA-A*2402 restricted peptides in vitro using IFN-y release ELISpot assay. The effects of effector CD8+ T-cells pulsed with PASD1 peptides against different cancer cells were detected in cytolytic and granzyme-B release ELISpot cytotoxicity assays. PASD1 mRNA expression was determined via RT-PCR on paired tumors and normal tissue samples from 25 CRC patients. Tissue expression of PASD1 was assessed via immunochemistry (IHC) in another 18 CRC samples. Four immunogenic PASD1 peptides predicted to bind to Major Histocompatibility Complex (MHC) Class 1 allele were identified using web-based algorithms. Peripheral blood mononuclear cells from CRC patients were used to investigate the immunogenicity of these HLA-A*2402 restricted peptides in vitro using IFN-y release ELISpot assay. The effects of effector CD8+ T-cells pulsed with PASD1 peptides against different cancer cells were detected in cytolytic and granzyme-B release ELISpot cytotoxicity assays.

Results

Gene expression of PASD1 was detected in 20% (5/25) CRC samples but not its protein expression. One of the four PASD1 peptides, PASD1(4), was shown to be immunogenic in CRC patients studied via the IFN-y release ELISpot assay. CD8-positive cytotoxic T cells from three patients raised against PASD1(4) were able to lyse HLA-A*2402 positive-SW480 cell line expressing endogenous PASD1 protein in all three effector/target ratios.

Conclusion

This is the first report of CD8-positive CTL response to PASD1 protein in CRC patients. Preliminary results show that PASD1 peptide is a probable immunogenic antigen and represents a potential target for peptide-based immunotherapy in CRC

Keywords: PASD1, colorectal cancer, HLA-A*2402, cancer immunotherapy, Cytotoxic T-cell

Poster 6.2

Mechanism of 1,4-Benzoquinone-Induced Genotoxicity in Hematopoietic Stem / Progenitor Cells: Linking Benzene to Hematological Disorders

Zariyantey Abdul Hamid1*, Paik Wah Chow1, Ramya Dewi Mathialagan1, Nor Fadilah Rajab1, Salwati Shuib2 and Siti Razila Abdul Razak3

1Faculty of Health Sciences, National University of Malaysia, Malaysia
2Medical Center, National University of Malaysia, Malaysia
3Cluster of Oncological and Radiological Science, Advanced Medical and Dental Institute, University of Science, Malaysia, Malaysia

Corresponding author: Dr. Zariyantey Abdul Hamid, zyantey@ukm.edu.my

Background

Hematopoietic stem / progenitor cells (HSPCs) are sensitive targets for benzene-induced leukemogenesis and hematotoxicity. This study aims to investigate the genotoxicity mechanism linking benzene exposure to targeting HSPCs using combined lineage-directed and genotoxicity strategies.

Methods

Freshly isolated mouse bone marrow cells (MBMCs) were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), at 1.25, 2.5, 5, 7, and 12 μM for 24 h. Then, the cells were seeded onto colony forming cell (CFC) assay specialized for myeloid, lymphoid Pre-B and erythroid progenitors, followed by colonies isolation from respective progenitors for chromosomal aberration and DNA damage analyses using karyotyping and Comet Assays; respectively.

Results

Relative frequency of chromosomal aberrations (CA) predominantly via Robertsonian translocation was significantly increased (p < 0.05) in MBMCs and all progenitors at all 1,4-BQ concentrations. Pre-B lymphoid progenitor demonstrated significantly higher (p < 0.05) CA than erythroid progenitor at 1.25, 2.5, and 7 μM ; followed by significantly higher (p < 0.05) CA than myeloid progenitor at 7 μM 1,4-BQ. 1,4-BQ exposure causes DNA damage on HSPCs in a dose-dependent manner. Differential responses were observed among the hematopoietic progenitors in term of the percentage of DNA in tail following exposure to 1,4 BQ. A significant (p < 0.05) increase in the percentage of DNA in tail in erythroid progenitors was only notable at 12μM 1,4 BQ as compared to myeloid and lymphoid pre-B progenitors of which the significant increase (p < 0.05) occurred at as lower as 7μM and 12μM of 1,4 BQ. A significant increase (p < 0.05) in the percentage of tail moment was only noted at 12 μM 1,4 BQ for all progenitors.

Conclusion

Conclusively, benzene could mediate hematological disorders and malignancies by causing genomic instability of HSPCs. The role of lineage specificity in governing the genotoxicity mechanism of 1,4-BQ deserves further investigation.

Keywords: 1,4-BQ, Hematopoietic progenitor cells, Benzene, Lineages, Genotoxocity

Poster 6.3

Tocotrienols: potential anti-cancer agent

Puvaneswari Meganathan1, 2*,  Ju Yen Fu2

1University of Malaya, Malaysia
2Malaysian Palm Oil Board, Malaysia

Corresponding author: Puvaneswari Meganathan,mpuvaneswari@gmail.com 

Background

Cancer is the second leading cause of death globally. The increasing cost of treatment and adverse effects often seen with conventional therapies have shifted the attention towards a multidisciplinary approach which includes the use of nutraceuticals as an alternative. Curcumin, resveratrol and tocotrienols are among the natural compounds with anti-cancer properties that have been studied in line with the concept “from bench to bedside”. Among these nutraceuticals, tocotrienols being a member of vitamin E family has been studied in depth in the last 3 decades. However, alpha-tocopherol which is a lesser potent isoform, is often mistaken as a general term for Vitamin E, thus making tocotrienols as the unsung heroes in anti-cancer research despite their beneficial biological properties. This poster is aimed to provide the overview of the anti-cancer effects of tocotrienols based on evidence from in vitro, in vivo and clinical trials.

Methods

Following numerous pre-clinical studies, two clinical trials involving cancer patients were conducted. The first pilot study was conducted in 240 women with early breast cancer in Kuala Lumpur General Hospital and the second study was carried out in 25 presurgical patients with pancreatic ductal neoplasia in H. Lee Moffitt Cancer Center and Research Institute, USA.

Results

Based on the pre-clinical studies, the anti-cancer activity of tocotrienols have been attributed to their anti-proliferative, anti-angiogenic, pro-apoptotic and immune enhancing effects. In addition, new self-emulsified formulations have improved the bioavailability of tocotrienols in humans that has been a debated issue in the past.

Conclusion

Despite emerging new studies showing evidence that circulating plasma tocotrienol levels are sufficient to evoke therapeutic response, more clinical studies are warranted to convince the scientific community, clinicians and healthcare authorities on the potential role of tocotrienols as an anti-cancer agent.

Keywords: bioavailability, Tocotrienols, Vitamin E, Cancer, Nutraceuticals

Poster 6.4

Cytotoxicity of Pogonatum cirratum extracts against CCL-119, a human T-cell acute lymphoblastic leukaemia cell line

Elda S. Latif1*, Nurul F. Kamaludin1 and Rajayaashini Rajasegaran1

1National University of Malaysia, Malaysia

Corresponding author:Dr. Elda S. Latif, elda.latif@ukm.edu.my 

Background

Acute lymphoblastic leukaemia (ALL), the most common form of childhood cancer, is characterised by abnormal differentiation, proliferation, and accumulation of lymphoid progenitor cells in the bone marrow and extramedullary sites. Given the various harmful side effects associated with current treatment regimens of ALL, natural products have become the focus of on-going search for alternative anti-acute leukaemic agents. Bryophyte mosses that possess medicinal values have yet been fully explored. Thus, this study aimed to investigate phytochemicals and cytotoxic activity of Pogonatum cirratum subsp. macrophyllum extracts against CCL-119, a human T-cell acute lymphoblastic leukaemia cell line.

Methods

P. cirratum was extracted using three solvents with different polarity; aqueous, methanol and petroleum ether. Each extract was then subjected to chemical screening of phytochemical contents. CCL-119 cells were treated with each extract at different concentrations ranging from 0-200µg/mL for 48 hours. Dexamethasone (Dex) was used as positive control. Cytotoxic effects and apoptotic activities of the extracts against CCL-119 ALL cell line were assessed using colorimetric MTT assays and Annexin V/ Propidium iodide staining, respectively.

Results

The percentage yield of aqueous, methanol and petroleum ether extracts were 1.4%, 6.25% and 0.38%, respectively. The extracts were tested positive for flavonoid, glycoside, phenol and saponin. P. cirratum exerted cytotoxic effects and apoptotic activities against CCL-119 cells. Aqueous extract of P. cirratum was the most potent (IC50= 4.570.33µg/mL) followed by methanol (IC50= 6.00.29µg/mL) and petroleum ether (IC50= 20.51.51µg/mL) extracts. Primary cell death was mediated via apoptosis with percentage of 90.52.85%, 82.73.03% and 69.71.23% in aqueous, methanol and petroleum ether extracts-treated cells, respectively. Dex-treated cells showed IC50 at 0.08×10-22.85µM, and cell death was mediated via apoptosis (44.12.11%) and necrosis (9.00.67%).

Conclusion

P. cirratum-induced cytotoxicity by apoptosis in cultured acute leukaemic cells could serve as the scientific basis for its usefulness as potential safer chemotherapeutic agent, particularly for childhood ALL.

Keywords: bryophyte pogonatum, medicinal value moss , childhood T-cell ALL, Cytotoxicity, Apoptosis

Poster 6.5

The Antioxidant Properties and Anticancer Effect of Medjool Dates (Phoenix dactylifera L.) on Human Breast Adenocarcinoma (MCF-7) Cell Lines: In vitro Study

Ismail Rozila1*, Najwa M. Abdul Manap1, Liana N. Ghazali1, Nurkhalida Kamal1, Waheedah Abdul Hakeem1, Nur Fariha Mohd Manzor2, Shiplu R. Chowdhury3 and Shamima Abdul Rahman1

1Department of Pharmaceutical Sciences, Faculty of Pharmacy, Cyberjaya University College of Medical Sciences, Malaysia
2Department of Biochemistry, Faculty of Medicine, Universiti Sains Islam Malaysia, Malaysia
3Tissue Engineering Centre, UKM Medical Centre,, Malaysia

Corresponding author: Dr. Ismail Rozila, rozila@cybermed.edu.my

Background

Breast cancer is a prevalent malignancy among women, especially in Malaysia. A large number of anticancer agents with natural origin have been reported. Hence, natural sources such as dates may play an essential role in prevention and treatment of cancers. Phoenix dactylifera L. cum Medjool dates is a sunnah food locally known as king of dates has been described in the traditional and alternative medicine to provide several benefits including antioxidant, anti-inflammatory and anticancer effects, but many properties remain to be scientifically validated. The purpose of this study was to evaluate the antioxidant and anticancer effect of methanolic extract of the commercial Medjool dates (P.dactylifera L.) on human breast adenocarcinoma (MCF-7) cell in-vitro.

Methods

200 g of Medjool dates were extracted using methanol solvent. The methanolic extract of Medjool dates (MEMD) was diluted to five different concentrations which were 0.01 mg/ml, 0.1 mg/ml, 1 mg/ml, 10 mg/ml and 100 mg/mL. The antioxidant properties of MEMD were analysed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. The MCF-7 cells were treated with MEMD for 24 until 72 hours. The structural of putative compounds in MEMD were detected by using Nuclear Magnetic Resonance (NMR) spectroscopy. To evaluate the cytotoxic effect of MEMD, cell viability test was conducted using CellTiter-Blue® assay. Then, the concentrations of 10 mg/ml and 100mg/ml of MEMD were used for determination of anticancer effect such as apoptosis assay using Muse™ Annexin V and Dead Cell assay. The morphology of the cells were observed using inverted microscope. In this study, untreated cells were used as the negative control and cells treated with doxorubicin (1.5 µg/ml) as the positive control.

Results

MEMD at 100 mg/ml showed the highest radical scavenging activity against DPPH free radicals (93.02%) and the highest cytotoxicity activity (IC50 = 64.75 mg/ml) which inhibit MCF-7 proliferation from 45.13 ± 6.6 % to 21.94 ± 5.42% at 72 hours. This was supported by the apoptosis assay which demonstrated the percentage of total apoptotic cells treated with 100 mg /ml of MEMD (77.16%) showed two-fold higher than the percentage of total apoptotic cells treated with 10 mg/ml of MEMD (33.45%). The morphological changes of MCF-7 cells treated with 100 mg/ml MEMD showed that cell undergoing apoptosis with cell shrinkage and rounding with the cytoplasm appeared dense. This is comparable with the morphological changes showed in the MEMD cells that were treated with doxorubicin (positive control group). The 1H NMR data of MEMD was analysed, and chemical fingerprints of compounds such as sugars, phenolic aliphatic compounds were detected. The 1H NMR spectrum detected the presence of sugar units at region 3-5 ppm. A doublet signal at δH 4.89 for the anomeric proton of a galactose unit, and a coupling constant of 3.67 Hz for the anomeric proton indicated its α-configuration. Another anomeric doublet signal at δH 4.26, and a coupling constant of 7.76 Hz for the ano-meric proton indicated its β-configuration of glucose unit. The upfield shift of the aromatic proton signals at the 6.19 and 6.57 ppm region suggested the presence of electron withdrawing groups such as hydroxyl substituents. The proton signals 0.00 to 2.90 ppm indicated the presence of aliphatic compounds in MEMD.

Conclusion

MEMD has significant antioxidant properties and anticancer activity against MCF-7 cells in-vitro study which might derived from phenolic compounds and sugars detected in the extract.

Keywords: Antioxidant, Anticancer effect, Apoptosis, MCF-7 (breast), Phoenix dactylifera l.

Poster 6.6

Uncovering the Landscape of Somatic Mutations in Malaysian Colorectal Cancer Patients via Whole Genome Sequencing

Ryia Illani Mohd Yunos1, Nurul-Syakima Ab Mutalib1, Jia-Shiun Khoo2, Sazuita Saidin1, Muhiddin Ishak1, Nadiah Abu1, Najwa F. Mohd Yusof1, Norshahidah Mahamad Nazir1, Isa M. Rose3, Ismail Sagap4, Luqman Mazlan4 and Rahman Jamal1*

1UKM Medical Molecular Biology Institute (UMBI), Malaysia
2Codon Genomics, Malaysia
3Department of Pathology, Faculty of Medicine, National University of Malaysia, Malaysia
4Department of Surgery, Faculty of medicine, University of Malaya, Malaysia

Corresponding author: Prof. Rahman Jamal, rahmanj@ppukm.ukm.edu.my

Background

Over the past few years, colorectal cancer (CRC) remains as the third most common cancer worldwide and the incidence are increasing continuously in some area of the world, including Malaysia. The expected rise of CRC burden in Malaysia underlines the importance of pushing ahead the pursuit of understanding this cancer by genetically profilling the genome of local CRC patients. In this study, we aimed to characterise the landscape of somatic mutations using whole genome sequencing approach, and to provide a better insight of the key molecular networks involved in Malaysian CRC patients.

Methods

Whole genome sequencing, with at least 30X coverage was performed on genomic DNA obtained from 28 CRC tissues. Identified variants were validated using Sanger sequencing. Expression of RNF43 gene in CRC tissues and cell lines was determined using qPCR.

Results

All types of somatic mutations were detected and they included single nucleotide variants (SNVs), insertions and deletion in both coding as well as in non-coding region of the genome. With the mutation rate that ranged between 1 to 26 per 106, an average of 29, 318 SNVs in tumour samples were obtained. It was found that TP53 (10/13), APC (9/13) and KRAS (5/13) remained as the most commonly mutated genes in patients. Wnt signalling pathway was identified as the major affected pathway. At least one actionable somatic alteration was identified in all our patients. RNF43 mutation was likely to co-exist with BRAF V600E mutation in patient with MLH1 deficiency. RNF43 expression was upregulated in both CRC cell lines and tumour tissues; it was, however, downregulated in CRC tissue with RNF43 mutations (p.G156Afs and p.P192Gfs) when compared to the wild type.

Conclusion

This mutational signature analysis had illustrated the genomic landscape of CRC. Alternative treatment targeting Wnt signalling pathway could be beneficial to CRC patients carrying RNF43 mutations.

Keywords: colorectal cancer, whole genome sequencing, Molecular landscape, precision medicine, Wnt Signaling Pathway

Poster 6.7

Influence of mitochondrial DNA alterations on cisplatin chemoresistance in oral squamous cell carcinoma

Amnani Aminuddin1, Pei Yuen Ng1, Chee-Onn Leong2, 3 and Eng Wee Chua1*

1Drug and Herbal Research Centre, Faculty of Pharmacy, University Kebangsaan Malaysia, Malaysia
2School of Pharmacy, International Medical University, Malaysia
3Centre for Cancer and Stem Cell Research, International Medical University, Malaysia

Corresponding author: Dr. Eng Wee Chua, cew85911@ukm.edu.my

Background

Cisplatin is a commonly used chemotherapeutic agent in the treatment of various solid tumours. To date, the development of secondary resistance towards cisplatin remains a major obstacle to the success of cancer chemotherapy including oral squamous cell carcinoma (OSCC). Recently, it has been reported that mitochondrial DNA (mtDNA) alterations could promote tumourigenesis and chemoresistance. Herein, we aimed to evaluate the genetic basis of cisplatin chemoresistance through comprehensive mtDNA profiling.

Methods

Cisplatin sensitivity of two OSCC cell lines, SAS and H103, and cancer stem-like cells (CSCs) derived from SAS were tested using the MTS assay. Variation in mtDNA abundance was measured by a real-time polymerase chain reaction (qPCR) assay. Sequencing of the mitochondrial genome was performed with the nanopore sequencing technology (MinION™) to screen for mtDNA point mutations. An in-house bioinformatics workflow was established for mutation discovery, and all detected mtDNA mutations were validated by Sanger sequencing

Results

H103 was more cisplatin-resistant than SAS-CSCs and SAS. In keeping with the assessment of cisplatin sensitivity, qPCR analysis showed that mtDNA was less abundant in H103 than SAS-CSCs and SAS. Interestingly, the nanopore sequencing identified a mutation in the D310 (displacement loop) regions of both SAS-CSCs and SAS, but the mutation was absent in H103. The displacement loop is a non-coding region that regulates mtDNA replication and gene transcription.

Conclusion

We demonstrated that enhanced cisplatin resistance in H103 could be influenced by mtDNA abundance. The absence of an activating mutation in the displacement loop region could have accounted for the lower abundance of H103’s mitochondrial genome when compared with SAS-CSCs and SAS. We suggest that alterations in mtDNA abundance might be used as a marker of cisplatin responsiveness in the treatment of OSCC. Future work may focus on investigating the mechanisms that underpin variation in mtDNA content and therefore cisplatin response.

Keywords: mitochondrial DNA, cisplatin resistance, Oxford Nanopore Technology, Mitochondrial DNA content, Point Mutation

Poster 6.8

Ternary Copper (II) Complex Induced Cell Cycle Arrest and Apoptosis in Human Breast (MDA-MB-231) and Colon (HT-29) Cancer Cells

Jhi Biau Foo1*, Xian Wei Teo1, Zheng Yang Lee1, Chee Hong Leong1, Faris b. Norizan1, Sathiavani a. Arikrishnan2, Yin Sim Tor2, May Lee Low3 and Chew Hee Ng3

1Faculty of Health and Medical Sciences, School of Pharmacy, Taylor’s University, Malaysia
2Faculty of Health and Medical Sciences, School of Biosciences, Taylor’s University, Malaysia
3Department of Pharmaceutical Chemistry, School of Pharmacy, International Medical University, Malaysia

Corresponding author: Dr. Jhi Biau Foo, foojhibiau@gmail.com

Background

The use of copper complexes for cancer treatment has been widely explored as cancer cells are reported to take up greater amounts of copper than normal cells. Our previous study had successfully synthesised and characterised the Cu(phen)(L-tyr)Cl]·3H₂O copper complex. Nevertheless, the mode of cell death induced by this copper complex against cancer cells remains unclear. Therefore, the current study investigated the mode of cell death induced by Cu(phen)(L-tyr)Cl]·3H₂O against human breast and colon cancer cell lines.

Methods

The inhibitory and morphological changes induced by Cu(phen)(L-tyr)Cl]·3H₂O against human breast (MDA-MB-231) and colon (HT-29) cancer cell lines were determined by the MTT assay and inverted light microscopy, respectively. Induction of cell cycle arrest and apoptosis were determined by flow cytometry analysis.

Results

The growth of cancer cell lines was inhibited by Cu(phen)(L-tyr)Cl]·3H₂O in a dose- dependent manner with IC50 value of 3.5 µM against these two cancer cell lines. The copper complex was 9 and 1.5 folds more potent than cisplatin in inhibiting the growth of HT-29 colon and MDA-MB-231 breast cancer cells, respectively. It was found that Cu(phen)(L-tyr)Cl]·3H₂O induced S phase cell cycle arrest on HT-29 colon and MDA-MB-231 breast cancer cells. Characteristics of apoptosis such as cellular shrinkage, membrane blebbing and apoptotic bodies were seen under inverted light microscope, indicating that Cu(phen)(L-tyr)Cl]·3H₂O induced apoptosis in the cells. The observation was further ascertained using Annexin-V/PI flow cytometry analysis.

Conclusion

Our present study provided evidence regarding the anticancer effects and underlying mechanisms of Cu(phen)(L-tyr)Cl]·3H₂O against human breast and colon cancer cells. These findings shed new light on the potential application of Cu(phen)(L-tyr)Cl]·3H₂O as anticancer agent.

Keywords: Copper Complex, Colon Cancer, breast cancer, Apoptosis, cell cycle arrest

Poster 6.9

Identification of Differentially Expressed Genes in CaSki Cervical Cancer Cells Treated with a Selected Diarylpentanoid

Rakesh Naidu1*, Felicia Paulraj1, Faridah Abas2, 3, Nordin H. Lajis3 and Iekhsan Othman1

1Jeffrey Cheah School of Medicine and Health Sciences (JCSMHS), Malaysia
2Faculty of Science, Putra Malaysia University, Malaysia
3Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, Malaysia

Corresponding author: Dr. Rakesh Naidu, kdrakeshna@hotmail.com

Background

Diarylpentanoids are curcumin analogues with a 5-carbon linker between its aryl rings, showed greater growth inhibitory activity over curcumin. Structural modifications of curcumin, an active component from the root of Curcuma longa has shown to exhibit significant growth-suppressive effect in a wide range of cancer cell lines but its effects on cervical cancer has not been well studied. Our preliminary findings on cytotoxicity and apoptosis indicate that diarylpentanoid (MS17) is able to induce apoptosis and demonstrate anti-proliferative effect in human papillomaviruses (HPV) infected cervical cancer cells (CaSki-HPV-16). However, the molecular mechanism underlying this effect is not fully understood and warrants further investigation. The aim of the present study was to identify differentially expressed genes (DEGs) in CaSki following 6µM MS17 treatment for 24 hours.

Methods

Gene expression profile was performed using Agilent SurePrint G3 Human gene expression array. Functional classification and pathway analysis were performed on the DEGs.

Results

We had identified 104 significantly regulated (p<0.05) DEGs, 45 up- and 59 down-regulated genes in response to MS17 treatment. Functional classification analysis revealed that the up- and down-regulated genes were associated with cell adhesion, cell cycle, chromosome organisation, cytoskeleton organisation, DNA damage response, signal transduction, transcription regulator activity and transporter activity. Our data also revealed that among the DEGs, some up-regulated genes were exclusively associated with oxidation reduction, protein binding, tumour suppressor and unfolded protein response while down-regulated genes were associated with chromosome segregation. Pathways significantly modulated by MS17 were Mitotic Roles of Polo-Like Kinase, Cell Cycle: G2/M DNA Damage Checkpoint Regulation, ATM Signaling, Cyclins and Cell Cycle Regulation, and GADD45 Signaling.

Conclusion

The modulation of these genes and associated molecular pathways suggest that MS17 may exhibit its anticancer therapeutic potential activity through cell cycle regulation, growth inhibition and apoptosis on cervical cancer cells.

Keywords: cervical cancer, diarylpentanoid, Gene Expression Profiling, Curcumin analogue, Microarray

Poster 6.10

Cancer-associated Fibroblasts Promote Endometrial Cancer Cell Migration in 3D Environment 

Yap Chee Voon1, 2, Intan Sofia Omar1, 2, Nur Akmarina B.M. Said3, Ivy Chung1, 2*

1Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
2University of Malaya Cancer Research Institute, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia,
3Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

Corresponding author: Ivy Chung, ivychung@ummc.edu.my

Background

Cell motility is a critical step in metastasis cascade, yet the role of cancer-associated fibroblasts (CAFs) from tumour microenvironment in facilitating endometrial cancer (EC) cell motility remains unclear.  In this study, we studied the interaction of CAFs and EC cells in an in vitro 3D model.

Methods

3D culture was established using Matrigel in tumour-fibroblasts coculture. Live cell imaging was performed to examine cell velocity and projection formation. Confocal microscopy and 3D reconstruction was performed to analysis spheroid structures. qPCR was performed to investigate RhoA and ROCK mRNA expression. ROCK inhibitor, Y-27632 was utilised to validate RhoA/ROCK signaling in CAF-mediated EC motility and spheroid formation.

Results

We demonstrated that CAFs increased the motility of EC cell line (ECC-1) compared to ECC-1 monoculture (3-fold increase, p<0.05). CAFs formed cell projections which served as contact guidance for EC cell locomotion in the spheroid formation process. Molecular analysis revealed that RhoA/ROCK signalling activation was in part contributing to CAFs-mediated EC cell migration. Presence of ECM increased RhoA mRNA expression in ECC-1 monoculture. RhoA mRNA was also upregulated in CAFs monoculture and ECC-1 and CAFs coculture on Day 14. RhoA downstream effector, ROCK1 mRNA expression was only upregulated in coculture on Day 14. Treatment with Y-27632 attenuated CAF motility (p<0.05) but not ECC-1in monoculture and significant inhibition (p<0.0001) in ECC-1 motility in ECC-1 and CAFs coculture. Subsequently, tumour spheroid formation was inhibited in due to stress fiber inhibition in CAFs.

Conclusion

RhoA/ROCK1 signaling pathway is required for CAFs to serve as cellular vehicle for EC tumour cells to migrate and form spheroids in a 3D environment.

Keywords: Uterine cancer, tumor microenvironment, cancer-associated fibroblast, motility, Rho family of GTPases

Poster 6.11

Local β-Glucan Source from Oyster Mushroom (Pleurotus flabellatus) as Potential Usage in Medical Applications

Shaiful A. Mohamad1*

1Malaysian Nuclear Agency, Malaysia

Corresponding author: Dr. Shaiful A. Mohamad, shaifulazuar@gmail.com

Background

Many applications of mushroom polysaccharides have been reported such as in drug delivery system, skin applications and as immunomodulatory agents. The polysaccharides can be obtained from the fruit body, mycelium, culture broth and spores of mushroom. In this study, oyster mushroom (Pleurotus flabellatus) culture was produced by submerged culture fermentation due to its higher productivity and faster fermentation rate.

Methods

The biomass obtained from submerged culture fermentation was extracted by using hot water extraction. The polysaccharides were tested using β-glucan kit and also studied using small angle X-ray scattering (SAXS) equipment. Then the polysaccharides were separated using column chromatography before determining the molecular weight using gel permeation chromatography-multi angle laser light scattering (GPC-MALLS).

Results

Both endopolysaccharide and exopolysaccharide obtained have shown the presence of β-glucan (1,3;1,6). The polysaccharides also showed the presence of high molecular weight (~3×106 Da) and low molecular weight components. SAXS results showed that the maximum agglomerate size was ~27 nm and the average size of globular was 2.6 nm.

Conclusion

The presence of high molecular weight β-glucan showed potential application as immunomodulatory agents. The polysaccharides can be classified as nanobiomaterials. These studies supported potential usage of local compounds in medical applications.

Keywords: beta glucan, Pleurotus flabellatus, Submerged culture fermentation, Immunomodulation, Small angle X ray scattering

Poster 6.12

Anti-cancer potential of methyl gallate isolated from Bambangan (Mangifera pajang) in MCF-7 cell line

Azlen Che Rahim1, Mohd Fadzelly Abu Bakar1*, Nur Kartinee Kassim2, Johnson Stanslas2 and Wan Nor I’zzah Wan Mohamad Zain

1Universiti Tun Hussein Onn Malaysia, Malaysia
2Putra Malaysia University, Malaysia
3Universiti Teknologi MARA, Malaysia

Corresponding author:Dr. Mohd Fadzelly Abu Bakar, fadzelly@uthm.edu.my

Background

Mangifera pajang or widely known as Bambangan is an indigenous fruit of Borneo Island. The kernel part of this fruit has been reported to display anti-cancer potential. This study was conducted to isolate the major bioactive compound from the kernel of Bambangan and determine the cytotoxic activity and apoptosis-inducing ability of the compound in MCF-7 cell line.

Methods

Methyl gallate (C8H8O5) has been successfully isolated from the methanol crude extract of Bambangan kernal. Identification of methyl gallate was done using spectroscopic method analysis such as infrared (IR), mass spectrometry (MS), nuclear magnetic resonance (NMR) and compared to reported data. Cytotoxic activity of methyl gallate was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of methyl gallate on cell cycle and apoptosis induction was assessed using flow cytometry analysis.

Results

The results showed that methyl gallate induced strong cytotoxic activity in MCF-7 cell line with IC50 value of 54.7 µM. Methyl gallate increased cell population in sub-G1 phase indicating apoptosis and cell cycle arrest at G0/G1. In addition, staining with Annexin V-FITC and propidium iodide revealed that total apoptotic cells were increased after incubation with the compound in a time dependent manner.

Conclusion

As a conclusion, methyl gallate isolated from Bambangan induced anti-cancer potential in breast cancer cell. Study on the mechanism of action as well as the efficacy of this compound in in vivo is suggested moving forward

Keywords: Mangifera pajang, Methyl gallate, MCF-7, Apoptosis, cytotoxic

Poster 6.13

Protective Effect of Andrographolides against Docetaxel-induced Cognitive Impairment in Rats Bearing Mammary Tumours

Nasir I. Khatab1, Dahiru Sani1, Brian P. Kirby2, Norhafizah, Muhtarrudin1, Ho G. Fuang3, Johnson Stanslas1* and Hamidon Basri1*

1Putra Malaysia University, Malaysia
2Royal College of Surgeons in Ireland, Ireland
3University Malaya Medical Centre, Malaysia

Corresponding author: Prof. Johnson Stanslas, jstanslas@yahoo.co.uk and Prof. Hamidon Basri, hamidonbasri@gmail.com

Background

Andrographolide (AGP) and 14-deoxy-11,12-didehydroandrographolide (DDAG) are two main diterpenoid constituents of Andrographis paniculata, which have shown several therapeutic effects. The aim of this study was to examine the protective effects of AGP and DDAG against cognitive impairment (CICI) caused by chemotherapeutic agent, docetaxel (DTX), on rats with mammary tumours.

Methods

A two-day Morris Water Maze (MWM) protocol, which involved training with a visible platform on day one followed by testing with a hidden platform on day two, was performed. Ten groups of female Sprague Dawley rats were used. The rats were injected with LA7 cells into the mammary gland pad to form tumours and further divided into groups of normal control (NC), cancer control (CC), treatment with DTX (single dose 5 mg/kg i.p.), three doses (0.25, 0.50 and 1.0 mg/kg) of AGP or DDAG and a vehicle. Subsequently, AGP, DDAG or vehicle was administered intraperitoneally for seven consecutive days followed by DTX injection after day one of visible platform trial. Levels of pro-inflammatory cytokine interleukin IL-1, lipid peroxidation marker (thiobarbituric acid-reactive substances (TBARS) and reactive oxygen species (ROS)) were measured.

Results

After 24 hours of DTX injection, the escape latency of DTX had significantly (p<0.001) increased to 58.1 sec compared to 24.7 sec in CC. AGP and DDAG significantly (p<0.001) reduced this DTX-induced latency to about 10-16 sec. AGP and DDAG significantly (p<0.001) reduced the elevated IL-1β (78-91%), IL-6 (55-75%), TNF-α (63-73%), TBARS (52-83%) and ROS (22-50%) levels when compared to DTX. AGP and DDAG did not affect the anti-cancer activity of DTX.

Conclusion

AGP and DDAG, if given prior to DTX, may be protective against the chemotherapy-induced cognitive impairment.

Keywords: AGP, DDAG, DOCETAXEL, Morris water maze, cognitive impairment

Poster 6.14

Tualang Honey Inhibited H23 Human Lung Adenocarcinoma Cell Growth by Modulating the Expression of Functional Apoptotic Proteins

Nazirah Amran1, Puteri Shafinaz Abdul-Rahman1* and Hwee-Ming Cheng1

1University of Malaya, Malaysia

Corresponding author: Dr. Puteri Shafinaz Abdul-Rahman, terisar@um.edu.my 

Background

Tualang honey (TH) is a multiflora rainforest honey from Malaysia and produced by wild giant bees (Apis dorsata) that built their hive on the Tualang tree. Recently, honey has been shown to have anti-cancer properties on various cancer lines. The purpose of this study is to investigate the effect of TH on the molecular mechanism of apoptosis and proliferation in H23 human lung adenocarcinoma cell line.

Methods

3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to evaluate the anti-proliferative effect of TH in H23 cells for 24 and 48h. Detection of apoptosis was carried out by measuring annexin V-FITC apoptosis assay. Apoptosis protein profiling was detected by RayBio® Human Apoptosis Array. The quantitative label-free LC-MS/MS strategy was performed to characterize the proteomes of H23 cell in response to TH-treatment

Results

The MTT assay indicated that TH was able to inhibit the proliferation of H23 cells in a dose- and time-dependent manner with the IC50 value of 3.6±0.6 % and 2.7±0.2 %(v/v) for 24 and 48h, respectively. TH induced apoptosis in H23 cells by upregulating the expression of caspase-3, cytochrome c, Bad, Bax, p53 and BCLAF1 proteins (p<0.05). TH treatment significantly (p<0.05) decreased the expression of HSPB1. TH also inhibit the expression of CREB1, HDAC, PCNA and 14-3-3 proteins.

Conclusion

TH inhibited cell proliferation and enhanced apoptosis in H23 human lung adenocarcinoma cells by modulating the proteins involved in mitochondrial pathway-induced apoptosis.

Keywords: p53, caspase-3, PCNA, 14-3-3 protein, Tualang honey

Poster 6.15

Differential protein expression of SKOV3 ovarian cancer cells treated with standardised extract of Eurycoma longifolia (SEEL30)

Nor Datiakma Mat Amin1*, Nurhanan M. Yunos1* and Muhamad Haffiz Jauri1

1Forest Research Institute Malaysia (FRIM), Malaysia

Corresponding authors: Nor Datiakma Mat Amin, nordatiakma@frim.gov.my and Dr. Nurhanan M. Yunos, hanan@frim.gov.my 

Background

Ovarian cancer is the leading cause of mortality amongst women in urban countries with 225,500 new cases and 140,200 estimated deaths reported worldwide. The long-term survival of ovarian cancer patients is still poor, with less than 40% across all stages. Hence, there is an urge to search for new potential anti-ovarian cancer drug candidates. The standardised extract of E. longifolia (SEEL30) have shown active anti-cancer effect against SKOV3 ovarian cancer cell line with an IC50 of 17.26±0.11µg/mL. The acute toxicity tests showed no adverse and alarming behavioral response with LD50 > 2000mg/ kg body weight of male and female Sprague Dawley rats. Recent advances have enabled the study of cancer proteome. Proteins are the main components of the physiological metabolic pathways of cells as they provide structure, catalysing almost all the reactions that take place in a living cell. The current study therefore aimed to observe the differential expression of SKOV3 ovarian cancer cells treated with standardised extract of Eurycoma longifolia (SEEL30).

Methods

Ovarian cancer cells (SKOV3) were treated with or without (control) standardised extract of E. longifolia (SEEL30) using the IC50 value. Then, protein profiling was conducted using 2-Dimensional Electrophoresis (2-DE). The gels were stained with Coomassie Brilliant Blue-G200 (CBB-G200). Digital images of the analytical gels were acquired and analysed quantitatively for differentially expressed proteins using ImageMaster 2D Platinium 7.0 analysis software.

Results

In total, at least 900 protein spots were successfully being separated and detected in each 2-DE profiles. Only 62 proteins were however, differentially expressed in 2-DE profiles of SKOV ovarian cancer cells treated with SEEL30 as compared to the control (selection criteria: Anova p value<0.05 and protein fold change >1.5). Of the 62 differentially expressed proteins, 29 proteins were up-regulated and 33 proteins were down-regulated.

Conclusion

The differential proteins obtained will be identified and networks of the involved proteins will be elucidated.

Keywords: Eurycoma longifolia, 2-Dimensional electrophoresis, Proteomics, Ovarian cancer cell line, Standardised extract

Poster 6.16

Synergistic Effects between 9-Methoxycanthin-6-one
when Combined with Selected Chemo-drugs in
Inhibiting the Proliferations of Ovarian Cancer Cells 

Nurhanan M. Yunos1*, Nor Datiakma Mat Amin1, Muhamad Haffiz Jauri1, Kiong S. Ling1, Norhasnida Hassan1, Asiah Osman1 and Mohd Hafidz Hadi Abdullah1

1Forest Research Institute Malaysia (FRIM), Malaysia

Corresponding author:Dr. Nurhanan M. Yunos, hanan@frim.gov.my 

Background

The main modality of treatments for advanced ovarian cancer is still chemotherapy. However, significant numbers of patients still could not survive due to drug resistance and toxicity problems. The combination treatments are becoming one of the improved method to increase drug efficacy and reduce these problems clinically. However, more new drug candidates with different modes of action and positive druglikeness activities are still needed since different patients have different susceptibility on the drug uptake. An alkaloid compound had been isolated from the roots of Eurycoma longifolia and characterized as 9-methoxycanthin-6-one (EL50). EL50 was found to have anti-proliferative effects when tested in vitro and in vivo in our previous studies. The objective of this study was to evaluate if there is any synergism on the anti-proliferative activities when EL50 was combined with either cisplatin, paclitaxel, gemcitabine or doxorubicin.

Methods

The compound was used to treat ovarian cancer cell line (SKOV-3), either alone or in combination with chemotherapy drug (either cisplatin, paclitaxel, doxorubicin or gemcitabine). The combination treatments were performed either by simultaneous addition or sequential addition (either EL50 was added first, followed by the drug four hours later or vice versa). The results were evaluated after performing Sulforhodamine B assay using IC50 values for single treatment or combination index (CI) at different inhibition concentrations for binary treatments. CI values <1, =1 and >1 indicate synergism, additivity and antagonism in combined drug action, respectively.

Results and Conclusion

Initial studies had found that EL50 gave IC50 values were 1.70 ± 0.20, 1.65 ± 0.06 and 1.72 ± 0.18 µg/mL, respectively when treated in A2780, A2780cisR and SKOV-3 ovarian cancer cells. All the combination treatments were found to have synergistic effects (CI < 1) in inhibiting the proliferations of ovarian cancer cells when EL50 was added simultaneously with each of the mentioned drug, in which, highest synergism was found when EL50 was combined with paclitaxel. For sequential addition, it was found that highest synergism was shown when EL50 was added four hours before gemcitabine

Keywords: simultaneous addition, Combination index (CI), ovarian cancer, Eurycoma longifolia, , 9-methoxycanthin-6-one (EL50)

TRACK 7: INFECTIOUS DISEASES

Poster 7.1

Antibacterial activity of newer Quinazolin-4(3H)-one derivatives

Gopal Natesan1*, Appala Raju1, Nelyica Su Yean Tze1 and Soon An Nie1

1Faculty of Pharmacy, Mahsa University, Malaysia

Corresponding author: Dr. Gopal Natesan,  gopal@mahsa.edu.my

Background

Heterocyclic chemistry has gained remarkable attention recently due to its pharmacological diversity in medicinal chemistry research. Quinazolin-4(3H)-one pharmacophore is one of the most important heterocycles, which possess wide spectrum of biological properties like antimicrobial, anticonvulsant, anti-inflammatory and anticancer activities. Hence, this pharmacophore has been considered as a privileged structure to introduce many bioactive moieties to its nucleus for creating new prospective medicinal agents.

Methods

Quinazolinone derivatives were synthesized by reacting anthranilic acid with benzoyl chloride/4-methoxy benzoyl chloride followed by condensation with hydrazine hydrate, which was further subjected to Schiff base reaction with the different aromatic/heterocyclic aldehydes. The structures of all newly synthesized compounds were characterized by using FT-IR, H1 NMR Spectra. Ten (10) final compounds and two (2) intermediates were evaluated for their antibacterial property against gram-positive (Staphylococcus aureus ATCC 29213 and Bacillus cereus ATCC11778) and gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) using agar well diffusion method at a concentration of 50 µg/ml and 100 µg/ml using Norfloxacin as reference drug.

Results

The parent 2-phenyl-3-amino quinazoline-4(3H)-one (IIa) showed moderate activity against both gram-positive bacteria and gram-negative bacteria whereas substitution of –OCH3 in the phenyl position at the 2nd position of quinazolinone reduces the activity against both strains. The same pattern of the results has been observed among the final Schiff bases. Among the Schiff bases, increase in substitution of –OCH3 group at benzylidene nucleus alters the antibacterial activity. Lower number of methoxy groups exhibited moderate activity against all microorganism whereas increase in number of methoxy group substitution at benzyl-inde nucleus decreases the antibacterial activity against B. cereus and P. aeruginosa and diminishes the activity against S. aureus and E. coli. On the other hand, substitution of a heteryl group at the aldimine in 3-amino quinazolinone pharmacophore significantly increases the antibacterial activity against all the tested bacterial pathogens.

Conclusion

From the present study, it has been concluded that 2-Phenyl-3-[1-(furfurylidene amino)]-quinazolin-4-(3H)-one (IIIe) showed superior activity among the synthesized compounds. Replacement of benzene ring with heteryl group or substitution of electron donating group in the benzylidene nucleus displayed good antibacterial effects.

Keywords: Quinazolin-4(3H)-one, Schiff base, Antibacterial, benzoxazinone, Benzylidene

Poster 7.3

Anti-Leptospiral Potential of Zingiber zerumbet (L.) Smith Crude Extracts against Leptospira spp.

Asmah Hamid1*, Nurul A. Abdul Aziz1, Latifah Ibrahim2, Farah W. Ibrahim1 and Nurul F. Jufri1

1School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, National University of Malaysia, Malaysia
2Institute for Medical Research, Ministry of Health, Malaysia

Corresponding author: Dr. Asmah Hamid, asmah0901@ukm.edu.my

Background

Leptospirosis is one of the emerging infectious diseases that the number of cases had been increasing progressively in Malaysia. Numerous attempts have been made to control the disease by using chemoprophylaxis but only showed limited success. The present study investigated the anti-leptospiral potential of hexane, ethyl acetate and methanol extracts of Zingiber zerumbet

Methods

The extracts were assayed for anti-leptospiral activity using broth microdilution method towards Leptospira interrogans (serovar Batavie, Canicola, Australis) and Leptospira biflexa serovar Patoc. The DNA damaging properties of Z. zerumbet on DNA of Leptospira sp. was assessed by incubating the DNA of Leptospira sp. in the presence or absence of Z. zerumbet crude extracts and subsequently analyzed by electrophoresis.

Results

The anti-leptospiral activity of Z. zerumbet hexane extracts showed an IC₅₀ value of 248 μg/ml towards L. interrogans serovar Canicola, IC₅₀ value 125 μg/ml towards L. interrogans serovar Australis, IC₂₅ value 15.63 μg/ml towards L. interrogans serovar Batavie and IC₅₀ value 109 μg/ml towards L. biflexa serovar Patoc. However, ethyl acetate and methanol extracts exhibited a weak/mild anti-leptospiral activity. Since the hexane extract of Z. zerumbet displayed strong anti-leptospiral activity, the DNA damaging properties of this extract was tested at their IC₅₀ and IC25 values that were specific to each serovars. The result showed that hexane extract do not possess DNA damaging properties towards L. interrogans serovar Canicola and Bataviae as there was no smearing or fragmentation of DNA band. For L. biflexa serovar Patoc and L. interrogans serovar Australis there were DNA damage which was evidenced by the appearance of fragmented DNA on agarose gel.

Conclusion

The present study showed that Z. zerumbet hexane extract has the potential to inhibit the growth of Leptospira spp. by damaging the bacterial DNA.

Keywords: Anti-leptospiral, Zingiber zerumbet, Leptospira, DNA Damage, extract

Poster 7.4

Evaluation of antibacterial activity and chemical constituents of Melicope glabra (DC.) T. G. Hartley

Hafizah Mohd Zaini1*

1Putra Malaysia University, Malaysia

Corresponding author: Hafizah Mohd Zaini, fizazaini07@gmail.com

Background

Melicope glabra is an edible plant which belongs to the Rutaceae family. It has been traditionally used in treating several illnesses including stomach pain, high blood pressure and to revitalize the body. In the present study, the antibacterial activity of ethyl acetate extract from the bark of M. glabra was evaluated against two oral pathogenic bacteria, Enterococcus faecalis and Streptococcus mutans. E. faecalis and S. mutans are oral pathogens that have been recognized as an important etiological agent in human dental caries. It has been suggested that E. faecalis virulence may be related to resistance to intracanal medicaments while S. mutans is a potent initiator of caries that play an important role in caries formation.

Methods

A new flavonoid compound was isolated from the ethyl acetate extract of M. glabra stem bark and its structure was elucidated by performing various spectroscopic techniques (nuclear magnetic resonance, mass spectroscopy, infrared spectroscopy) and compared with related literature data. The antimicrobial activities were evaluated by TLC-bioautography and minimum inhibitory concentration (MIC).

Results

The ethyl acetate extract exhibited good inhibition zones by TLC-bioautography method while the MIC assay showed that the extract possessed significant antibacterial activity against E. faecalis and S. mutans with the values of 5.0 mg/ml and 0.625 mg/ml respectively. Assay guided isolation and purification of ethyl acetate extract successfully isolated a new flavonoid compound; 7-hydroxy-2-(4’-hydroxy-3’-methoxy-phenyl)-3, 5-dimethoxy-chromen-4-one.

Conclusion

Ethyl acetate extract from M. glabra stem bark exhibited significant antibacterial activity and therefore can serve as a potential plant-based antimicrobial agent.

Keywords: Melicope glabra, Bioassay guided isolation, Antimicrobial activity,, Streptococcus mutans, Enterococcus faecalis

Poster 7.6

Hemolymph of Baikal Endemic Crustacean Eulimnogammarus Verrucosus as a Source for Novel Natural Products

Denis Axenov-Gribanov1, 2*, Eugenii Protasov1, Polina Drozdova1, Kseniya Vereshchagina1, 2, Yuliya Lubyaga1, 2, Zhanna Shatilina1, 2, Maxim Timofeyev1 and David Lam3

1Irkutsk State University, Russia
2Baikal Research Center, Russia
3Faculty of Pharmacy, National University of Malaysia, Malaysia

Corresponding author: Mr. Denis Axenov-Gribanov, Denis.axengri@gmail.com

Antibiotic resistance and targeted therapy are one of the most important and worldwide issues. Extreme and unusual ecosystems such as Lake Baikal and its inhabitant are considered as potential tools for the discovery of novel natural products with biological activities. The aim of this study was to assess the possibility to use Baikal endemic amphipods as a source for screening of novel natural products. Analysis of hemolymph from an amphipod Eulimnogammarus verrucosus by HPLC-MS was performed for the first time and allowed us to find a number of natural products with new molecular mass values for Baikal endemic macroinvertebrates. We identified a compound as chrysin by comparison of MS1, MS2 profiles and molecular masses of natural products registered in Dictionary of Natural Products and MZ-cloud databases. Also, we found a new compound with a mass of 493.80277 Da and a number of antimicrobial peptides circulating in the amphipod hemolymph. Thus, we confirmed that the hemolymph of Baikal endemic crustacean E. verrucosus is a source of novel natural products and could have biotechnological value for pharmacy and biomedicine.

Keywords: Baikal, amphipods, endemics, Natural Products, Eulimnogammarus verrucosus

Poster 7.7

Immunomodulatory Activities of Carica papaya Leaf Juice in AG129 Mice Infected with a Clinical Dengue Virus Isolate

Mohd Ridzuan Mohd Abd Razak1*, Nor Azrina Norahmad1, Nur Hana Md Jelas1, Norazlan Mohmad Misnan1, Amirrudin Muhammad1, Bazilah Jusoh1, Tiffiny Ho Chau Dee1, Adiratna Mat Ripen1, Ravindran Thayan1 and Ami Fazlin Syed Mohamed1

1Institute for Medical Research, Ministry of Health (Malasiya), Malaysia

Corresponding author: Dr. Mohd Ridzuan Mohd Abd Razak, eezoo79@gmail.com

Background

Carica papaya leaf has been associated with immunomodulatory activities by several studies but limited to chemically induced inflammatory in vitro and in vivo models. Therefore, this study was initiated to investigate the immunomodulatory effect of the C. papaya leaf juice in a mouse model of dengue virus (DENV)-infection.

Methods

A mouse model of DENV-infection was established by intraperitoneal inoculation of clinical DENV-2 isolate in AG129 mice, lacking type I and II interferon receptors. The infected mice were orally treated with 500 mg/kg/day and 1000 mg/kg/day of freeze-dried C. papaya leaf juice for three days starting on day 1 post infection. The platelets and leukocyte components were counted by microscopy technique. Plasma cytokines and chemokines levels were determined by multiplex immunoassay technique. The level of viral RNA, cytokines and chemokines in the liver, kidney, spleen and brain tissues were determined by quantitative reverse transcription PCR

Results

The inoculation of DENV-2 clinical isolate caused a symptomatic but non-lethal illness to AG129 mice accompanied by the production of proinflammatory cytokines and chemokines in the plasma and organs tissues. Treatments with 500 mg/kg and 1000 mg/kg of freeze-dried C. papaya leaf juice increased the platelet and leukocyte counts in DENV-2 infected AG129 mice. A significant decrease (p<0.05) in viral RNA level was detected in the liver and kidney of infected AG129 mice treated with 1000 mg/kg of freeze-dried C. papaya leaf juice. C. papaya treatment also significantly decreased (p<0.05) the levels of certain cytokines and chemokines in plasma, liver and kidney tissues of infected AG129 mice.

Conclusion

The findings suggest the beneficial effect of C. papaya leaf juice in dengue treatment could be due to its immunomodulatory properties. Hence, identification of compounds that are responsible for the immunomodulatory activities of C. papaya leaf is in need.

Keywords: Carica papaya, Antiviral activity, dengue virus infection, AG129 mice, Immunomodulatory activity, cytokine

Poster 7.8

Antibacterial Effects of Gallic Acid Loaded Graphene
Oxide (GAGO) against Methicillin-Resistant
Staphylococcus aureus (MRSA)

Nurhuda Elias1, Seri Narti Edayu Sarchio2, Faizah Md Yasin3, Shafinaz Abd Gani1 and Suhaili Shamsi1*

1Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Malaysia
2Department of Biomedical Sciences, Faculty of Medicine and Health sciences, University of Putra Malaysia, Malaysia
3Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, Malaysia

Corresponding author: Dr. Suhaili Shamsi, sh_suhaili@upm.edu.my

Background

Staphylococcus aureus strains known as “superbugs” are exceptionally rigid and can quickly develop protective mechanisms against synthetic antibiotics. One increasingly common superbug is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to most available antibiotics. With the ongoing dearth in antibiotics development, the design and identification of alternative classes of antibacterial agents with new modes of action that can effectively overcome multidrug resistance (MDR) is more pressing than ever. The present study aims to investigate the potential of graphene oxide (GO), a biocompatible and cost-effective graphene derivative, with promising antibacterial properties, and gallic acid, a phenolic compound with myriad health benefits, including antibacterial activity, combined together by means of nanoformulation (GAGO) to reverse the MDR in MRSA, hence enhancing antibacterial efficacy. The present study evaluated the interaction of GAGO with MRSA, and its capacity to exert antibacterial activity against MRSA.

Methods

GAGO was synthesized by physical mixing of GO and GA at 1: 5 ratio (GO: GA) and was characterized for morphology, chemical characteristics and GA content. The interaction of GAGO with MRSA was investigated by evaluating its antibacterial activity through disc diffusion and minimum inhibitory concentration (MIC) assays.

Results

GA was successfully loaded onto GO (GAGO) with a loading efficiency of 29.11% that resulted in a GA loading of 886 mg/g of GAGO nanocomposite. The GAGO obtained was in the form of black thin film and FESEM image showed the thickness of the GAGO structure, as compared to only GO which indicates the loading of GA. The antibacterial activity of GA against MRSA increased significantly at lower concentration when loaded onto GAGO nanoformulation (MIC: 150 µg/mL to 100 µg/mL) due to the presence of GO.

Conclusion

Fundamentally, the results demonstrated the potential of GAGO as a promising antibacterial agent against MRSA.

Keywords: graphene oxide, Gallic Acid, Antibacterial, multi-drug resistance, MRSA

Poster 7.9

Umbilical Cord Blood of CRP, Procalcitonin And IL-6 Cord Related with Funisitis in Premature Rupture of Membrane (Consideration for Early Treatment with Empiric Antibiotic)

Julian Dewantiningrum1, 2*, Herman Kristanto1, 2, Besari A. Pramono1, 2, Dik Puspasari1, 2 and Agoes O. Poerwoko1, 2

1Diponegoro University, Indonesia
2Dr. Kariadi Hospital, Indonesia

Corresponding author: MD. Julian Dewantiningrum, lia.percy@gmail.com

Background

Premature rupture of membrane (PROM) occurs in about 1% of all pregnancy and 30% of all preterm deliveries, and its exact etiology has not been completely elucidated. PROM is related to high incidence of funisitis which could result in neonatal sepsis. Funisitis is inflammation of the umbilical cord which is a sign of FIRS (Fetal Inflammatory Response Syndrome). FIRS is caused by microbial invasion. Microbial invasion of chorion and amnion will cause fetal invasion involving funisitis. The incidence of funisitis is not known. A study showed the incidence of funisitis in 88 of 1316 subjects.(1) Funisitis is often associated with long-term complications, such as chronic lung disease and neurodevelopmental disorders in the neonate.(2,3) In addition, it is also associated with short-term complications of neonatal infection, treatment in the NICU and a low apgar score.(4) Administration of antibiotics will reduce the incidence of chorioamnionitis which will further reduce the incidence of funicitis.(5) So it is important to examine biomarker on cord blood which not invasive for neonates and can be obtained in a relatively short time than when waiting for the results of histopathological examination or culture. Biomarkers such as high sensitivity C-Reactive Protein (hsCRP), procalcitonin and Interleukin 6 (IL-6) were hypothesized playing critical role in funisitis. This study was aimed to investigate whether those biomarkers are related to funisitis.

Methods

An observational cross sectional study of all pregnant women with PROM who gave birth at the Obstetric and Gynocology department of dr. Kariadi General Hospital and Diponegoro University’s network hospital on a period of time that met inclusion and exclusion criteria. Inclusion criteria were singleton and 34 weeks of gestation. Exclusion criteria were fetal anomaly and medical problem during pregnancy. As soon as the baby was born, cord blood and umbilical cord were taken. Cord blood was examined for procalcitonin, hsCRP and IL-6. The cord is histopathologically examined to find out whether there was funisitis or not.

Results

The study sample included 88 pregnant women with PROM. Table 1 and 2 showed characteristics of subjects hsCRP and IL-6 blood concentrations were shown to be increased in the funisitis group, but not procalcitonin on table 3. Compared to non-funisitis group, procalcitonin umbilical blood level in the funisitis group was 1,08(0,45) vs 0,86(0,57) ng/mL (p = 0,051), IL-6 level was 63,55 (9,34-454,82) vs 2,53 (0,09-18,22) pg/mL (p < 0,001), hsCRP level was 1,61 (0,07-10,05) vs 0,17 (0,03-10,5) mg/L (p < 0,001).

Discusion

Funisitis was associated with an increase of IL-6 and hsCRP, but it was not associated with procalcitonin levels. Yoon BH et al found the relationship between increased CRP and funisitis.(6) Tasci Y et al states that increased IL-6 was also associated with funisitis.(7) However, this study is different from the study of N joram et al which showed an increase in procalcitonin with funisitis.(8) The weakness of this study was intraamnion bacterial invasion has not been demonstrated by amniotic fluid culture.

Conclusion

hsCRP and IL-6 umbilical blood levels could be used as biomarkers for funisitis. These markers might be used for early detection of infection, so early treatment with empiric antibiotic can be done. Procalcitonin was not related with funisitis.

Keywords: IL-6, PROM (pregnancy), hsCRP, Umbilical cord (UC), Procalacitonin

Poster 7.10

Isolation and Antimicrobial Activity of Probiotic Strains for Caries Prevention

Nor Zaihana Abdul Rahman1*, Rohazila Mohd Hanafiah1, Siti Aisyah Abdul Ghafar1and Nurul Iffah Abdul Rashid1

1Department of Basic Sciences & Oral Biology, Faculty of Dentistry, Universiti Sains Islam Malaysia, Malaysia

Corresponding author:Dr. Nor Zaihana Abdul Rahman, zaihana@usim.edu.my 

Background

Probiotics can be described as live microorganism which when consumed in adequate amounts, it will confer health benefits to the host. The benefits arising from probiotics has been mostly shown on gut health. However, its function to oral health is still poorly investigated. The aim of this study was to isolate lactic acid bacteria (LAB) from local fermented food and to determine its antibacterial activity, in order to select candidate probiotic strains for preventing caries.

Methods

The probiotic strains were isolated from five local fermented food which are tapai ubi, tapai pulut, rebung, thosai and taucu. Ten-fold serial dilutions of the fermented food samples were made in sterile peptone water before plating on de Man Rogosa and Sharpe (MRS) agar. The pure cultures were then randomly picked and biochemically identified. Then, the antibacterial activity of LAB against Streptococcus mutans were assessed by using disk diffusion method.

Results

A total of 120 LAB were isolated from five different fermented foods. The morphology of the isolates were circular, convex, dull opaque white or translucent white. Gram-staining identification showed that the isolates were Gram-positive rods. Of the 120 LAB isolates, 6 strains displayed moderate to strong antibacterial activity against S. mutans with the inhibition zones ranging from 7-12 mm.

Conclusion

The antibacterial activity demonstrated from LAB isolated from local fermented foods suggests that the isolates from local fermented foods possess antibacterial properties against pathogen responsible for causing dental caries. These strains are currently investigated in depth to assess whether they can be fully characterized as probiotics.

Keywords: Probiotics, Lactic acid bacteria, Dental Caries, Streptococcus mutans, Oral Health

Poster 7.11

Recombinant Antibodies Targeting HIV-1 Capsid Protein (p24): The Development of Single-Chain Variable Fragment

Siti Aisyah Mualif1, 2*

1University of Technology Malaysia, Malaysia
2School of Biomedical Engineering & Health Sciences, Faculty of Engineering, University of Technology Malaysia, Malaysia

Corresponding author: Dr. Siti Aisyah Mualif, aisyahmualif@utm.my

Background

Human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS) disease. The increasing morbidity and mortality due to HIV/AIDS in the past decades has sparked an interest to combat HIV/AIDS. However, ongoing research against the HIV/AIDS pandemic has failed to completely eradicate the infection. Introduction of highly active anti-retroviral therapy (HAART) in early 1990s has reduced the death rate of HIV/AIDS, but it has also resulted in the development of drug-resistant strains of HIV. Therefore, there is a pressing need to develop new and improved therapeutic modalities. HIV-1 capsid protein (p24) plays important roles in both early and late stages of HIV-1 replication. Small molecule inhibitors and peptides targeting p24 have shown to inhibit viral infection. However, rapid clearance and toxicity are major drawbacks associated with the aforementioned therapeutic modalities. The potential of monoclonal antibodies (mAbs) targeting p24 was discovered and found out that p24-targeting antibodies can be developed into novel therapeutic modalities. However, natural or hybridoma-derived mAbs are large molecules and difficult to engineer. Recombinant DNA technology allows the engineering of antibodies in multiple formats. Therefore, the aim of this study was to generate, characterize, and evaluate recombinant antibodies targeting HIV-1 p24.

Methods

Recombinant anti-p24 antibodies were generated from a combinatorial library of variable domains cloned from a hybridoma cell line and subsequently expressed on the surface of filamentous bacteriophage. Recombinant scFvs reacting specifically with HIV-1 p24 were isolated, expressed, and characterized.

Results

Out of 50 clones, three specific binders were identified via initial ELISA screening. Specificity of the binders was confirmed through competition studies and the selected clones were expressed in E. coli. The recombinant scFvs markedly inhibited p24 polymerization in vitro and HIV replication in Jurkat T cell lines when expressed as intracellular antibodies (intrabodies).Out of 50 clones, three specific binders were identified via initial ELISA screening. Specificity of the binders was confirmed through competition studies and the selected clones were expressed in E. coli. The recombinant scFvs markedly inhibited p24 polymerization in vitro and HIV replication in Jurkat T cell lines when expressed as intracellular antibodies (intrabodies).

Conclusion

The anti-p24 scFvs engineered in this study have potential to be developed into novel antibody-based therapeutics against HIV.

Keywords: HIV-1 p24, scFv, Phage display antibody library, anti-p24 scFv, therapeutic

Poster 7.12

Phytochemical investigation and evaluation of antimicrobial activity of different extracts of Swietenia macrophylla barks

Velaga V. Appalaraju1, 2* and Lam Kok Wai3*

1Mahsa University, Malaysia
2Faculty of Pharmacy, Mahsa University, Malaysia
3Faculty of Pharmacy, National University of Malaysia, Malaysia

Corresponding authors: Dr. Velaga V. Appalaraju, velagaappalaraju@gmail.com and Dr. Lam Kok Wai, david_lam@ukm.edu.my

Background

The plant Swietenia macrophylla possesses therapeutic uses like antibacterial, antifungal, antidiarrhoeal, anti-inflammatory, hypoglycaemic, antioxidant, cytotoxic, antimalarial and antidiabetic activities. The purpose of this study is to determine the phytochemical composition and antimicrobial activity of two different extracts of S. macrophylla bark against six bacterial strains.

Methods

Swietenia macrophylla bark powder was extracted by Soxhlet extraction method by using acetone and methanol. The extract was characterised by Shinoda test, Fehling’s test, Wagner’s test and Biuret test. Different concentration (10 and 20 mg/mL) of plant extracts were tested by agar well diffusion method by using bacterial strains Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus vulgaris and compared with tetracycline (0.05 mg/mL).

Results

Extracts of S. macrophylla barks revealed the presence of flavonoids, alkaloids, phenolic compounds, carbohydrates, proteins, fats and oils. The most susceptible bacterial strains were P. aeruginosa and S. aureus for methanol extract and acetone extract respectively. Antibacterial screening with acetone and methanol extracts showed both significant (p<0.05) and not significant (p>0.05) zone of inhibition. The MIC was found in the range between 0.63 to 2.50 mg/mL. Extracts of S. macrophylla barks revealed the presence of flavonoids, alkaloids, phenolic compounds, carbohydrates, proteins, fats and oils. The most susceptible bacterial strains were P. aeruginosa and S. aureus for methanol extract and acetone extract respectively. Antibacterial screening with acetone and methanol extracts showed both significant (p<0.05) and not significant (p>0.05) zone of inhibition. The MIC was found in the range between 0.63 to 2.50 mg/mL.

Conclusion

Swietenia macrophylla bark showed that it has broad spectrum of antimicrobial activity that appear as potential alternative therapeutic agent against certain microorganisms in future drug development.

Keywords: Swietenia macrophylla, Antimicrobial activity, Agar well diffusion, Zone of inhibition, minimum inhibitory concentration

TRACK 8: MENTAL HEALTH

Poster 8.1

Evaluation of Opioid Withdrawal After Drug Cessation in Morphine and Mitragynine Dependent Rats 

Rahimah Hassan1, Sharif M. Mansor1 and Zurina Hassan1*

1University of Science, Malaysia, Malaysia

Corresponding author: Dr. Zurina Hassan, zurina_hassan@usm.my

Background

Mitragynine is an indole alkaloid found in Mitragyna speciosa or known as Kratom. It is native in Southeast Asia and originally used for its therapeutic effects and as a substitution in self-treatment of opiate addiction. However, mitragynine consumption develops drug dependence. Hence, the present study aims to evaluate drug dependence after drug cessation via spontaneous withdrawal symptoms in morphine and mitragynine dependent rats.

Methods

Morphine dependent rats were developed by increasing the doses (mg/kg and shown in bracket) twice daily for 6 consecutive days: 1st day (0, 10), 2nd day (10, 10), 3rd day (20, 20), 4th day (30, 30), 5th day (40, 40), 6th day (50, 50). Morphine treatment was disrupted on day 7 in order to induce spontaneous opiate abstinence. For mitragynine dependent rats, 30 mg/kg were given daily for 14 days and disrupted on day 15. The assessment of physical dependence of morphine, mitragynine and control groups were based on motor and vegetative signs. The signs of spontaneous withdrawal symptoms were recorded for 30 minutes in an open field test chamber, 24 hours after the last dose for 28 days. The ‘counted signs’ and ‘checked signs’ were multiplied with the respective ‘weighing factors’ for the evaluation of the severity of abstinence syndrome.

Results

The morphine dependent rats develop opioid withdrawal by significantly increase the mean aggregate value of spontaneous withdrawal symptoms. The morphine withdrawal symptoms last for 27 days after drug cessation. However, the spontaneous withdrawal symptoms of mitragynine dependent rats reduced by half on the 2nd day after drug cessation and last for 12 days.

Conclusion

Spontaneous withdrawal symptoms in mitragynine dependent rats was found to be resolved in shorten period compared to morphine dependence rats.

Keywords: mitragynine, Morphine, Opioid withdrawal, Drug cessation, spontaneous withdrawal symptoms

Poster 8.2

Students and Depression: What Do They Know?

Nadiatul Azra Ahmad Mazlan1* and Chee Ming Chua1

1Faculty of Pharmacy, Mahsa University, Malaysia

Corresponding author:  Dr. Nadiatul Azra Ahmad Mazlan, azra@mahsa.edu.my

Background

Studies found young populations at the age of 16-24 years old undergo the supreme rate of acute and chronic suicide which is approximately 10-26%. Low to minimal level of mental health literacy and stigmatisation may lead to higher depression prevalence. This study aimed to evaluate MAHSA undergraduate pharmacy students’ knowledge on depression.

Methods

A cross-sectional survey was conducted using self-administered pre-validated questions. The questionnaire consists of knowledge on symptoms of depression, pharmacological and alternative medicine and source of information.

Results

There was a total of 183 respondents with two-third were aged 21 – 24 years old and 72.7% were females. Only 29.5% believed that they had at least 25% chances of suffering from depression. Majority of the students only able to identify three out of seven typical symptoms of depression stated by WHO that is “Being sad, down or miserable” (80.9%), “Thinking I’m worthless” (69.9%) and “Being unhappy or depressed” (69.4%). Highest number of respondents (74.3%) agreed that psychotherapy was effective in treating depression, with 48.1% believed in herbs and one-fifth believed in vitamins. More than 80% agreed that meditation will certainly help in treating depression. However, despite the increasing trends of suicidal act among students, slightly half of the students (51.9%) ever looked for information about depression and majority of them (74.7%) preferred to search through internet which may be related to uneasiness to discuss this topic in public.

Conclusion

In summary, moderate level of knowledge on depression was noticed among MAHSA undergraduate pharmacy students. Most believed that interpersonal disharmony will create life imbalance which may lead to depression. More than two thirds gave positive response to alternative treatment.

Keywords: Pharmacy, MAHSA, Depression, knowledge, Students

Poster 8.3

Different Concentration of Standardized Ethanolic Orthosiphon stamineus Extract induced Different Behavioral Alterations in Gestationally-stressed Postpartum Rats

Nabilah Othman1 and Nur Hidayah Kaz Abdul Aziz1*

1School of Pharmaceutical Sciences, University of Science Malaysia, Malaysia

Corresponding author: Dr. Nur Hidayah Kaz Abdul Aziz, hidayahkaz@usm.my

Orthosiphon stamineus (OS) has been traditionally used for many ailments but for psychiatric disorders. Rosmarinic acid (RA) is one of the phenolic compounds contained in OS, and it has been shown to have positive antidepressant-like effect on rats in other studies. The objective of this study is to investigate the effects of standardized ethanolic OS extract on the behaviors of rat dams exposed to gestational stress, which is linked to be a factor of postpartum depressive-like behaviors in rodents. Female Sprague Dawley rats were mated and divided into two groups – stressed and non-stressed. The stressed groups were subjected to chronic mild stress procedure after mating until the day of parturition, whereas non-stressed groups were left undisturbed. After parturition, the stressed group were further divided into 4 groups – non-treated, amitriptyline-treated 20mg/kg, OS-treated 100mg/kg (OS100) and OS-treated 200mg/kg (OS200). Treatments were given via oral once daily until the end of the behavioral tests. After weaning of pups, all rat dams were tested for locomotor activity and anxiety-like behavior in open field test, whereas depression-like behavior was assessed in forced swim test (all groups n: 7-10). In open field test, one-way ANOVA analysis showed that treatments with amitriptyline and both OS concentrations significantly increased the locomotor activity of gestationally-stressed postpartum rats, similar to non-gestationally stressed postpartum rats (p<0.05). No significant anxiety-like behavior was observed in any groups. In forced swim test however, only treatments with amitriptyline and OS100 decreased the immobility behavior similar to the control group (p<0.0001). Standard ethanolic OS extract showed ability to reverse the adverse effect of gestational stress on rats depending on the dose given, hence does have the potential as a treatment for depression at postpartum period.

Keywords: Orthosiphon stamineus (Benth.), Open field test (OFT), Forced swim test (FST), gestational stress, Postpartum

Poster 8.4

Lactobacillus plantarum LAB12 enhances cognitive function via modulation of neurotransmitters and neuropeptides in antibiotic- and LPS-challenged rats

Noor Azman Muhammad Syukri 1, 2, Siong Meng Lim1,2, Fei Tieng Lim2 and Kalavathy Ramasamy1,2*

1Faculty of Pharmacy, University Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
2Collaborative Drug Discovery Research (CDDR) Group, Pharmaceutical and Life Sciences Community of Research, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor Darul Ehsan, Malaysia

Corresponding author: Dr. Kalavathy Ramasamy, kalav922@gmail.com

Background

Based on the notion that probiotic lactic acid bacteria (LAB) could potentially correct dysbiosis of microbiota and ameliorate neuroinflammation via the Gut-Brain Axis, this study assessed the effects of LAB12 on memory, expression of major neurotransmitters and neuropeptides.

Methods

Sprague Dawley rats (male, 3 months old) were being administered with an antibiotic cocktail (imipenem, vancomycin, ampicillin, ciprofloxacin and metronidazole) before being challenged with 0.25mg/kg LPS. The rodents were divided into four groups (n=6/group): wild-type, LPS control, antibiotics (ABX) +LPS and ABX+LAB12+LPS.  The rats were subjected to the Morris Water Maze Test. Rat hippocampi were harvested and subjected to biochemical analyses and metabolic assays.

Results

LAB12 improved spatial learning and memory in antibiotic- and LPS-challenged rats.  ABX+LAB12+LPS group was presented with reduced escape latency and escape distance when compared to the ABX+LPS counterpart. In terms of neurotransmitters in the hippocampus, increased serotonin (5HT) and gamma-aminobutyric acid (GABA) was observed in ABX+LAB12+LPS when compared to ABX+LPS. There were also increased neurotensin (NT) and neuropeptide Y (NPY) levels in the hippocampus of ABX+LAB12+LPS when compared to ABX+LPS, indicating enhanced cognitive processing. Whilst mitochondrial complex enzymes (II, III and IV) were impaired in the hippocampus of ABX+LPS rats, supplementation of LAB12 ameliorated this effect.

Conclusion

The present findings implied that L. plantarum LAB12 could potentially improve memory impairment via modulation of neurotransmitters and neuropeptides in the CNS.

Keywords: Lactobacillus plantarum LAB12, memory enhancing, neurotransmitters, neuropeptides

TRACK 9: MISCELLANEOUS II

Poster 9.1

Increased EMMPRIN Serum Levels in Vascular Dementia Positively Correlate with Matrix Metalloproteinase 2

Yuek Ling Chai1*, Christopher P Chen1, Edward HM Koo1 and Mitchell KP Lai1

1National University of Singapore, Singapore

Corresponding author: Yuek Ling Chai, yuekling@u.nus.edu

Background

Vascular dementia (VaD) is primarily attributed to stroke or cerebrovascular disease, and is characterized by changes in blood vessels and surrounding extracellular matrix (ECM) in the brain. Matrix metalloproteinases (MMPs) play an important role in vascular remodeling. However, elevated MMPs in the brain may worsen vascular injury by disrupting the blood-brain barrier, attacking myelin fibers and promoting neuroinflammation. Several stress signals and pathways may trigger the secretion of MMPs, one of which is the extracellular matrix metalloproteinase inducer (EMMPRIN or CD147). While MMPs levels such as MMP-2 has been found to be increased in the cerebrospinal fluid of patients with vascular cognitive impairment, circulating levels of the MMPs as well as EMMPRIN in VaD remain unknown.

Methods

29 non-cognitive impaired (NCI) controls and 38 VaD subjects were included in the present cross-sectional analyses. Comprehensive clinical, neuropsychological and neuroimaging assessments were performed for all subjects. Blood samples were collected, and serum MMP-2, MMP-9 and EMMPRIN levels were measured using sandwich ELISAs.

Results

Serum MMP-2 and EMMPRIN levels were significantly higher in VaD as compared to NCI. After adjusting for vascular risk factors, association between higher EMMPRIN levels and VaD remained significant. Furthermore, serum EMMPRIN levels were positively correlated with MMP-2 levels, among all subjects as well as within VaD subjects only.

Conclusion

Increased EMMPRIN and MMP-2 serum levels suggest ECM and vascular injury, and thus may be a useful clinical biomarker and a potential therapeutic target for VaD.

Keywords: vascular dementia (VaD), Matrix metalloprotease (MMP), MMP-2, MMP-9, EMMPRIN (CD147)

Poster 9.2

Swelling Kinetic Study of Poly(Methyl Vinyl Ether-Co-Maleic
Acid) Hydrogels as Vehicle Candidates for Drug Delivery

Rubhan Chandran1, Eusni Mohd. Tohit1*, Mansor Ahmad2, Tuan Mazlelaa Tuan Mahmood3 and Johnson Stanslas2

1Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia
2Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia
3Faculty of Pharmacy, National University of Malaysia, Malaysia

Corresponding author:Dr. Eusni Mohd. Tohit, eusni@upm.edu.my

Background

Hydrogels have been widely used for various biomedical and pharmaceutical applications due to their biocompatibility, high water content and rubbery nature, which resemble natural tissue. Polyethylene glycol (PEG) crosslinked poly(methyl vinyl ether and maleic acid) (PMVE/MA) hydrogel is widely studied as a vehicle for various types of drug delivery. It has been reported that swelling and diffusion property of hydrogel are important features for their effectiveness. Higher swelling of PMVE/MA hydrogel facilitates greater amount of drug to be delivered. However, delivery of high molecular weight drugs such as ovalbumin and bevacizumab is still a challenge with existing formulation of PMVE/MA hydrogels. This study aims to optimise PMVE/MA hydrogel formulations and determine the swelling kinetics of different hydrogel formulations.

Methods

PMVE/MA hydrogels were prepared by inducing esterification reaction with PEG. Each formulation of hydrogel consists of different concentration and molecular mass of PMVE/MA and PEG. Swelling kinetics of each formulation were studied by calculating % swelling and second order kinetic model was used to calculate the swelling rate constant (Ks) and degree of swelling at equilibrium (Seq). The effect of different foaming agents (Na2CO3 and NaHCO3) on the swelling of hydrogel was also studied.

Results

Our results shows that hydrogels synthesised from higher molecular weight 15% (w/w) PMVE/MA and 7.5 % (w/w) PEG 12,000 have 2200% swelling. The swelling of hydrogel decreased with increasing concentrations of PMVE/MA and PEG. Hydrogel mixture containing PEG 12,000 with longer polymer chains resulted in better swelling compared to PEG 10,000. Meanwhile high concentration of foaming agents (up to 3% w/w) has a positive effect on hydrogel swelling.

Conclusion

The hydrogels formulation containing 15% (w/w) PMVE/MA and 7.5 % (w/w) PEG 12,000 in this study yielded 1.28 times greater swelling compared to previously reported formulation. It is proposed that, this hydrogel would serve as a better vehicle candidate for macromolecular drug delivery.

Keywords: Hydrogel, Poly(ethylene glycol), Swelling kinetic, Drug delivery, poly(methyl vinyl ether and maleic acid)

Poster 9.3

Evaluating the Performance of MM/PBSA for Binding
Affinity Prediction Using G Protein-Coupled Receptor Crystal
Structures

Mei Qian Yau1, Abigail Emtage2 and Jason S. Loo1

1School of Pharmacy, Taylor’s University, Malaysia
2School of Pharmacy, University of Nottingham Malaysia Campus, Malaysia

Corresponding author: Dr. Jason S. Loo, jasonsiauee.loo@taylors.edu.my

Background

The diverse G protein-coupled receptors (GPCRs) are major drug targets. The recently solved GPCR crystal structures offer opportunities to utilize various structure-based computational methods for drug design. Among these methods is Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA). As an end-state method, MM/PBSA provides a compromise of speed and accuracy between docking and free energy perturbation methods in predicting binding free energies. In this study, we evaluated the performance of MM/PBSA, which has been shown to be system-specific, using GPCR crystal structures.

Methods

Crystal structures of 15 Class A GPCRs were obtained from the Protein Data Bank. For each structure, a set of ligands with similar scaffold to the co-crystallized ligand and pKi values ranging from 5-10 were obtained from the ChEMBL database. The ligands were docked into the structures using Schrödinger’s Induced Fit Docking protocol. Molecular dynamics simulations were then performed using GROMACS with the AMBER ff99SB*-ILDN force field. MM/PBSA calculations were subsequently performed using g_mmpbsa, with the solute dielectric constants varied between 1, 2 and 4 to obtain the best correlation.

Results

The Pearson (rp) and Spearman (rs) correlations obtained between the MM/PBSA-predicted and experimental binding free energies ranged from -0.23-0.78 and -0.32-0.79 respectively. With the exception of a single target (5XRA: rp=0.78, rs=0.79), correlations produced by MM/PBSA were generally poor. MM/PBSA improved the correlations in only 7 out of 15 targets when compared to docking scores, which provided correlations in the range of rp -0.43-0.43 and rs -0.44-0.50.

Conclusion

The performance of MM/PBSA in predicting binding free energies using GPCR crystal structures remains relatively weak overall, but may provide good predictions for certain targets and offer marginal improvements over docking scores for some others. When using MM/PBSA to predict binding free energies of GPCR ligands, validation of the method for each target is therefore recommended.

Keywords: MM/PBSA, GPCR, binding free energy, Induced fit docking, molecular dynamics

Poster 9.4

Alginate-based microcapsules facilitate targeted release of Lactobacillus plantarum LAB12 in vivo

Muhamad Fareez Ismail ¹,²,³, Siong Meng Lim ¹,², and Kalavathy Ramasamy ¹,²*

¹Faculty of Pharmacy, University Teknologi MARA (UiTM), 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
²Collaborative Drug Discovery Research (CDDR) Group, Pharmaceutical and Life Sciences Community of Research, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor Darul Ehsan, Malaysia
³Dental and Biomedical Research (DBR) Group, Faculty of Dentistry, MAHSA University, Bandar Saujana Putra, 42610 Jenjarum, Selangor Darul Ehsan, Malaysia

Corresponding author: Kalavathy Ramasamy, kalav922@gmail.com

Background

Microencapsulation can essentially provide probiotic lactic acid bacteria (LAB) a protective barrier against harsh gastrointestinal conditions. Although cell liberation in the intestines is deemed a mandatory prerequisite for effective application, in vivo validation of the delivery of LAB to the target site and subsequent digestibility of encapsulating matrix remains limited.

Methods

This study validated the release and fate of orally administered L. plantarum LAB12 encapsulated in a unique blend of alginate (Alg) and pea protein isolate (PPi) in Sprague Dawley rats (male; 4-6 weeks). Fate of the ingested microencapsulated LAB12 was examined at 1 and 4 hours post-ingestion in the stomach and the large intestines, respectively, by means of confocal imaging. The release of microencapsulated LAB12 were quantitatively assessed in terms of the proportion and relative abundance of the L. plantarum in various gut sections by qPCR.

Results

The microcapsules started to disintegrate in the large intestines 4 hours post-ingestion as evident by the presence of rod-shaped LAB12 and small pieces of polymeric matrix. qPCR confirmed that ingesta harvested from the stomach exhibited very minimal traces (<1 log CFU g-1) of L. plantarum which was unlikely associated with cell burst release from Alg-PPi microcapsules. Relative abundance of L. plantarum indicated an almost complete release of LAB12 from capsules after 16 hours post ingestion at target sites which included the ileum, caecum and colon.

Conclusion

Alg-PPi could serve as an ideal wall matrix that protects LAB12 from harsh gastric conditions and facilitate their release in the target site within the intestinal tract.

Keywords: Lactobacillus plantarum LAB12, microencapsulation, Alginate, Pea protein isolate, Targeted release

Poster 9.5

Kava Lactones Sub-Acute Hepatopathy Alone or in Combination with EtOH

Hemn H. Othman1, 2

1University of Sulaymaniyah, Iraq
2Putra Malaysia University, Malaysia

Corresponding author: Dr. Hemn H. Othman, hemnho759604@gmail.com

Background

Kava is a herbal supplement, known for its recreational use as a mood enhancer, relaxation, as well as pain relief, for centuries. Kava extracts were considered safe alternatives to anxiolytic drugs, documented as possessing numerous other health benefits, and was one of the leading dietary supplements. Presently, kava toxicity appears to be “idiosyncratic.” Despite these warnings, kava continues to be used in the Pacific Island countries as it is believed that the adverse effects are mild or negligible, except for the occurrence of skin lesions.

Methods

The objectives of this study were to examine the sub-acute toxicity effects of different doses of 70% kavalactones gavaged orally in rats, as well as to elucidate the mechanisms of toxicity alone and in combination with ethanol. Thirty three Sprague-Dawley rats, aged 7-8-week-old, weighing about 200±25gm, were administrated with kava (alone and in combination with EtOH) by oral gavage for 21 consecutive days. Rats were equally allotted into eleven groups comprising three animals each, namely control groups {(NCx), (PCx), (EtOH)}, KL alone groups {(200), (400), (600) and (800) mg/kg)} and KL in combination with EtOH groups {(E+200KL), (E+400KL), (E+600KL) and (E+800KL)}.

Results

The most common side effects observed were, abnormal breathing, ataxia, lethargy, loss of appetite, indigestion and loss of coordination, especially in the 800 mg/kg b.w dosage of kava treatment group alone, and in combination with alcohol. In the sub-acute study, there were dose-related decreases in the rates of body weight, feed intake, and water consumption. Gross and histopathological findings revealed that, the liver was abnormal in color, size, consistency and the liver weight significantly increased at a dose of 800 mg/kg, with kava alone and a greater increase in combination with EtOH. Hepatocellular hypertrophy (HP) and necrosis with KCs hyperplasia were observed in the periacinar zone of all rats with kava (800 mg/kg) alone, and severe changes in combination with alcohol. The periportal (Z1) and mid-zonal (Z2) areas of the hepatocytes were less affected as compared to the periacinar zone.

Conclusion

Our results conclude that, ethanol aggravated the sedative and hypnotic activity of kava, and evidently increased toxicity. The histopathological results supported the clinical and biochemical findings, and the severity of hepatic injury is a dose-dependent manner.

Keywords: Kava lactones, Hepatotoxicity, alcohol, Sub-acute, Sprague-Dawley rat

Poster 9.6

Aripiprazole-loaded in Soft Lipidic Non-Lamellar Liquid Crystalline Nanostructures

Intan Diana Mat Azmi1* and Lai Y. Khen1

1Putra Malaysia University, Malaysia

Corresponding author:Dr. Intan Diana Mat Azmi, intandiana@upm.edu.my

Background

Non-lamellar liquid crystalline (LC) nanostructures have recently received attention in application in drug delivery systems, due to their unique well-defined and tunable internal nanostructures. The rich polymorphism of LC nanostructures are considered as a key factor in improving loading efficiency of different types of poorly soluble therapeutic agents. This project is focused on developing Aripiprazole-loaded non-lamellar LC nanostructures, consisted of a binary mixture of soy phosphatidylcholine (SPC) and citric acid ester of monoglyceride (citrem). Aripiprazole is a psychotropic drug that used to treat schizophrenia and bipolar disorder, which showed poor aqueous solubility of less than 0.3 μg/ ml. It is utmost interest to see the potential of non-lamellar LC nanoparticles in improving the loading efficiency of Aripiprazole by entrapping the drugs into their well-order internal nanostructures.

Methods

The investigated drug-free and drug-loaded nanodispersions were prepared based on a lipid ratio of SPC:Citrem (1:1 wt/wt) at 5 wt% lipid concentration via high energy ultrasonication. The characterizations were done to determine the particle size, stability, morphology, encapsulation efficiency and the interaction between the drugs and the nanodispersions.

Results

The results show that Aripiprazole, at three different concentrations (0.1, 0.2, and 0.25 wt%), were successfully loaded into LC nanoparticles where the encapsulation efficiency was all above 92%. The particle size of LC nanoparticles produced was in the range of 154.1-183.4nm, with polydispersity index, PDI 0.103-0.172, and zeta potential of -26.0mV to -29.1mV. Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR) showed that there was a chemical interaction between Aripiprazole and LC nanoparticles, while Transmission Electron Microscopy (TEM) was used to see the morphology and overall size distribution of drug-free and drug-loaded nanodispersions.

Conclusion

This study suggests the potential use of non-lamellar LC nanodispersions as a promising nanocarrier for Aripiprazole delivery, which is expected to improve the pharmacokinetics and stability of this drug.

Keywords: Liquid crystalline nanostructures, aripiprazole, Drug delivery, Schizophrenia, Nanoparticle (NP)

Poster 9.7

Anti-biofilm and Anti-adherence Activities of Spilanthes acmella Leaves Extracts Towards Streptococcus mutans

Rohazila Hanafiah1*, Siti Sarah Azhar1, Luqman Atif Elias1 and Siti Aisyah Abd Ghafar1

1Universiti Sains Islam Malaysia, Malaysia

Corresponding author:Dr. Rohazila Hanafiah, rohazila@usim.edu.my

Background

Streptococcus mutans is the main causative agents of dental caries by developing biofilm and increase adherence activity. Dental caries have becoming more common due to antibiotic resistance towards Streptococcus mutans. Spilanthes acmella is well-known as anti-toothache plant with high medicinal usages. It has been recognized as an important medicinal plant with high demand worldwide. Therefore, the objective of this study is to establish antimicrobial activities, including anti-biofilm and anti-adherence activity of Spilanthes acmella on cariogenic Streptococcus mutans.

Methods

Spilanthes acmella leaves extracts were prepared by serial extraction with n-hexane, dichloromethane, acetone and methanol. Antibacterial activities of these extracts were conducted using disc diffusion assay, anti-biofilm and anti-adherence activities methods.

Results

The result indicates that n-hexane and methanol leaves extracts are endowed with anti-streptococcus activity. Both methanol and n-hexane extracts inhibit moderately Streptococcus mutans growth as indicated in the disc diffusion assay. Methanol and a n-hexane extracts inhibit the biofilm formation and adherence activity of Streptococcus mutans.

Conclusion

This study highlights the potential of Spilanthes acmella leaves extract as a new promising anti Streptococcus mutans agents.

Keywords: Spilanthes acmella, Streptococcus mutans, Anti-biofilm, Anti-adherence, anti-bacteria

Poster 9.8

Agrobacterium rhizogenes-mediated hairy root culture of Solanum mammosum (‘terung susu kambing’) for an improved solasodine production through elicitation

Chai Theam Ooi1, 2*, Johnson Stanslas3 and Mahmood Maziah2, 4

1Faculty of Dentistry, Mahsa University, Malaysia
2Institute of Bioscience, University Putra Malaysia, Malaysia
3Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia
4Faculty of Biotechnology and Biomolecular Sciences, Putra Malaysia University, Malaysia

Corresponding author:Dr. Chai Theam Ooi, campbellskysky@gmail.com

Background

The aglycone of steroidal alkaloid, solasodine, which is present in Solanum mammosum, can be readily converted to 16-dehydropregnenolone, a key intermediate for the high-revenue chemical synthesis of useful steroid hormones such as progesterone and cortisone in the pharmaceutical industries. Different strains of Agrobacterium rhizogenes were used to infect S. mammosum, with the expectation to cause alteration to the phenotype and host plant secondary metabolism through the expression of root loci (rol) genes, to generate transformed hairy roots that could produce higher amount of solasodine. Two most responsive transformed hairy root lines both in the aspect of higher biomass growth and solasodine production, hairy root line-ATCC31798 and hairy root line-A4, which were separately induced by A. rhizogenes strain ATCC31798 and A4, were then used for enhancement of solasodine production through elicitation.

Methods

Different concentrations of two separate elicitors, methyl jasmonate (0 to 400 µM) and salicylic acid (0 to 200 µM), were added separately into the liquid culture medium for respective hairy root line to evaluate for the solasodine production.

Results

The addition of methyl jasmonate on day 20 of the culture gave higher growth indices with better solasodine productivity of 3.9 ± 0.4 mg/g dw and 4.1 ± 0.6 mg/g dw for hairy root line-ATCC31798 and line-A4 respectively, when compared to the other time frames tested. Similar observation has also been observed for the additon of salicylic acid; however, the overall solasodine productivity from the two transformed hairy root lines showed 30% lesser than those obtained from methyl jasmonate treatment. Interestingly, the solasodine content obtained from the transformed hairy roots after elicitation was at least ten times higher than the untransformed roots.

Keywords: Agrobacterium rhizogenes, elicitor, Hairy root, Solanum mammosum L., solasodine

Poster 9.9

Characterization of Nano-graphene Oxide Stabilized by Pluronic as a Nanocarrier

Suhaili Shamsi1*, Faizah Md Yasin2, Seri Narti Edayu Sarchio3 and Dharshini Perumal1

1Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Malaysia
2Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, Malaysia
3Department of Biomedical Sciences, Faculty of Medicine and Health sciences, University of Putra Malaysia, Malaysia

Corresponding author:Dr. Suhaili Shamsi, sh_suhaili@upm.edu.my

Background

Since its first discovery, graphene oxide (GO) has emerged as a new carbon-based nanoscale particles due to its extraordinary properties such as high dispersibility, hydrophilicity, large surface area and low production cost. The biological application of GO is however plagued by its poor physical and chemical instability, with a significant toxicity profile in biological milieu. The functionalization of GO with surfactants and polymers are necessary for enhanced stability and biocompatibility. Often, the functionalization of GO involves a complicated series of reactions, organic solvents and harsh synthesis condition, which could limit the yield, increase production cost and aggravate its biocompatibility. The present study aims to develop a simple and cost-efficient, yet stable GO functionalized with Pluronic F127 by means of physical mixing to further advance the potential of GO in biological applications.

Methods

GO functionalized Pluronic F127 (PF-GO) was synthesized by physical mixing of GO and Pluronic F127 at different ratios. This method was preferred for its simplicity, low cost, yield of small nanosized structure and potential for scale up production. The PF-GO dispersion was characterized for size, size distribution, zeta potential, morphology, chemical characteristics and Pluronic content. The stability of the formulation in physiologically relevant media was also investigated.

Results

PF-GO showed a discrete structure with a mean size of 235.9 ± 4.2 nm and a narrow size distribution. The functionalization of GO was successfully observed with the increased zeta potential value of -48.3 ± 0.5 nm to -3.79 ± 0.23 nm, and by the FTIR and UV-Vis analyzes. The loading efficiency of Pluronic F127 was found to be 89 ± 5.6 % with batch to batch consistency. The PF-GO was also stable upon storage at room temperature and dispersion with physiologically relevant media.

Conclusion

The collective data demonstrated the effective stabilization of GO with Pluronic F127 copolymers, which could result in a greater potential for PF-GO as nanocarrier for biological applications.

Keywords: graphene oxide, Pluronic F127, stability, nanocarriers, characterization

Poster 9.10

Antibacterial activity of silver nanoparticles Clinacanthus nutans (Agnp-CN) against Streptococcus mutans

Siti Aisyah Abd Ghafar1*, Nor Baitie Adura Muhd Fudzi1, Wan Nur Farhanah Wan Sulaiman1, Rohazila Mohamad hanafiah1 and Vuanghao Lim2

1Department of Basic Sciences & Oral Biology, Faculty of Dentistry, Universiti Sains Islam Malaysia, Malaysia
2Cluster of Integrative Medicine, Advanced Medical and Dental Institute, University of Science, Malaysia, Malaysia

Corresponding author: Dr. Siti Aisyah Abd Ghafar, aisyahghafar@usim.edu.my

Background

Clinacanthus nutans was found to possess anti-venom, anti-inflammatory, analgesic, anti- diabetic, anti-rheumatism, antiviral and antioxidant properties. Silver nanoparticles are nanoparticles between 1nm to 100nm in size and play significant role in medicinal fields. Silver nanoparticles exhibit distinctive properties, such as good conductivity, chemical stability, catalytic and antibacterial activity. Streptococcus mutans is commonly found in human oral cavity and the main contributor to tooth decay. There is no study on antibacterial effect of silver nanoparticles Clinacanthus nutans (AgNp-CN) against Streptococcus mutans reported to date. Therefore, objective of this study is to investigate antibacterial properties of silver nanoparticles Clinacanthus nutans against Streptococcus mutans.

Methods

Streptococcus mutans was subcultured in brain heart infusion (BHI) broth and agar. AgNp-CN with different concentrations was tested against Streptococcus mutans via disc diffusion assay, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Amoxicillin was used as positive control while DMSO and blank silver nanoparticles were used as negative control.

Results

Disc diffusion assay shows AgNp-CN inhibit Streptococcus mutans growth. AgNp-CN shows the greatest inhibition properties (15.3±1.53mm) in comparison to Clinacanthus nutans leaves extract (6.0±0.00mm) and blank silver nanoparticles (6.0±0.00mm). MIC value for AgNp-CN is 2.5mg/mL while amoxicillin is 25mg/mL. Whereas MBC values for AgNp-CN is 2.5mg/mL. Results are concentration dependent, with higher concentration shows better inhibition property.

Conclusion

It can be concluded AgNp-CN possesses bactericidal properties against Streptococcus mutans.

Keywords: Tooth decay, Antibacterial, silver nanoparticles, Streptococcus mutans, Clinacanthus nutans

Poster 9.11

Toxicity level of polyherbal product containing Physta® Tongkat Ali (Eurycoma longifolia) and SLP+® Kacip Fatimah (Labisia pumila) in rodent

Bee P. Teh1*, Elda Nurafnie Ibnu Rasid1, Nor Azlina Zolkifli1, Umi Rubiah Sastu@Zakaria1, Amirrudin Muhammad1, Bazilah Jusoh1, Nor Liyana Mohamed Yusoff1, Azlina Zulkapli1, Juo Y. Sia1 and Norzahirah Ahmad1

1Institute for Medical Research, Ministry of Health (Malasiya), Malaysia

Corresponding author: Bee P. Teh, tehbp@imr.gov.my 

Background

Capsule blend containing Physta® Tongkat Ali (TA) and SLP+® Kacip Fatimah (KF) extracts is proposed to improve general health and well-being. Both Eurycoma longifolia & Labisia pumila plants are found in Malaysia and traditionally used to improve blood circulation, promote general health, overcome fatigue, and increase body strength. We aimed to evaluate the no-observed-adverse effect level (NOAEL) of this polyherbal formulation.

Methods

The sub-acute 28-day study was conducted according to Organization for Economic Cooperation and Development principles of Good Laboratory Practice. Both gender Wistar rats served as a control group (received water) while the other three groups were given the polyherbal formulation (TA and KF) at doses 125, 500 and 2,000mg/kg body weight (BW) administered daily via oral gavage for 28 days, respectively. On day 29, blood sampling was done for hematological & biochemical analysis and the rats were sacrificed and the organs collected for histopathological examination.

Results

The general, detailed and functional clinical observations of the rats showed no signs of toxicity. There were no significant changes in BW, food & water intake, gross examination on the bodies & organs, and biochemical & hematological parameters (p>0.05). Histopathological examination of the organs also did not reveal any dose related toxicity. However, an early response to exogenous toxicity was found in a number of organ tissues of some rats in the treated groups.

Conclusion

This study shows the polyherbal formulation does not cause acute toxicity but was found to cause an early response of exogenous toxicity at the higher doses (not all treated rats) after 28 days of treatment. Therefore, the NOAEL for the formulation is suggested to be not more than 2,000 mg/kg/day. The early response to exogenous toxicity highly suggests for longer dosing duration study to be conducted to investigate any further toxicity effect of the formulation.

Keywords: Toxicity, Eurycoma longifolia, Labisia pumila, Physta®, SLP+®

Poster 9.13

Exploring In silico Properties of Human 5-HT3 Receptor As A Potential Target For Smoking Cessation

Muhammad Harith Zulkifli1, Lam Kok Wai1, Shuhaila Mat Shaarani2, Malina Jasamai1 and Nor Syafinaz Yaakob1*

1Drug and Herbal Research Centre, Faculty of Pharmacy, University Kebangsaan Malaysia, Malaysia

2Malaysia Genome Institute, Malaysia


Corresponding author: 
Dr. Nor Syafinaz Yaakob, nsy@ukm.edu.my 

Background

The serotonin or 5-hydroxytryptamine subtype 3 receptor (5-HT3R) is a potential target for the treatment of drug addiction. This study explores possibility of using the 5-HT3R antagonists for the treatment of nicotine abuse by understanding it at the molecular level. However, the structure of human 5-HT3R is not yet fully understood, therefore this study first aimed to construct an in silico functional model of 5-HT3R to have a deeper understanding on the receptor binding and activation/inhibition with ligands such as nicotine and 5-HT3R antagonists like palonosetron and the natural bioactive compound gingerol.

Methods

Homology model of human 5-HT3A receptor was constructed using Swiss Model approach. The model was submitted for scoring by Ramachandran Plot, Verify3D, ERRAT and ProSA. Following preorientation with respect to the lipid bilayer, serotonin docking and addition of charges, lipid bilayer, water molecules and ions forming a system, it was sent for molecular dynamic (MD) simulation using Gromacs 2016. For docking studies, nicotine, palonosetron and 6-gingerol were docked individually into the orthosteric binding site using AutoDock Vina.

Results

Scores of the model were 86.2% (Ramachandran Plot), 45.39% (Verify3D), 93.38 (ERRAT) and -5.51 (ProSA), indicating that the model was good for MD simulation. Docking studies showed that cation-π interaction between nicotine and tryptophan group, multiple aromatic and hydrophobic interactions at tricyclic ring of palonosetron, and interactions of 6-gingerol with R87 and N123 residues were respectively observed similar to other conducted studies.

Conclusion

Construction of an in silico functional model of 5-HT3R is useful in order to understand the interactions between the receptor and various ligands. The model allows elucidation of nicotine action on 5-HT3R as an antagonist, which can possibly be competed with other 5-HT3R antagonists such as palonosetron and gingerol. This positioned the 5-HT3R as a good potential target for smoking cessation strategies either as an alternative or adjunct treatment.

Keywords: Serotonin, 5-HT3 receptor, Nicotine, Gingerol, Palonosetron

 

Poster 9.14

The Lead Poisoning Control in Zamfara and Niger States,
Nigeria: A 2010-2018 Review

Nasir Tsafe Umar-Tsafe1, 2*, Adebola T Olayinka3, 4, Saad Ahmed4, Muhammad S Shehu4, Gaby Poggensi2, Abdulrazaq Habib5, Kabir Sabitu2, 4, Patrick M Nguku2, Abubakar Jafiya2, Mairo Kachalla2, Aishatu Binu Gubio2, Hawwa Inna Muhammad2, Sagir Aliyu6, Bashir Idris1, Bara’atu Shehu1, Abdulrahman Isah1, Halilu Ahmad1, Yusuf Madaro1, Rabi Usman1, Ibrahim Halilu1, Habibu Yalwa1, Hauwa Kolo7, Endie Waziri2, Saheed Gidado2, Mahmud Dalhat2, Benjamin J Mwangombe8, Ruth Olabiyo8, Gbemisola Oloruntuyi8, Abdullahi Zakariyya Yauri8, Balkisu A Shinkafi9, Nasir Sani-Gwarzo7, Zubairu Iliyasu5, Aisha Indo Mamman4, Hassan S Isah4, Shehu Akuyam4, John I. Anetor10 and Mary Jean Brown11

1Ministry of Health, Zamfara State, Nigeria
2Nigeria Field Epidemiology and Laboratory Training Program (NFELTP), Nigeria
3Nigerian Centre for Disease Control (NCDC), Nigeria
4Faculty of Medicine, College of Health Sciences, Ahmadu Bello University, Nigeria
5Bayero University Kano, Nigeria
6Zamfara Environmental Sanitation Agency, Nigeria
7Federal Ministry of Health (Nigeria), Nigeria
8Medicines Sans Frontiers (Nigeria), Nigeria
9School of Science, Federal University of Technology, Nigeria
10College of Medicine, University of Ibadan, Nigeria
11National Center for Environmental Health (CDC), United States

Corresponding author:Dr. Nasir Tsafe Umar-Tsafe, untsafe@gmail.com 

Background

The lead poisoning (LP) disasters in Zamfara (2010) and Niger States (2015), Nigeria, were described as largest in modern times by scope and magnitude. LP due to artisanal gold-ore processing activities, affected children less than five years old (U5) with acute-severe outbreaks. This review provides an update on magnitude, scope, environmental, clinical, safety and other interventions applied to control and prevent further menace.

Methods

Secondary data reviewed on reports, publications from LP outbreaks and related studies (2010-2018): house-to-house cross-sectional, scoping-chain-referral and cluster sampling surveys. These covered 14 and 1 local government areas of Zamfara and Niger States, respectively. Standard interventions were applied by stakeholders based on Lead contamination (LC) values: >400 ppm defined elevated soil lead levels (ESLL), ≥5 and ≥10 µg/dL defined elevated blood lead levels (EBLL), confirming LP in U5 and animals, respectively. LCs were analysed in blood samples from U5s/animals, soil, water, food-items/crops, air, gold-ore materials and other environmental samples, using lead care II, X-ray fluorescence, atomic absorption and inductively-coupled-mass spectrometers. Data were analysed using SPSS, OpenEpi 2.3 and Epi-Info 7.

Results

The highest ESLL, EBLL were >150,000ppm, >700µg/dL (2010) and >550,000ppm, 300µg/dL (2015), in Zamfara and Niger States, respectively. These reduced to 10,000ppm, 30µg/dL and 1,000ppm, 29.7µg/dL (2018). Highest animal-EBLL were >300µg/dL (Zamfara,2010) and >270µg/dL (Niger,2015). LC Levels in other samples were significantly above their respective US-EPA standards. The strongest risk-factor associations between U5s requiring chelation therapy and environmental LCs were significant (OR: 5.8, 95% CI: 1.7, 19.1, P<0.01, Zamfara) and (OR: 32.8, 95% CI: 7.6, 141.9, P<0.001, Niger). Over 10,200 and 281 U5s were screened, with >7,200 and 180 successfully treated of LP, in Zamfara and Niger States, respectively (2010-2018).

Conclusion

LP-based U5 mortality (734-Zamfara and 28-Niger) has ceased. Zamfara LP morbidity-prevalence effectively reduced from >97%(2010) to <3%(2018), Niger’s reduced from 98%(2015) to <1%(2018). LP control program in Niger was so successful, handing-over by December 2018, whereas in Zamfara, >3,000 LP U5s are continuously exposed to LCs. Zamfara disaster remains an emergency, due to ineffective re-contamination control, access to low-cost intervention mechanisms, inadequate Government responses. Recommendations: environmental remediation, chelation therapy, safer mining practices/health education, continuous surveillance, other control and prevention measures.

Keywords: Lead Poisoning, Chelation Therapy, Soil remediation, Zamfara, Niger, Nigeria, EBLL, Safer mining

Poster 9.15

Enhanced Stability and Bioavailability Systems Composed of
Curcumin as a Natural Anticancer Compound

Mansoureh Nazari1*, Ibrahim Aldeeb1 and Amin Malik Shah Abdul Majid1

1University of Science, Malaysia, Malaysia

Corresponding author: Dr. Mansoureh Nazari, nazarimansoure@gmail.com

Background

In recent decade, abundant improvements have been made in order to develop novel drug delivery system especially for phytoconstituents. Curcumin is a phytoconstituent with wide range of antimicrobial and anticancer activities. However its bioavailability in plasma is very low because of its limited intestinal uptake and fast metabolism. The aim of this review is to identify different types of delivery systems to improve curcumin oral bioavailability.

Methods

This article reviews different types of nanotechnology-based delivery systems such as liposomes, inclusions, micelle and emulsions to increase the bioavailability and pharmacological efficacy of curcumin. Such delivery systems can be made using thin film method, extrusion method, homogenizer and super-critical method, freeze-drying method and ultrasonic method.

Results

Different types of curcumin delivery systems in this review have been discussed. Liposomal vesicle of curcumin using lipids has shown promising results in its delivery. In vivo pharmacokinetics analysis using liposomal vesicle of curcumin revealed 4.96 times enhanced bioavailability of curcumin in plasma with more stability compared with non-formulated curcumin.

Conclusion

The review demonstrated that liposomal vesicle of curcumin among other types of delivery systems is the easiest and more economic system to prepare in order to enhance its bioavailabity and pharmacological efficacy.

Keywords: Curcumin, stability, Liposome, Administration, Cancer

Poster 9.16

Co-delivery of Ruthenium Polypyridyl Complex (RPC) and Cisplatin for the treatment of cancer

Haslina Ahmad ¹*, Nur Aininie Yusoh ¹, Siti Norain Harun ¹ and Chia Suet Lin 2 

1Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
²Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43000 UPM Serdang, Selangor, Malaysia

Corresponding author: Haslina Ahmad, haslina_ahmad@upm.edu.my

Background

Ruthenium Polypyridyl Complex (RPC), which is a metal complex has been investigated as a potential anti-cancer agent for the treatment of various cancer. However, information pertaining to the effects of combining RPC with existing chemotherapeutic drugs remain scarce. This study aimed to investigate the possible synergistic cytotoxic effects of using RPC in combination with cisplatin on different cancer cell lines.

Methods

A549, MCF7, Hela and T24 cells were treated with different concentrations of RPC or cisplatin alone, as well as different combinations of these two agents at a fixed ratio 1:1 over the course of 72 hr to assess their individual and combination effects. Cell viability was analysed using MTT assay. The combination index (CI) was calculated based on the Chou Talalay Method.

Results

Single-agent treatment at 72 hr with RPC or cisplatin led to dose-dependent decreases in the viability of the A549, MCF7, Hela and T24 cells at 72 hr. Furthermore, increasing the concentrations of the combinations up to four folds of half maximal inhibitory concentration (IC50) statistically decrease the cell survival rates of A549 and MCF7 cells thus, displayed synergistic effects.

Conclusion

Treatment of MCF7 and A549 cells with a combination of RPC and cisplatin showed a synergistic effect and thus are promising as an alternative for cancer treatment.

Poster 9.17

Association between Sphingosine-1-Phosphate and Bone Characteristics in Men and Post-menopausal Women in Malaysia

Nasrin Shahifar1, Subashini C. Thambiah1, Siti Yazmin Zahari Sham1, Salmiah Md Said2, Swan S. Yeap3, 4, Fen L. Hew3, 4 and Intan Nureslyna Samsudin1*

1Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia
2Department of Community Health, Faculty of Medicine and Health Sciences, Putra Malaysia University, Malaysia
3Puchong Specialist Clinic, Malaysia
4Subang Jaya Medical Centre, Malaysia

Corresponding author: Dr. Intan Nureslyna Samsudin, intanlyna@upm.edu.my

Background

Prevention of osteoporotic fracture requires identification of individuals at high risk of fracture. There remains an inadequate predictive discrimination ability of current tools to identify those at risk. Sphingosine-1-phosphate (S1P) being a novel marker of bone metabolism may complement the current model. We determined the association between plasma S1P with sociodemographic factors, clinical characteristics, bone mineral density (BMD) and blood tests for bone profile among healthy adult residents of Puchong and Kajang, Malaysia.

Methods

A cross-sectional study was carried out involving Chinese subjects aged 50-90 years old with no previous history of osteoporosis who attended a health screening program in Puchong Specialist Clinic. Each subject had a BMD determined by a dual-energy x-ray absorptiometry (DXA) scan. Fasting blood samples were analysed for 25-hydroxyvitamin D, parathyroid hormone (iPTH), calcium, phosphate, procollagen type 1 amino-terminal propeptide (P1NP), carboxy-terminal collagen crosslinks (CTX) and S1P. Pearson’s and Spearman’s correlation tests were used to determine the associations between S1P with sociodemographic factors and bone characteristic parameters.

Results

There were 45 (34.4%) males and 86 (65.6%) post-menopausal women with median age of 65 (IQR=17) years old. Osteopaenia and osteoporosis were noted in 46.6% and 29.0% of study subjects, respectively. The S1P levels in men (2.12±0.75 µmol/L) and post-menopausal women (1.96±0.68 µmol/L) were not statistically different (p=0.235). S1P had a moderate negative correlation with iPTH (r=-0.232; p=0.008) but not with the other laboratory parameters, age, body mass index and individual BMD values at various sites in both men and post-menopausal women.

Conclusion

Osteoporosis was diagnosed in almost 30% of subjects. S1P levels did not differ between genders. It did not correlate with either of the bone turnover markers (P1NP and CTX) nor with any BMD values at various sites. S1P levels in the subjects were however low which may suggest that the risk of future osteoporotic fracture is low.

Keywords: Sphingosine-1-phosphate, Osteoporosis, osteopenia, Men, post-menopausal women

Poster 9.18

Estrogen-like Activity and LC-MS Characterization of Traditionally Used Achyranthes aspera L. in Gynecological Problems in Bangladesh

AKM M. Huq1*, Johnson Stanslas2, Maulidiani Maulidiani3, Faridah Abas3 and Jamia A. Jamal4

1Southeast University, Bangladesh
2Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia
3Laboratory of Natural Product, Institute of Bioscience, University Putra Malaysia, Malaysia
4Faculty of Pharmacy, National University of Malaysia, Malaysia

Corresponding author: Dr. AKM M. Huq, moyeenul@siswa.ukm.edu.my

Background

Hormone replacement therapy is typically prescribed in postmenopausal symptoms mediated due to estrogen hormone deficiency. However, it has the possible risk of breast cancer development. Achyramthes aspera is traditionally used in gynecological problems, but its therapeutic potential in terms of estrogenic activity has not been well studied.

Methods

This study was aimed to investigate the estrogenic property of A. aspera on estrogen-responsive MCF-7 breast cancer cell line. Estrogen-dependent cell proliferation was done by MTT cell proliferation assay. The estrogen-responsive marker trefoil factor-1 (TFF1) and progesterone receptor (PGR) gene expression were also performed. Phytochemical characterization was carried out by liquid chromatography-mass spectroscopic (LC-MS) method.

Results

In cell proliferation assay, the MeOH fraction showed the highest proliferation of MCF-7 cells at 100 µg/mL (137.7%, p<0.001) with relative proliferation effect (RPE) value of 96.53% compared to 17β-estradiol at 0.01 µM (136.79%; 100% RPE; p<0.001). The MeOH fraction also significantly (p<0.001) stimulated the expression of estrogen marker genes TFF1 (20.14-23.94 folds) and PGR (10.83-14.84). LC-MS analysis identified the presence of phenolic acid, flavonoid and its glycosides and sterols in the MeOH fraction of A. aspera.

Conclusion

The results indicate that A. aspera possesses estrogenic activity and supports the traditional use in gynecological problems. More study is required to evaluate its potential use in postmenopausal disorders.

Keywords: Estrogenic activity, Achyranthes aspera L., MCF-7 cell, TFF1 and PGR genes, LC-MS Analysis