Current Issue

 

Volume 27; 2019

 

Effects of methanolic plant extracts on cell proliferation and HIF activity under hypoxic condition in vitro
Pheik-Sheen Cheow, Norazizah Shafee, Sien-Yei Liew, Muhajir Hamid
APJMBB 27 (1): 1-9 
Click here to download [PDF] [Supplementary Data]
Low oxygen tension is termed as hypoxia. Hypoxia will lead to transcription of hypoxia-inducible factor (HIF) andregulation of downstream gene expression. Underexpression or overexpression of HIF was found to be responsible for various diseases. Proper regulation of this transcriptional factor will aid in treatment of the related diseases. Nowadays, many different approaches are used to modulate HIF, including the usage of naturally-derived plant extracts. Plant extracts are widely accepted compared to other treatments as they are less harmful to the patient and are widely available. In this study, the cytotoxicity of eight different plant extracts under two different gaseous conditions, hypoxic and normoxic, were examined. We also examined the HIF activity shown by the cells under treatment of various concentrations of plant extracts. All eight plants were dried, blended, extracted using methanol, and evaporated to form crude plant extracts. MTT assay was performed by treating the cells with different concentrations of plant extracts and cell viability was determined. Meanwhile, HIF activity of the cells was evaluated by using single luciferase reporter assay. Relative cytotoxicity shown by the cells was different for each plant extract under the various concentration. Pereskia bleo, Orthosiphon aristatus, and Clinacanthus nutans showed high cell viability, 80% of cell viability, within the range of concentration tested. In contrast, Gynura procumbens, Hydrocotyle sibthorpiodies, Pereskia grandifolia, Strobilanthes cripus, and Melastoma malabathricum showed low cell viability. Most of the cells showed activation of HIF activity when treated with different concentrations of the plant extracts. When cells were treated with high concentrations of plant extracts, inhibition of HIF activity were seen and was correlated with low cell viability after treatment. The most notable part of the study was that more than 100% HIF activation was observed for Clinacanthus nutans. However, the cell viability remained high. This might indicate that Clinacanthus nutans is a promising candidate to activate HIF at a transcriptional level with minimal cytotoxicity.

 

The effect of Malaysian stingless bee, Trigona spp. honey in promoting proliferation of the undifferentiated stem cell
Mohd Amin Marwan Mohamad, Muhammad Alif Mazlan, Muhammad Ibrahim, Afzan Mat Yusof, Shamsul Azlin Ahmad Shamsuddin, Nik Fakhuruddin Nik Hassan, Hussin Muhammad, Muhammad Lokman Md. Isa
APJMBB 27 (1): 10-19 
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Stem cells provide various potential applications in regenerative medicine through its ability of self-renewal and differentiation. Among the various stem cells, dental pulp stem cells (DPSCs) have shown encouraging results in their ability to regenerate. Honey has been used in traditional culture as a natural medicine in supporting wound healing. Yet, very few studies on honey were conducted for its potential as a proliferative agent for stem cells. The aim of this study is to evaluate the stability of two Trigona spp. honeys (1 and 2) added in culture media and its proliferative effect on DPSCs. Both honeys were diluted with standard culture medium through dilution process to prepare the concentrations of 0.01%, 0.04%, 0.10% and 0.25%. DPSCs were treated with the diluted honeys for 24 hours. The proliferative activity was determined through the images taken using an inverted microscope for every six hours. In addition, the MTT assay was conducted to determine the cell viability of DPSCs when treated with both honey 1 and 2 at various concentrations. The results showed a stable culture media added with honey for three days and a dose-dependent proliferative effect of both Trigona spp. honey samples on DPSCs. Optimum proliferative effects were observed at 24 hours for both Trigona spp. honey 1 and 2 on DPSCs. The optimum concentration of Trigona spp. honey 1 was from 0.04% to 0.10% and Trigona spp. honey 2 was below 0.01%. It is concluded that Trigona spp. honey has a promising proliferative effect on DPSCs.

 

Evaluation of acute and sub-acute oral toxicity of the aqueous extract of Aquilaria malaccensis leaves in Sprague Dawley rats
Redzuan Nul Hakim Abdul Razak, Suzanah Abdul Rahman, Asmah Hanim Hamdan, Roszaman Ramli, Muhammad Lokman Md. Isa, Hussin Muhammad, Nik Fakhuruddin Nik Hassan
APJMBB 27 (1): 20-32
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Aquilaria malaccensis or commonly known as ‘gaharu’ is a species of Aquilaria genus and belongs to the Thymelaeaceae family. It is widely distributed in Malaysia, Indonesia, and the Borneo Islands. Traditionally, its leaves were used to relieve bruises and studies have shown that they function as an antioxidant, aphrodisiac, and tranquilizer. Despite its proven beneficial medicinal properties, information regarding its toxicity is limited. Therefore, we performed a safety evaluation on the aqueous A. malaccensis leaves extract (AMAE) in Sprague Dawley rats. The assessment of acute toxicity based on the Organization for Economic Cooperation and Development (OECD) Guideline 420 revealed that AMAE did not influence mortality, clinical appearance, body weight gain, or necropsy findings at a dose of 2000 mg/kg body weight. In the sub-acute toxicity, all doses did not significantly modify the body weight and food and water intake. In male rats treated with 2000 mg/kg, there was a significant reduction in the relative weight of liver. Not only that, an increase in alkaline phosphatase and alanine transaminase was also observed in different groups among the female rats. A significant decrease in the creatinine level was also seen among male rats administered with different doses of AMAE. In both sexes, histopathological analysis had shown abnormalities in the liver and kidney of rats treated at the dose of 2000 mg/kg. In conclusion, the 50% lethal dose (LD50) of AMAE was estimated to be greater than 2000 mg/kg. In sub-acute duration, the findings suggested that AMAE administered orally is slightly toxic at higher doses (2000 mg/kg) and could provoke functional and structural changes in the kidney and liver of rats. Thus, the extract should be used with caution.

 

Impact of CYP3A4 and CYP3A5 single nucleotide polymorphisms on anastrozole-associated adverse events among Malaysian breast cancer patients
Murtala Abubakar, Huay Lin Tan, Venkata Murali Krishna Bhavaraju, Siew Hua Gan
APJMBB 27 (1): 33-42
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The catalytic activity of the cytochrome P450A (CYP3A4) enzyme is reportedly affected by the presence of single nucleotide polymorphisms (SNPs), leading to inter-individual variability in drug efficacy and adverse reactions. CYP3A4 polymorphisms can serve as potential biomarkers for predicting the efficacy of many drugs, including those used in breast cancer treatment. This study was conducted on 94 hormone receptor-positive postmenopausal breast cancer patients who received 1 mg of anastrozole per day. Anastrozole-associated adverse events (AAAEs), such as musculoskeletal adverse events (MSAEs), hot flashes, mood disturbance and vaginal dryness/dyspareunia, were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was performed to determine the allelic frequency of CYP3A4*4, CYP3A4*18A, CYP3A4*18B, CYP3A4*22 and CYP3A5*3. The frequencies of CYP3A4*18A T>C (rs28371759), CYP3A4*18B G>A (rs2242480) and CYP3A5*3 were 0.03, 0.48 and 0.64, respectively. However, no CYP3A4*4 A>G (rs55951658) or CYP3A4*22 C>T (rs35599367) alleles were detected. No significant association was observed between the alleles and the development of AAAEs. We have demonstrated for the first time that CYP3A4*18B G>A is highly prevalent among Malaysian breast cancer patients. The clinical relevance of CYP3A4*18B is currently under investigation by our group.

 

Expression of cytochrome P450 2C9 (CYP2C9) in Escherichia coli and its functional characterization
Boon Hooi Tan, Yan Pan, Uma Devi Palanisamy, Iekhsan Othman, Nafees Ahmed, Beow Chin Yiap, Chin Eng Ong
APJMBB 27 (1): 43-55
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This study aimed to express the major human hepatic drug metabolizing cytochrome P450 (CYP), CYP2C9, together with NADPH cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate its catalytic activities. Co-expression of CYP2C9 and OxR was achieved by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates valsartan and tolbutamide. Results from immunoblotting demonstrated the presence of CYP2C9 protein in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to previously published data. Kinetic parameters, Kmand Vmaxvalues determined from the valsartan and tolbutamide hydroxylase assays, were also concordant with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP2C9 suitable for drug metabolism and interaction studies.

 

Acute gamma irradiated Stevia rebaudiana Bertoni enhanced particular types of steviol glycosides
Miao Si Chiew, Kok Song Lai, Sobri Hussein, Janna Ong Abdullah
APJMBB 27 (1): 56-65
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Stevia rebaudiana Bertoni from Asteraceae family is commercially valuable for its steviol glycosides (SGs) contents, which is 300 times sweeter than commercial sugar. The bottleneck in Malaysia is the lack of suitable stevia varieties that are able to thrive well under her climatic conditions and still produce high SGs. Mutation induction including gamma irradiation is effective in generating genetic variations and developing new plant varieties with desired traits.  This study was aimed to determine the effects of acute gamma irradiation on phenotypic changes and enhancement of SGs contents of Stevia rebaudiana Bertoni variety AKH L1 (herein after will be designated as AKH L1). In vitro shoot tip explants of AKH L1 were subjected to a gamma doses regime of 10Gy to 50Gy, following which phenotypic changes of the irradiated explants and subsequent regenerated plantlets were observed. All irradiated explants exhibited different survival rates, with the lowest at 9.33±8.33% when subjected to 50Gy, while all the control (non-irradiated explants) survived. The LD50 was found to be at 23Gy. Subsequent irradiation of 900 shoot tip explants at 23Gy, produced 468 surviving shoot tips, which were all capable to develop and successfully sub-cultured until the fourth generation, M4. These M4 in vitro mutant plantlets exhibited significant increase in the numbers of leaf (16.07±5.19) and average leaf size (1.12±0.26cm x 0.54±0.15cm). HPLC analysis performed in parallel further revealed the mutant plants contained higher concentrations of stevioside (387.04ppm), rebaudioside A (670.18ppm) and rebaudioside D (106.26ppm) compared to the non-irradiated plantlets, which exhibited 96.87, 194.42 and 28.25ppm, respectively.

 

Effect of liquid medium on shoots amplification, in vitro flowering and ex vitro rooting of Oldenlandia umbellata L. - a dye yielding medicinal herb
Revathi Jayabal, Manokari Mani, Latha Rasangam, Priyadharshini Selvam, Mahipal Singh Shekhawat
APJMBB 27 (1): 66-74
Click here to download [PDF]
Oldenlandia umbellata L. gains importance due to its medicinal properties and the presence of anthraquinones based natural dyes in the roots. Present study describes the effect of Murashige and Skoog’s (MS) liquid medium (full strength) on in vitroregeneration, flower bud induction and ex vitrorooting in O. umbellata.  Shoot segments with 2-3 nodes (each node with 2 axillary buds) served as explants for establishment of cultures. The liquid medium augmented with 2.0 mg L-1 6-benzylaminopurine (BAP) with additives (50 mg L-1 of ascorbic acid and 25 mg L-1 each of arginine, adenine sulphate and citric acid) was effective for shoot bud induction (6.4±0.19 shoots per explant within 2-3 weeks). The shoots were further multiplied (89.3±1.07 shoots, 2-3 weeks) when the shoot clusters obtained from the culture initiation directly transferred to the full-strength MS liquid medium incorporated with 1.0 mg L-1 BAP and 0.5 mg L-1 indole-3 acetic acid (IAA) with additives. Flower buds were induced (12.0±0.15 buds per shoot) when the shoots were cultured on 1.0 mg L-1 BAP and kinetin (Kin, 6-furfurylaminopurine) and 0.5 mg L-1 of IAA at 45 µmol m−2s−1 SFPD (Spectral Flux Photon Density) light intensity for 14/10h (light/dark) photoperiod. The adventitious roots were induced on 1/4 strength MS medium supplemented with 1.5 mg L-1 indole-3 butyric acid (IBA). Ex vitro rooting was achieved (16.0±0.53 roots per shoot) by pulse treatment of the shoots with 300 mg L-1 IBA for 2 min. The in vitroproduced plantlets were acclimatized in the greenhouse and finally translocated to thein vivo conditions with 93% success rate. This is the foremost (use of liquid MS medium) and cost-effective method for large scale multiplication of O. umbellata.

 

Antifungal activities against oil palm pathogen Ganoderma boninense from seaweed sources
Syamimi Diyana Abdul Aziz, Nur Fazirah Jafarah, Suriana Sabri, Mohd Aswad Abdul Wahab, Zetty Norhana Balia Yusof
APJMBB 27 (1): 75-83
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Basal stem rot (BSR) disease is the most devastating disease in oil palm which is caused by a fungal pathogen, Ganoderma boninense. However, to date, there is no reliable control for this disease. This study investigated the antifungal potential of seaweed extracts against G. boninense and screening of the compounds possessing this antifungal activity. Four seaweed species namely cfSargassum oligocystumCaulerpa racemosaCaulerpa racemosa var. lamouroxii and cfHalimeda macrophysa were collected from Teluk Kemang, Port Dickson, Malaysia and their antifungal potential against G. boninense were evaluated. Two solvents with different polarities were used for crude extraction namely methanol and chloroform. Antifungal assay using crude methanolic and chloroform extracts from these seaweed species were carried out at various concentrations using the poisoned food technique. Caulerpa racemosa var. lamouroxii chloroform extract showed strong antifungal activity against G. boninense with 27.44% inhibition of the fungus followed by C. racemosa methanolic extract with 26.92% inhibition of the fungus at the lowest extract concentration of 0.25 mg/mL. The extracts were subjected to Gas Chromatography-Mass Spectrometry analysis and the dominant bioactive compounds detected in both extracts were phytol and l-(+)-ascorbic acid 2,6-dihexadecanoate which were also found in plant extracts showing antimicrobial activities in previous studies. The findings suggested that local Malaysian seaweed species have high potential as a source of antifungal compounds which could be useful specifically for the application in the oil palm industry.

 

Inoculation of fowlpox viruses coexpressing avian influenza H5 and chicken IL-15 cytokine gene stimulates diverse host immune responses
Abdul Razak Mariatulqabtiah, Nadzreeq Nor Majid, Efstathios S. Giotis, Abdul Rahman Omar, Michael A. Skinner
APJMBB 27 (1): 84-94
Click here to download [PDF]
Fowlpox virus (FWPV) has been used as a recombinant vaccine vector to express antigens from several important avian pathogens. Attempts have been made to improve vaccine strains induced-host immune responses by coexpressing cytokines. This study describes the construction of recombinant FWPV (rFWPV) strain FP9 and immunological responses in specific-pathogen-free (SPF) chickens, co-expressing avian influenza virus (AIV) H5 of A/Chicken/Malaysia/5858/2004, and chicken IL-15 cytokine genes. Expression of H5 (50 kD) was confirmed by western blotting. Anti-H5 antibodies, which were measured by the haemagglutinin inhibition test, were at the highest levels at Week 3 post-inoculation in both rFWPV/H5- and rFWPV/H5/IL-15-vaccinated chickens, but decreased to undetectable levels from Week 5 onwards. CD3+/CD4+ or CD3+/CD8+T cell populations, assessed using flow cytometry, were significantly increased in both WT FP9- and rFWPV/H5-vaccinated chickens and were also higher than in rFWPV/H5/IL-15- vaccinated chickens, at Week 2. Gene expression analysis using real time quantitative polymerase chain reaction (qPCR) demonstrated upregulation of IL-15 expression in all vaccinated groups with rFWPV/H5/IL-15 having the highest fold change, at day 2 (117±51.53). Despite showing upregulation, fold change values of the IL-18 expression were below 1.00 for all vaccinated groups at day 2, 4 and 6. This study shows successful construction of rFWPV/H5 co-expressing IL-15, with modified immunogenicity upon inoculation into SPF chickens.

 

About MSMBB

We are a non-profit organisation that was established in 1988 to promote molecular biology and biotechnology.

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Next Event

3rd International Conference on Molecular Biology & Biotechnology

in conjunction with the 26th  Scientific Meeting of the Malaysian Society of Molecular Biology & Biotechnology (MSMBB) & UCSI University's 2nd Applied Science Symposium

Organized by MSMBB & UCSI University

FOSTERING INNOVATION THROUGH RESEARCH

24-25th April 2019, UCSI University Kuala Lumpur

Visit http://bit.ly/3rdICMBB 

 

 

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