CURRENT ISSUE

April 2022

Malay. J. Biochem. Mol. Biol. (2022) 25 (1)

TABLE OF CONTENTS

Page 1- 7

Huynh Nguyen Que Anh, Le Pham Tan Quoc

MICROWAVE-ASSISTED EXTRACTION OF PHENOLIC COMPOUNDS FROM
Polyscias fruticosa (L.) Harms ROOT

Abstract 

Polyscias fruticosa (L.) Harms is an herbal plant that possesses many medicinal uses. The interest in this material and its applications have been increasing rapidly in recent years. However, changes in phytochemical components and antioxidant properties of P. fruticosa roots when using microwave-assisted extraction (MAE) methods have not been well understood. The aim of this study was to determine the best extraction conditions for total phenolic content (TPC) and antioxidant capacity (AC) of P. fruticosa roots using MAE method. Four factors of the extraction process (solvent/material (SM) ratio, solvent concentrations, microwave power, and extraction time) were investigated. DPPH method was used to determine free radical scavenging activities while TPC was estimated by the Folin-Ciocalteu assay. The results pointed to the optimal extraction parameters which were ethanol concentration of 50% (v/v), ethanol/material ratio of 60/1 (mL/g), microwave power of 265 W, and extraction time of 5 min. TPC and AC obtained were approximately 2.31±0.01 mg GAE/g DW and 76.62±0.23%. In addition, the initial material was completely destroyed under microwave treatment. The results also indicated that MAE could be a fast and reliable method for quantitative analysis of phenolic compounds in P. fruticosa roots.

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Page 8-17

Anatolii Onishchenko, Galina Gubina-Vakulyck, Oleksandr Knigavko, Ketino Sharashydze, Olena Pionova, Dmytro Butov, Hanna Polikarpova, Anton Tkachenko

INTAKE OF SEMI-REFINED CARRAGEENAN CAUSES LOW-GRADE COLONIC INFLAMMATION AND ALTERS EXPRESSION OF EPITHELIAL-MESENCHYMAL TRANSITION MARKERS

Abstract 

The aim of this research was to evaluate the effects of semi-refined carrageenan consumed orally on the amount of CD3 and CD68 positive cells residing in the colon and the expression of endothelial-mesenchymal transition (EMT) markers.
Materials and methods. To assess colonic expression of CD3+, CD68+, fascin, vimentin and E-cadherin, sections obtained from 8 rats orally exposed to semi-refined carrageenan (E407a) at a dose of 140 mg / kg of weight during 14 consecutive days and 8 control animals were immunostained with the corresponding antibodies. Levels of expression were assessed quantitatively.
Results. Oral exposure to semi-refined carrageenan resulted in an increase in CD3 and CD68 positive cells in the colonic lamina propria. Quantitative analysis of fascin, vimentin and E-cadherin immunostaining revealed changes in expression of these EMT markers both in the colonic stroma and epithelial cells. Vimentin and fascin were overexpressed in stromal and epithelial cells, while E-cadherin was upregulated in the stroma and downregulated in epithelia.
Conclusions. Our observations suggest that oral intake of semi-refined carrageenan results in the development of low-grade colonic inflammation accompanied by infiltration with CD3 and CD68 positive cells and changes in the expression of EMT markers.

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Page 18-27

Maryam Usman Ahmed, Isaac John Umaru and Ismaila Yada Sudi

TOXICOLOGICAL EVALUATION OF TWO FRACTIONS FROM Annona senegalensis
ROOT BARK

Abstract 

Annona senegalensis root bark is widely used as a medicinal plant for the treatment of a wide array of diseases. This study evaluated the safety of solvent fractions obtained from its aqueous root bark extract. Crude aqueous extract was partitioned into hexane, dichloromethane and ethylacetate fraction by solvent-solvent fractionation. Albino rats were administered 100, 200 and 400 mg/kg b.wt. Dichloromethane and ethylacetate fractions for 14 days. The effect of their administration on liver, kidney, antioxidant enzymes activities, lipid peroxidation and heamatological parameters was investigated. Both fractions significantly increase alanine transaminase activity, Na+ and K+ concentration, glutathione peroxidase activity and malondialdehyde concentration. They fraction significantly decreased urea concentration and serum superoxide dismutase activity. Dichloromethane fraction significantly increase the PCV while there was no significant difference in all heamatological parameters of rats treated with ethylacetate fraction from aqueous root bark (EFAR) when compared with the control. The two fractions are toxic and should be used with caution.

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Page 28-36

Fauziatul Fitriyah, Yora Faramitha, Dini Astika Sari, Irma Kresnawaty

MOLECULAR IDENTIFICATION OF LOCAL CYANOBACTERIUM AND SCREENING FOR BIOHERBICIDE ACTIVITY IN MODEL PLANT

Abstract 

Cyanobacteria produce a wide range of secondary metabolites with promising possible applications in agriculture, human health, or industry. This study aims to identify cyanobacterium using the rbcL gene as a molecular barcode and screen for the herbicidal activity of water and ethanol crude extract of the strain. DNA of cyanobacterium strain was extracted with modified CTAB method and then amplified using 1AB_rbcL primers and sent for sequencing. BLAST analysis showed that the strain belongs to Synechocystis sp. The strain was then cultured in 2 L of f/2 culture medium for harvesting. Harvested Synechocystis sp. was extracted in water and 70% ethanol with the ratio of 1:3 (w/v) in an 80°C water bath for 2 hours and ethanol extract was evaporated at room temperature. The herbicidal activity was assayed by germinating sorghum in 0.5% extract with glyphosate (Roundup) as positive control and water as a negative control in 5 replications. Germination rate, shoot, and root length were analyzed statistically with one-way ANOVA in GENSTAT software, Duncan post hoc analysis was performed. The local axenic isolate of cyanobacterium from Indonesia was identified as Synechocystis sp. based on the rbcL gene sequence. The ethanol extract of Synechocystis sp. promotes germination in sorghum seeds compared to water extract, but both were still lower compared to the sorghum seeds germination under glyphosate-based herbicide treatment. The ethanol extract also showed higher root growth inhibition compared to the water extract, but both extracts had no activity on shoot inhibition of sorghum seedling compared to the negative control.

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Page 37-50

Mei Wei Lai, Woon Ling Chia, Yee Soon Ling, Yen Min Lan, Sheh May Tam. Chung Cheng Richard Kong, Ming Hock Haw, Yang Mooi Lim

MORPHOLOGICAL CHARACTERISTICS, DNA BARCODING AND METABOLITE PROFILING OF Oldenlandia diffusa (Willd.) Roxb. AND Oldenlandia corymbosa L.

Abstract 

Oldenlandia diffusa (Willd.) Roxb. is a well-known traditional Chinese medicinal herb and frequently replaced by Oldenlandia corymbosa L. However, their roles and efficacies are not the same. Therefore, the purpose of this study was to distinguish these two plants based on morphology and DNA barcodes, and to reveal their putative compounds. Wild O. diffusa and O. corymbosa were collected from Johor and Negeri Sembilan in Malaysia, respectively. The morphological characteristics of both plants were recorded during plant collection. DNA barcodes of ribulose-1,5-bisphosphate carboxylase (rbcL), maturase K (matK), and nuclear ribosomal internal transcribed spacer (ITS) were used to identify these plants up to species level. Non-targeted metabolite profiling was performed to determine the putative compound of both plants. Results obtained for rbcL, matK, and ITS DNA barcoding analysis showed 100 % similarity of Oldenlandia corymbosa L. Results obtained for matK DNA barcoding analysis showed 99.64 % similarity of Oldenlandia diffusa (Willd.) Roxb. LC-MS/MS analysis revealed a total of 42 and 12 primary and secondary putative compounds detected in O. corymbosa and O. diffusa extracts, respectively. Overall, we currently recommend the use of both morphological and DNA barcoding for plant identification. In the future, we proposed to design a primer to increase primer specificity and further confirm the structure and biological function of potential putative compounds.

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Page 51-57

Muhammad Fazril Mohamad Razif, Boon-Hong Kong, Yeaw-Khim Yee, Noorain Zulkapli, Nik Nurmadihah Azma Nik Ampuan, Shin-Yee Fung

FERMENTED Morinda citrifolia LINN JUICE HAS REDUCED ANTIOXIDANT CAPACITY AND DEMONSTRATES CYTOTOXICITY TOWARDS HUMAN BREAST CANCER CELLS

Abstract 

Morinda citrifolia Linn (noni) is a plant belonging to the Rubiaceae family. Recently, fermented noni fruit juice has undergone a surge in popularity, driven by its supposed health benefits. The aim of this study was (i) to determine the nutrient composition of fermented noni fruit juice, and (ii) to compare the antioxidant and cytotoxic properties between unfermented (UNJ) and fermented (FNJ) noni fruit juice. The results showed that FNJ is rich in carbohydrates (81.3 g/100 g DW), but low in both protein (5.4 g/100g DW) and fat (4.0 g/100g DW). FNJ also contains high amounts of amino acids (AAs) glutamic acid, aspartic acid and leucine. UNJ exhibited higher free radical scavenging and antioxidant activity than FNJ. UNJ was found to exhibit comparable cytotoxicity across all cells tested. Notably, FNJ demonstrated higher toxicity against normal human breast (184B5) cells (up to 2.6-fold) compared to human breast cancer (MCF7 and MDA-MB-231) cells. To our knowledge, our study is the first to demonstrate that noni fruit juice (both fermented and unfermented) is toxic to normal breast cells. Their toxicity toward normal breast cells and their non-selective targeting may require in-depth in vivo study to reveal their potential therapeutic benefits. Caution is warranted as noni fruit juice products from other sources may possess different toxicological and pharmacological profiles.

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Page 58-67

Mohammad Faruq Abd Rachman Isnadi, Sivan Padma Priya and Tock Hing Chua

PATHOLOGICAL FEATURES OF Plasmodium knowlesi MALARIA INFECTION IN HUMANS AND MACAQUES

Abstract 

Plasmodium knowlesi is a simian malaria parasite and currently the dominant species in the Malaysian Borneo of Sabah and Sarawak. This parasite is transmitted by Anopheles balabacensis and the macaques of Macaca fascicularis and Macaca nemestrina, and monkeys of Presbytis melalophos are the reservoir hosts. The zoonotic disease, infection by P. knowlesi infection can cause a wide range of immunological responses in human comparable to other human malaria parasites including acute respiratory distress syndrome (ARDS), acute renal failure (ARF) and cerebral malaria (CM), leading to the pathological consequences in humans and macaques alike. Similar to other malaria species especially P. falciparum, pathological features such as sequestration of parasitised red blood cells (pRBC) and mononuclear cells-containing haemozoin, deposition of haemozoin on numerous tissues and petechial and/or focal haemorrhage could be observed in P. knowlesi infection. Diagnosis of P. knowlesi mainly involves microscopic examination on both thick and thin blood smears stained with Giemsa, nevertheless confirmation test by using Polymerase Chain Reaction (PCR) is compulsory. Most P. knowlesi cases are treated with artemisinin-combination therapy (ACT) or chloroquine in uncomplicated cases while artesunate followed by ACT in complicated or severe cases. Without accurate and timely means of diagnosis and treatment, the outcomes of death might happen. The pathological features of P. knowlesi malaria infection in multiple organs are described in this review.

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Page 68-76

Shivangi, Manish Kumar Mishra, Sachin Gupta, Konika Razdan, Shashi Sudan and Shelly Sehgal

ADVANCES IN MOLECULAR VIROLOGY AND DIAGNOSTICS OF HEPATITIS C VIRUS

Abstract 

The first and foremost step in the management of any infectious disease, viral or bacterial, is identifying the causative agent behind the disease, followed by determining the extent of incursion attained by it, so as to initiate the specific therapy against and control the exaggerated and self-damaging host reaction. The same applies to the Hepatitis C virus (HCV) infection which is still a global health burden. HCV infection is found worldwide with 71 million people affected globally exhibiting a greater risk of developing advanced-stage liver diseases like fibrosis, cirrhosis and hepatic carcinomas. However, this complex disease state is attained if only the infection is considerably prolonged and left untreated or mistreated. Thus, providing a means to intervene and stop its course, in favour of which an early and accurate diagnosis is required. The timely and correct diagnosis also helps to contain the spread of HCV infection. The basic assays prompting the development of diagnostics are the Enzyme-Linked Immunosorbent (ELISA) and Polymerase Chain Reaction (PCR). On the other hand, recent techniques like microarrays and next-generation sequencing have taken the field of diagnostics to a new phase. Here we present a compilation of all the approaches, recent and older, used in laboratory diagnosis of HCV infection. This review shall help the researchers decide the best method for HCV detection per their requirements and availability of resources.

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Page 77-87

Waqas Ahmad and Muhammad Naeem

ANALYSIS OF EFFICIENCY OF SEVEN GENOMIC DNA ISOLATION TECHNIQUES THROUGH APPLICATIONS OF DNA BARCODE, PCR AND NANODROP FROM FINS OF Notopterus notopterus (PALLAS, 1769)

Abstract 

Notopterus notopterus is a single species of Genus Notopterus. In Pakistan, N. notopterus is an important fish for food and ornamental trade. The unlimited catch and water pollution are major causes of rapid decline in N. notopterus population. However, genetic study is crucial for its conservation. DNA isolation is the first key step of genetic studies. With this objective, we compared the efficiency of seven genomic DNA isolation techniques. DNA was isolated from fins of N. notopterus because fins were used in small quantities and there was no detrimental effect on fish. Isolated DNA concentration and purity were measured with NanoDrop. PCR amplification and barcoding of mitochondrial COI gene were also used to analyse the purity of isolated DNA. Mitochondrial COI was selected because it is universally used genetic marker for genetic studies of species. Among all methods, GeneJET Genomic DNA Purification Kit was found significantly higher in terms of isolated DNA concentration (894 ng.μl-1), yield (178.3 μg.μl-1), purity (1.7 ng.μl-1), successful PCR amplification and barcode sequencing with 612 base pairs of N. notopterus as compared to investigated six traditional DNA isolation methods. GeneJET Genomic DNA Purification Kit was proved the best in terms of cost and labour and the least hazardous for a handler to perform. Present study has also revealed that the traditional DNA isolated methods are the secondary choice for isolation of DNA from fish fins. Moreover, information about the best genomic DNA isolation technique, from this study can be significant for many molecular techniques such as PCR amplification and gene barcode sequencing among others.

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Page 88-92

Yow Hui-Yin, Tang Yin-Quan and Lee Jyy-Shiuan

ANTIPROLIFERATIVE EFFECT OF ASPIRIN AND DICLOFENAC ON MDA-MD-231 TRIPLE-NEGATIVE BREAST CANCER CELLS

Abstract 

The role of nonsteroidal anti-inflammatory drugs in triple-negative breast cancer remains unclear. This study aimed to investigate the antiproliferative effect of aspirin and diclofenac on MDA-MB-231 cells using MTT assay. Aspirin (1 – 100 mM) inhibited cell proliferation at dose- and time-dependent manner. At 24 and 48 h treatment, 1 – 100 mM of aspirin exhibited significant antiproliferative effect (p < 0.05). Only ≥ 5 mM aspirin demonstrated significant effect at 72 h (p < 0.05). Similar trend was found with diclofenac (0.01 – 1 mM). This study highlights the potential of aspirin and diclofenac in the management of triple-negative breast cancer

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Page 93-103

Maulidi Firlandiana, Giyanto, Wilhelmus Terang Arga Sanjaya, Dwi Andreas Santosa

IN SILICO ANALYSIS OF GENE-ENCODED INVERTASE INHIBITOR (Sininh) FROM SEVERAL SUGARCANE VARIETIES

Abstract 

Invertase inhibitor gene fragments have been isolated from various cultivated plants including sugarcane. This article will review the structure of the invertase inhibitor gene (sininh) at 650 bp from multiple sugarcane varieties in Indonesia (PS 881, PS 882, PSJT 941, PS 862, BL/Bulu Lawang, KK/Kidang Kencana). Moreover, the primary structure, physicochemical properties, the secondary structure, 3D structure, and subcellular localization of invertase inhibitor protein (Sininh) were predicted utilizing bioinformatics tools. This information is beneficial for future research, especially in controlling sucrose accumulation at the post-translation level in sugarcane plants. The prediction of the Sininh consisting of 124-145 residues has different characteristics. The predicted Sininh sequences from PS 882 and BL varieties have the potency for translocation due to peptide signals. In addition, there are some Cys residues in both varieties associated with the formation of disulfide bonds for protein structure stability. Sustainable, functional conserved domain Plant invertase/pectin methylesterase inhibitors found in the Sininh protein variety BL/Bulu Lawang where they inhibit the activity of pectin methylesterase (PMEs) invertase with a complex formation. N-glycosylation motifs are also found in the protein Sininh varieties PS 881, PSJT 941, PS 862, BL/Bulu Lawang, KK/Kidang Kencana, which is related to potential stability and interaction with other proteins. These studies build the foundation for studying the structural aspects and the mechanism of the inactivation of invertase via its inhibitory proteins at the molecular level.

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Page 104-113

Siew Wei Lee, Joko Logis, Ayesha Fatima, Renee Lay Hong Lim, Szu Ting Ng, Chon Seng Tan, Shin Yee Fung, Nget Hong Tan, Amos Eng Liang Goh, Yong Hui Tan, Crystale Siew Ying Lim

DYE DECOLORISATION CAPACITY OF Lignosus rhinocerotis (Cooke) Ryvarden DIALYSED FRACTION AND ITS EXPRESSION OF NOVEL RECOMBINANT LACCASE IN Pichia pastoris

Abstract 

Azo dyes are widely used in the textile industry due to their bold colors and resistance to degradation. This has made azo dyes the global burden in wastewater treatment as conventional dye removal methods have their limitations. Laccase has emerged as an alternative due to its ability to decolorise a wide range of dyes, producing water as a by-product. In this study, we aim to assess the dye decolorisation capacity of the Basidiomycota, L. rhinocerotis dialysed fraction and produce recombinant laccase expressed in Pichia pastoris. Out of the three dyes tested, the dialysed fraction of L. rhinocerotis sclerotia was capable of decolorising around 90% of the Congo Red and Coomassie Brilliant Blue dye at the enzyme unit of 0.018 U/mL. The gDNA of Lac1 [MG210944] was successfully updated in GenBank and the sequence was used to design the pPICZαA/Lac vector, which was successfully cloned and the recombinant Lac1 was successfully expressed using the Pichia pastoris system. The recombinant Lac1 expressed contains the 6x His-tag for purification but the binding towards the Ni-NTA resin was weak, therefore the purification can be optimised through reducing Imidazole concentration to compensate for the weak binding. In conclusion, L. rhinocerotis extract showed promising results in decolorising CR and CBB, while recombinant Lac1 laccase successfully demonstrated its activity and can be further used for dye decolorisation in future studies.

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