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Fulltext Assessing product adulteration of Eurycoma longifolia (Tongkat Ali) herbal medicinal product using DNA barcoding and HPLC analysis

Abubakar BM, Salleh FM, Shamsir Omar MS, Wagiran A

Pharm Biol, 2018 Dec;56(1):368-377.
PMID: 30058427 DOI: 10.1080/13880209.2018.1479869

CONTEXT: Eurycoma longifolia Jack (Simaroubaceae) commonly known as Tongkat Ali is one of the most important plants in Malaysia. The plant extracts (particularly roots) are widely used for the treatment of cough and fever besides having antimalarial, antidiabetic, anticancer and aphrodisiac activities.
OBJECTIVES: This study assesses the extent of adulteration of E. longifolia herbal medicinal products (HMPs) using DNA barcoding validated by HPLC analysis.
MATERIALS AND METHODS: Chloroplastic rbcL and nuclear ITS2 barcode regions were used in the present study. The sequences generated from E. longifolia HMPs were compared to sequences in the GenBank using MEGABLAST to verify their taxonomic identity. These results were verified by neighbor-joining tree analysis in which branches of unknown specimen are compared to the reference sequences established from this study and other retrieved from the GenBank. The HMPs were also analysed using HPLC analysis for the presence of eurycomanone bioactive marker.
RESULTS: Identification using DNA barcoding revealed that 37% of the tested HMPs were authentic while 27% were adulterated with the ITS2 barcode region proven to be the ideal marker. The validation of the authenticity using HPLC analysis showed a situation in which a species which was identified as authentic was found not to contain the expected chemical compound.
DISCUSSION AND CONCLUSIONS: DNA barcoding should be used as the first screening step for testing of HMPs raw materials. However, integration of DNA barcoding with HPLC analysis will help to provide detailed knowledge about the safety and efficacy of the HMPs.
Fulltext Review: DNA Barcoding and Chromatography Fingerprints for the Authentication of Botanicals in Herbal Medicinal Products

Mohammed Abubakar B, Mohd Salleh F, Shamsir Omar MS, Wagiran A

Evid Based Complement Alternat Med, 2017;2017:1352948.
PMID: 28536641 DOI: 10.1155/2017/1352948

In the last two decades, there has been a tremendous increase in the global use of herbal medicinal products (HMPs) due to their claimed health benefits. This has led to increase in their demand and consequently, also, resulted in massive adulteration. This is due to the fact that most of the traditional methods cannot identify closely related species in a process product form. Therefore the urgent need for simple and rapid identification methods resulted in the discovery of a novel technique. DNA barcoding is a process that uses short DNA sequence from the standard genome for species identification. This technique is reliable and is not affected by external factors such as climates, age, or plant part. The difficulties in isolation of DNA of high quality in addition to other factors are among the challenges encountered using the DNA barcoding in the authentication of HMP. These limitations indicated that using DNA barcoding alone may ineffectively authenticate the HMP. Therefore, the combination of DNA barcoding with chromatographic fingerprint, a popular and generally accepted technique for the assessment and quality control of HMP, will offer an efficient solution to effectively evaluate the authenticity and quality consistency of HMP. Detailed and quality information about the main composition of the HMPs will help to ascertain their efficacy and safety as these are very important for quality control.
Fulltext Integrated Approach for Species Identification and Quality Analysis for Labisia pumila Using DNA Barcoding and HPLC

Tarmizi AAA, Wagiran A, Mohd Salleh F, Chua LS, Abdullah FI, Hasham R, et al.

Plants (Basel), 2021 Apr 07;10(4).
PMID: 33917172 DOI: 10.3390/plants10040717

Labisia pumila is a precious herb in Southeast Asia that is traditionally used as a health supplement and has been extensively commercialized due to its claimed therapeutic properties in boosting a healthy female reproductive system. Indigenous people used these plants by boiling the leaves; however, in recent years it has been marketed as powdered or capsuled products. Accordingly, accuracy in determination of the authenticity of these modern herbal products has faced great challenges. Lack of authenticity is a public health risk because incorrectly used herbal species can cause adverse effects. Hence, any measures that may aid product authentication would be beneficial. Given the widespread use of Labisia herbal products, the current study focuses on authenticity testing via an integral approach of DNA barcoding and qualitative analysis using HPLC. This study successfully generated DNA reference barcodes (ITS2 and rbcL) for L. pumila var. alata and pumila. The DNA barcode that was generated was then used to identify species of Labisia pumila in herbal medicinal products, while HPLC was utilized to determine their quality. The findings through the synergistic approach (DNA barcode and HPLC) implemented in this study indicate the importance of both methods in providing the strong evidence required for the identification of true species and to examine the authenticity of such herbal medicinal products.
Fulltext Authenticity Testing and Detection of Eurycoma longifolia in Commercial Herbal Products Using Bar-High Resolution Melting Analysis

Fadzil NF, Wagiran A, Mohd Salleh F, Abdullah S, Mohd Izham NH

Genes (Basel), 2018 Aug 12;9(8).
PMID: 30103564 DOI: 10.3390/genes9080408

The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) and the nuclear ribosomal region- internal transcribed spacer 2 (ITS2), PCR amplification and HRM analysis using saturated Eva green dye as the source of fluorescence signals, was accomplished by employing a real-time cycler. The results were further validated by sequencing to identify unknown sequence from Genbank database and to generate phylogenetic tree using neighbour joint (NJ) analysis. Both of the DNA markers exhibited a distinguishable melting temperature and shape of the normalised curve between the reference and the adulterants. In the case of species identification, ITS2 was more successful in differentiating between species. Additionally, detection of admixture sample containing small traces of targeted E. longifolia DNA (w/v) can be detected as low as 5% for rbcL and less than 1% for ITS2, proving the sensitivity and versatility of the HRM analysis. In conclusion, the Bar-HRM analysis is a fast and reliable technique that can effectively detect adulterants in herbal products. Therefore, this will be beneficial for regulatory agencies in order to regulate food safety issues.
Fulltext Combination of Plant Growth Regulators, Maltose, and Partial Desiccation Treatment Enhance Somatic Embryogenesis in Selected Malaysian Rice Cultivar

Ming NGJ, Binte Mostafiz S, Johon NS, Abdullah Zulkifli NS, Wagiran A

Plants (Basel), 2019 May 30;8(6).
PMID: 31151227 DOI: 10.3390/plants8060144

The development of efficient tissue culture protocol for somatic embryo would facilitate the genetic modification breeding program. The callus induction and regeneration were studied by using different parameters i.e., auxins, cytokinins, and desiccation treatment. Scanning electron microscopy and histological analysis were performed to identify the embryogenic callus for regeneration. The callus percentage results showed that MS (Murashige and Skoog) basal medium supplemented with 3 mg/L 2, 4-D and 30g/L maltose were the optimal callus induction medium for MR220 (80%) and MR220-CL2 (95%). The morphology of the embryogenic callus was confirmed by the SEM (Scanning Electron Microscopy) (presence of extracellular matrix surface network) and later by histological analysis. Finally, MS media supplemented with 0.5 mg/L NAA (Naphthalene Acetic Acid), 2 mg/L kin, and 1 mg/L BAP were selected as the optimum regeneration media treatment while callus desiccated for 48 h was proved to produce more plantlets in MR220 (60%) and MR220-CL2 (73.33%) compared to control treatment (without desiccation). The protocol presented here showed the necessity for the inclusion of partial desiccation as an important step in the tissue culture protocol of Malaysian indica rice genotypes in order to enhance their regeneration potential.
Fulltext DNA Barcoding, Phylogenetic Analysis and Secondary Structure Predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana

Saidon NA, Wagiran A, Samad AFA, Mohd Salleh F, Mohamed F, Jani J, et al.

Genes (Basel), 2023 Mar 11;14(3).
PMID: 36980969 DOI: 10.3390/genes14030697

Nepentheceae, the most prominent carnivorous family in the Caryophyllales order, comprises the Nepenthes genus, which has modified leaf trap characteristics. Although most Nepenthes species have unique morphologies, their vegetative stages are identical, making identification based on morphology difficult. DNA barcoding is seen as a potential tool for plant identification, with small DNA segments amplified for species identification. In this study, three barcode loci; ribulose-bisphosphate carboxylase (rbcL), intergenic spacer 1 (ITS1) and intergenic spacer 2 (ITS2) and the usefulness of the ITS1 and ITS2 secondary structure for the molecular identification of Nepenthes species were investigated. An analysis of barcodes was conducted using BLASTn, pairwise genetic distance and diversity, followed by secondary structure prediction. The findings reveal that PCR and sequencing were both 100% successful. The present study showed the successful amplification of all targeted DNA barcodes at different sizes. Among the three barcodes, rbcL was the least efficient as a DNA barcode compared to ITS1 and ITS2. The ITS1 nucleotide analysis revealed that the ITS1 barcode had more variations compared to ITS2. The mean genetic distance (K2P) between them was higher for interspecies compared to intraspecies. The results showed that the DNA barcoding gap existed among Nepenthes species, and differences in the secondary structure distinguish the Nepenthes. The secondary structure generated in this study was found to successfully discriminate between the Nepenthes species, leading to enhanced resolutions.
Fulltext Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas)

Mohd Din AR, Iliyas Ahmad F, Wagiran A, Abd Samad A, Rahmat Z, Sarmidi MR

Saudi J Biol Sci, 2016 Jan;23(1):S69-77.
PMID: 26858569 DOI: 10.1016/j.sjbs.2015.10.022

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L(-1)) and NAA (2 mg L(-1)) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5-1.5 mg L(-1)), BAP, NAA and 0.5 mg L(-1) of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L(-1)), Kin (1.5 mg L(-1)), NAA (0.5 mg L(-1)) and TDZ (0.5 mg L(-1)) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots.