The Rise of Natural-Based Practices
The current lifestyle trend leans towards natural-based practices, including healthcare and medicine (Coutinho Moraes et al., 2015; Elkordy et al., 2021; Insuasti-Cruz et al., 2022). This shift is driven by a desire for holistic well-being and a growing recognition of the benefits of plant-derived substances. Plant-derived medicines offer sustainable alternatives to synthetic drugs (Elkordy et al., 2021), align with environmental concerns (Theodoridis et al., 2023), and reduce reliance on non-renewable resources (Verpoorte et al., 2008). Advances in scientific research further support the importance of plant-based medicines as they uncover bioactive compounds with potential therapeutic applications (Gautam et al., 2023; Nyakudya et al., 2020).
Eurycoma longifolia: A Prized Medicinal Plant
Eurycoma longifolia, commonly referred to as pasak bumi or tongkat ali in the Indonesian and Malay languages, is renowned for its medicinal properties in Southeast Asian countries such as Malaysia, Indonesia, and Vietnam (Bhat and Karim, 2010; Chua et al., 2005; Evans Schultes, 1980; Kassim et al., 2002). E. longifolia possesses a diverse array of bioactive compounds, including quassinoids, β-carboline alkaloids, canthin-6-one alkaloids, triterpene-type tirucallane, derivatives of squalene, eurycolactone, eurycomalactone, laurycolactone, biphenyl neolignan, and bioactive steroids (Ang et al., 2000; Kuo et al., 2004; Mahfudh and Pihie, 2008; Miyake et al., 2009; Tran et al., 2014). These compounds contribute to the diverse pharmacological activities of E. longifolia, making it a subject of great interest for research and development of herbal products.
Challenges in Sustainable Utilization
This plant species grows naturally in Cambodia, Myanmar, and Thailand. In addition to its antimalarial, antipyretic, antiulcer, cytotoxic, and aphrodisiac properties (Bhat and Karim, 2010; Chua et al., 2005; Evans Schultes, 1980; Kassim et al., 2002), the root extracts have been traditionally used to enhance testosterone levels in men. The significant value and affordability of this species has led to an increased market demand and intensive trade, both domestically and internationally (Sihotang and Rahmawati, 2019). Unfortunately, this high demand has led to illegal export practices and the potential overexploitation of E. longifolia in Indonesia (Susilowati et al., 2021). The sustainable utilisation of this species is compromised by the persistence of current practices.
Molecular Authentication: A Solution for Ensuring Safety
Consequently, it is imperative to establish systems capable of monitoring and delineating the origins of plants and their derived products, as demonstrated by DNA barcoding (Abubakar et al., 2018). Molecular identification techniques, particularly DNA-based methods, play a pivotal role in the authentication and traceability of herbal products derived from E. longifolia. DNA markers have proven to be powerful tools for validating the authenticity and traceability of herbal and food products (Ganie et al., 2015) as in the case of Korean ginseng (Jung et al., 2014), manuka honey (McDonald et al., 2018), Orthosiphon stamineus herbal medicine (Liow et al., 2021), and processed hairtail fish products (Abdullah et al., 2019). These methods provide a reliable means of verifying the presence and authenticity of plant materials in various commercial products, including dietary supplements, traditional medicine, and herbal preparations (Fadzil et al., 2018; Gao et al., 2010; Han et al., 2016; Howard et al., 2019; Noh et al., 2018).
Molecular Identification of E. longifolia Products
By analyzing specific genetic markers such as DNA barcodes, it is possible to differentiate E. longifolia from other related species and detect any adulteration or substitution that may occur in the market (Gao et al., 2010; Han et al., 2016). Molecular identification methods have been widely used to confirm the presence of E. longifolia in commercial products. Studies have successfully employed techniques such as DNA sequencing, polymerase chain reaction (PCR), and DNA barcoding to assess the quality and authenticity of E. longifolia-based products (Abubakar et al., 2018; Vejayan et al., 2018). These molecular techniques provide accurate and reliable results, allowing for better regulation and control of the trade of E. longifolia products and ensuring consumer safety.
Adulteration and Substitution Concerns
Although previous studies have authenticated E. longifolia products, the derivative products of this species have become increasingly diverse (Abubakar et al., 2018). In Indonesia, raw materials are extracted from the wild because of the species’ lack of cultivation or domestication (Pasaribu et al., 2021; Suwardi et al., 2022). This has led to a decrease in authentic raw materials, prompting the use of species falsely branded as E. longifolia (Li et al., 2013; Mohammed Abubakar et al., 2017). Consequently, the need for broader authentication efforts has been emphasized. Moreover, the diversity of adulterated materials varies between localities, regions, and countries based on the occurrence of complementary species (Soares et al., 2017), necessitating specific authentication methods tailored to each geographical context.
Research Methodology and Findings
This study aimed to authenticate the products or derivatives of E. longifolia (pasak bumi) produced, marketed, and consumed in Indonesia using molecular identification techniques. A total of 37 leaf samples of E. longifolia were collected from the Sumatran mainland and Riau Islands, Indonesia (Table 1). Leaf samples were used as the primary reference for authentication, serving as crucial benchmarks for comparison with claimed derivative products of E. longifolia, including wood and roots.
The molecular analyses were performed at the Laboratory of Molecular Systematics, National Research and Innovation Agency, Indonesia. Two molecular markers, namely trnL-trnF and the whole internal transcribed spacer (ITS) regions, were selected for PCR amplification and DNA sequencing. The results revealed that all leaf samples were indeed E. longifolia based on the markers used, with the six products, only the herbal tea product (sample code TCPB) was most likely derived from E. longifolia based on the two regions, suggesting that not all products labelled as E. longifolia in the market are authentic.
The results also indicated that several other plants species are used as substitutes or adulterants, including Simaba spp., Simarouba spp., Homalolepis spp., Vernonia gigantea, Elephantopus scaber, Gymnanthemum amygdalinum, Cyanthillium spp., Potentilla lineata, Ailanthus altissima, Geijera paniculata, Hannoa chlorantha, and Dalbergia spp. Klebsiella pneumoniae bacteria were also identified in this study on the outer wooden cup of E. longifolia products.
Recommended DNA Markers for Authentication
The study demonstrated that the trnL-trnF and ITS regions are promising DNA barcodes for authenticating E. longifolia herbal medicinal products (HMPs) because of their short sizes. An ideal marker should be short enough to amplify degraded DNA, produce high-quality sequences, and possess robust variability for sample discrimination (Li et al., 2015; Vijayan and Tsou, 2010). The ITS region, confirmed by various researchers, including (Gao et al., 2010; Han et al., 2016), stands out as one of the best DNA barcode regions for the identification and authentication of medicinal plants.
Implications for Quality Control and Consumer Safety
The findings of this study have demonstrated that herbal products of E. longifolia available in the Indonesian market are subject to adulteration or substitution potentially stemming from inaccuracies in species identification. This study strongly advocates the prompt integration of DNA barcoding as a mechanism for identifying instances of species adulteration to improve the production of high-quality herbal products. Consequently, DNA barcoding should be included in the quality control measures of government certification bodies to facilitate a comprehensive evaluation of product authenticity and to ensure the safety of the herbal products consumed.
Conclusion
This study marks the pioneering use of DNA barcode molecular identification technology to identify E. longifolia and its adulterants, thereby extending the application of trnL-trnF and ITS sequences to medicinal plants, particularly in Indonesia. By providing nuanced insights into the safety and efficacy of these products, this approach will contribute to ensuring consumer safety and restoring trust in the herbal supplements market.
