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. 2023 Feb 28;9(3):e14130. doi: 10.1016/j.heliyon.2023.e14130

HPLC and DNA barcoding profiles for identification of the selected twelve Mucuna species and its application for detecting prohibited aphrodisiac Mucuna products

Aekkhaluck Intharuksa a, Jessada Denduangboripant b, Sunee Chansakaow a, Kannika Thongkhao c,d, Suchada Sukrong e,∗
PMCID: PMC10006835  PMID: 36915514

Abstract

Aphrodisiac herbal products originated from various plants including Mucuna species. In Thai folklore, Mucuna macrocarpa Wall. and M. pruriens (L.) DC. have long been consumed and utilized for their aphrodisiac properties. Consumption of these plants can lead to serious adverse effects caused by l-dopa. The plants have been legally banned for use as foods, dietary supplements, or nutraceuticals by the FDA of several countries. To protect consumers, methods for the identification of illicit plants or herbal products are needed. This study aimed to identify the selected twelve Mucuna species and examine the aphrodisiac herbal products containing M. macrocarpa and M. pruriens by using HPLC analysis of l-dopa coupled with DNA barcoding profiles of ITS, matK, rbcL, and trnH-psbA. The results showed that l-dopa could be found not only in the seeds of M. macrocarpa and M. pruriens but also in associated allied Mucuna species. Then, a DNA barcode was introduced to support in HPLC profiling to identify the plants. DNA barcodes of twelve Mucuna species found in Thailand were established and used to reconstruct a phylogenetic tree. In this study, ITS2 sequences showed the highest interspecific variability and could be used to differentiate all Mucuna species. The results of ITS2 sequence coupled with HPLC analysis revealed that all the purchased aphrodisiac products originated from M. pruriens only. Therefore, the integration of HPLC analysis and DNA barcoding profile was an efficient method for the identification of prohibited Mucuna species for safety monitoring of herbal supplements and protecting customer safety. Regulatory agencies should raise awareness and restrain the use of these commercial products.

Keywords: Mucuna, Aphrodisiac, Herbal product, HPLC, DNA barcode, ITS2

Highlights

  • •

    Mucuna macrocarpa Wall. and M. pruriens (L.) DC. were banned as food and health supplements because the consumption of their seeds may cause death.

  • •

    The combination of HPLC detectionand DNA barcoding was effective for the identification of M. macrocarpa and M. pruriens.

  • •

    Aphrodisiac herbal products containing banned Mucuna species are still prevalent, and the FDA should control them.

1. Introduction

Plants in the genus Mucuna Adans. (family Fabaceae) consist of approximately 105 species and are distributed globally in the pantropical zone, with the highest diversity observed in Asia, followed by Oceania, the Americas, and Africa [1]. Seventeen Mucuna species are present in Thailand [2,3]. Species of this genus are lianas with trifoliate leaves and pendent inflorescences of conspicuous flowers (Fig. 1a-l). The pod surfaces are ornamented by lamellae and/or thickly covered with bristles. They exhibit taxonomically informative characters that allow the discrimination of species within the genus based on fruit characteristics. However, some species are difficult to identify due to the ambiguity of taxonomic descriptions at the species [4] and subspecies levels and the similarity of morphological characters, especially in the vegetative stage [5]. Some species of Mucuna plants have been used for various applications in several countries where they have been introduced. For example, M. pruriens and M. bracteata have been used as green manure or covered crops in agriculture [4,6]. M. macrocarpa has been consumed to boost male potency and cure erectile dysfunction [7]. M. pruriens has long been used to treat Parkinson's disease and utilized for its aphrodisiac properties in Indian traditional medicine as well as Thai folklore [8]. It was reported that M. pruriens acts on the central nervous system via dopamine and the reproductive tract through adrenaline and noradrenaline [9] to improve sperm count and motility, as well as semen quality, in infertile men [10].

Fig. 1.

Fig. 1

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Inflorescences and fruits of the twelve Mucuna species collected in Thailand (a) M. pruriens var. pruriens (L.) DC.; (b) M. pruriens var. utilis (Wall. ex Wight) Baker ex Burck; (c) M. bracteata DC. ex Kurz; (d) M. gigantea (Willd.) DC.; (e) M. gracilipes Craib; (f) M. hainanensis Hayata; (g) M. interrupta Gagnep.; (h) M. macrocarpa Wall.; (i) M. monosperma DC. ex Wight; (j) M. revoluta Wilmot-Dear; (k) M. thailandica Niyomdham & Wilmot-Dear; (l) M. warburgii Lauterb. & K. Schum.

In addition to Mucuna plants, there are medicinal plants in other genera that have aphrodisiac activity. The use of these plants, for example, Boesenbergia rotunda (L.) Mansf. [11], Eurycoma longifolia Jack [12], Kaempferia parviflora Wall. ex Baker [13], and Tribulus terrestris L. [14], has been chosen as an alternative approach for solving male reproductive problems. All of these plants have been widely sold as raw materials, food supplements, and herbal product forms in Thai herbal markets, drug stores, e-commerce, and direct-sale businesses. However, according to the announcement of the Thai FDA, certain species, including M. macrocarpa and M. pruriens, have been legally banned for use as food, dietary supplements, or nutraceuticals [15]. Furthermore, M. pruriens has been placed in the list of prohibited plants to be used in or as part of the composition of health supplements regulated by the ASEAN traditional medicine and health supplement scientific committee [16] and European Food Safety Authority [17]. The consumption of seeds without inadequate processing methods can have adverse effects caused by l-dopa (the precursor of dopamine), such as nausea, anorexia, and vomiting [18]. There are some previous reports of l-dopa toxicity caused by ingestion of Mucuna beans, for example, 203 cases of acute toxic psychosis in Mozambique [19], a hospitalized 27-year-old woman with gastrointestinal toxicity in Hawaii [20], and a fatal case in Malawi [21]. Recently, there was one tragic report of an individual who consumed a M. pruriens product and died shortly after in Thailand (Supplementary Fig. S1) [22]. This case has once again brought the attention of the Thai FDA and consumers to safety issues associated with herbal products. However, the commercial herbal products of M. macrocarpa and M. pruriens that claim to promote male sex drive have been widely advertised. Therefore, the identification of illicit herbal products is necessary for safety monitoring of herbal supplements and for protecting customer safety including pharmacovigilance applications. In this study, we aimed to use a major chemical constituent incorporated with DNA barcoding techniques to identify the banned species, M. macrocarpa and M. pruriens. Phylogenetic relationships based on nucleotide sequences for DNA barcoding were reconstructed for the identification of these prohibited species. The methods of HPLC and DNA barcoding of ITS2 were also applied to identify the botanical origin of aphrodisiac herbal products for food safety implementation.

2. Materials and methods

2.1. Plant materials

With permission from the Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, the twelve Mucuna species distributed in Thailand were collected (Supplementary Table S1). Seeds of seven Mucuna species, namely, M. pruriens var. pruriens, M. bracteata, M. gigantea, M. macrocarpa, M. monosperma, M. interrupta, and M. revoluta (Fig. 2a-g), out of 12 species were collected and assembled (Supplementary Table S2) and used for chemical profile analysis. Seeds from five species were not able to be collected. Fresh leaves of twelve Mucuna species were collected and desiccated in silica gel for DNA extraction and further used in DNA barcoding method. Samples were identified based on the morphological characters of flowers and fruits (Fig. 1a-l) by Wannaree Charoensup, a botanist at the Faculty of Pharmacy, Chiang Mai University, and Wittaya Pongamornkul, a botanist in charge of the Queen Sirikit Botanic Garden (QBG) Herbarium. Voucher specimens were deposited at the Herbarium of Faculty of Pharmacy, Chiang Mai University (CMU) and Forest Herbarium (BKF) under the Department of National Parks, Wildlife and Plants Preservations, Ministry of Natural Resources and Environment.

Fig. 2.

Fig. 2

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HPLC fingerprint of the l-dopa reference standard and seven Mucuna seeds: (a) M. pruriens, (b) M. bracteata, (c) M. gigantea, (d) M. interrupta, (e) M. macrocarpa, (f) M. monosperma, and (g) M. revoluta. The scale bar in each figure indicates a length of 1 cm. The arrows indicate the l-dopa peak.

2.2. HPLC detection and quantification of l-dopa in Mucuna seeds

HPLC detection and quantification of l-dopa in seeds of Mucuna species (Supplementary Table S2) were conducted following the protocol of Dhanani and colleagues with minor adjustments [23]. Seeds of seven Mucuna plants, namely, M. bracteata, M. gigantea, M. interrupta, M. macrocarpa, M. monosperma, M. pruriens var. pruriens, and M. revoluta, were analyzed. Dried mature seeds were pulverized using an electric grinder and sieved through a 100 mesh. The powder was mixed with petroleum ether to separate the liquid in the seeds and refluxed. The solid residue was added to deionized water (pH 2.5) and refluxed for 5 h. Liquid extract was separated from solid residue by vacuum filtration and concentrated using a vacuum rotary evaporator (EYELA, Japan). Extracts of Mucuna seeds were evaluated by comparison with standard l-dopa (Sigma–Aldrich, USA) using HPLC with a UV/Vis detector (Shimadzu, Japan). The chromatographic separation was performed on a Supelcosil® LC-18 column (250 × 4.6 mm2, 5 μm) (Sigma–Aldrich, USA). The mobile phase was a mixture of 0.1 N formic acid and methanol (98:2). The solvent flow rate was 1.0 mL/min. The injection volume was 10 μL, and the run time was 20 min throughout the analysis. The photodiode array detector wavelength was 280 nm. The method validation was undertaken before the analysis in Mucuna seed extracts. The intraday and interday precision levels were determined by analysing the l-dopa standard on the same and three consecutive days, respectively. The validation parameters, including linearity, limit of detection (LOD) and limit of quantitation (LOQ), and accuracy were assessed. To evaluate the quantity of l-dopa in the Mucuna seed extracts, the linear regression equation of the l-dopa standard curve was applied to calculate the concentration in the extracts. Peak areas corresponding to l-dopa were determined.

2.3. Genomic DNA extraction

Total DNA was extracted from approximately 100 mg of leaf specimens using a DNA Plant Mini Kit (Qiagen®, Germany) following the manufacturer's protocol with minor modifications. For herbal products, 20 mg of sample powder was extracted by using cetyltrimethylammonium bromide (CTAB) [24], and the obtained genomic DNA was purified using a GENECLEAN® Kit following the manufacturer's protocol.

2.4. PCR amplification and sequencing

The PCR system contained 100–120 ng of total DNA template mixed with 12.5 μL of 2x PCR Buffer for KOD FX Neo (ToYoBo®, Japan), 0.4 mM of each deoxyribonucleotide triphosphate (dNTP), 0.15 μM of each universal primer (Supplementary Table S3) and 0.5 unit of KOD FX Neo DNA polymerase. PCR amplification was carried out in a DW-K960 gradient PCR thermal cycler (Drawell, China) using cycling conditions of 94 °C for 2 min, followed by 35 cycles of 98 °C for 10 s, 57 °C for 30 s, and 72 °C for 1:30 min (for rbcL and matK) or 45 s (for ITS and trnH-psbA) and a final elongation at 72 °C for 10 min. The PCR product was examined by 1.8% agarose gel electrophoresis and visualized by staining with RedSafe™ nucleic acid staining solution (iNtRON Biotechnology, Korea) under UV light radiation using a Gel Doc™ EZ Imager (Bio-Rad, USA). The PCR products were subsequently sequenced by Sanger sequencing (Biobasic. Inc, Canada) using the amplification primer for each region (Supplementary Table S3).

2.5. Analysis of DNA sequences

The successful bidirectional sequences of the four regions were aligned by using Clustal W [25] and MUSCLE [26] in Molecular Evolutionary Genetics Analysis (MEGA)X version 10.0.5 software [27] with manual adjustment. Assessment of genetic distance was performed by the Kimura 2-Parameter (K2P) distance model [28]. Intraspecific and interspecific divergences were evaluated by using MEGA software. The discriminatory power and variation in interspecific distance were compared among DNA barcoding regions. The consensus sequences were deposited in the DDBJ/EMBL/GenBank database with their accession numbers (Supplementary Table S1).

2.6. Phylogeny reconstruction

Nucleotide sequences of the twelve Mucuna taxa were used as ingroups in this study. Additionally, sequences of Pueraria candollei Benth., the plant in Fabaceae were retrieved from the GenBank database to be used as an outgroup. The ITS, ITS2, matK, rbcL, and trnH-psbA sequences of all Mucuna specimens were edited and separately aligned using the MUSCLE algorithm implemented in MEGA version 7.0.26 software [29]. To assess congruence between the chloroplast and nuclear data sets, a partition homogeneity test was performed with an incongruence length difference (ILD) test in PAUP* version 4.0a166 software [30]. The test was conducted using a branch-and-bound search and run for 1000 replicates.

Phylogenetic analysis was performed using two different approaches: maximum parsimony (MP) and maximum likelihood (ML). The MP and ML analyses were performed using the PAUP* program with branch-and-bound searching. Statistical supporting values of each node were obtained from bootstrapping with 1000 replications. For the ML analysis, the program JModelTest version 2.1.10 [31] with Akaike Information Criterion (AIC) was used to suggest the best nucleotide substitution model for likelihood setting.

2.7. Herbal product testing

Ten aphrodisiac herbal products (P1-10) were randomly purchased from online stores and used to identify the plant of origin. HPLC analysis was carried out following the protocol mentioned earlier. DNA barcoding analysis of the ITS2 region, the most suitable DNA barcoding region for the identification of Mucuna species, was undertaken as described in the preceding sections. The obtained nucleotide sequences were authenticated using a BLASTn search in the DDBJ/EMBL/GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The best hits with a match of 97% or above from the BLASTn results were considered. The ITS2 nucleotide sequences of the products and the twelve authentic Mucuna species were constructed via a neighbor-joining tree with 10,000 bootstrap replicates using MEGA X version 10.0.5 software. Nucleotide sequences of leguminous plants containing l-dopa, including Alysicarpus rugosus (Willd.) DC. (KX057832 and LC377385), Bauhinia purpurea L. (MH548399 and MH813044), Canavalia gladiata (Jacq.) DC. (MZ198558 and OK090453), Parkinsonia aculeata L. (KF379226), Prosopis chilensis (Molina) Stuntz (JX139106), Senna floribunda (Cav.) H.S. Irwin & Barneby (MN264653), Senna hirsuta (Cav.) H.S. Irwin & Barneby (KT279733 and KX057898), and Vicia faba L. (MW463321 and OK090459) [32], were retrieved from GenBank and included in the phylogenetic analysis.

3. Results

3.1. HPLC fingerprinting and quantification of l-dopa in Mucuna seeds

To ensure the reliability of the analytical method, the method validation was assessed in this work. The linearity, limit of detection (LOD), limit of quantitation (LOQ), and precision were examined as validation parameters. In the l-dopa concentration range of 6.250–800 mg/L, the calibration curve of the reference l-dopa standard showed good linearity with correlation coefficients (R2 = 1.000). The parameters LOD and LOQ, which indicate the sensitivity of this method, were 3.125 and 6.250 mg/L, respectively. The intraday and interday precision levels of retention time expressed as the percentage of relative standard deviation (%RSD) were both 0.11%. The accuracy of the procedure was determined by observing the recovery of the l-dopa standard. The % recovery value of the l-dopa standard was within the range of 98–102%, and the % RSD value was less than 2%.

The detection of l-dopa in the seed extracts of seven Mucuna species was performed based on the retention time of the reference l-dopa standard. The total run time for l-dopa was 15 min, and the spectral peak detected at 280 nm had an HPLC retention time of approximately 5.4 min. Comparison of the HPLC chromatograms of the reference l-dopa standard with those of seven Mucuna species showed a distinct single peak of l-dopa in all the Mucuna species (Fig. 2B). HPLC analysis showed that the quantity of l-dopa varied among Mucuna species. The l-dopa content in the seed extract of M. bracteata was highest at 99.07 mg/100 mg, followed by M. pruriens var. pruriens at 72.52 mg/100 mg, M. gigantea at 68.02 mg/100 mg, M. macrocarpa at 52.59 mg/100 mg, M. revoluta at 13.53 mg/100 mg, and M. interrupta at 6.64 mg/100 mg, with the lowest value observed for M. monosperma at 2.90 mg/100 mg.

3.2. DNA barcoding analysis

The DNA barcode regions ITS, ITS2, matK, rbcL, and trnH-psbA were established from forty-two Mucuna specimens representing the twelve species (Supplementary Figs. S2–5). The nucleotide sequence of each region generated in this study was identical within the same species; then, the represented nucleotide sequences of selected plant samples were deposited into the DDBJ/EMBL/GenBank database (Supplementary Table S1). The features of all sequences were calculated, and the discriminatory ability among the five DNA barcode regions was compared (Table 1). Amplification by using our designed primers (Supplementary Table S3), the total lengths of the nucleotide sequences were 672, 217, 778, 327, and 720 bp for ITS, ITS2, matK, rbcL, and trnH-psbA, respectively. The sequences obtained from ITS2 had the highest percentage of variable and parsimony-informative sites (40.55% and 34.09%), followed by ITS (26.1% and 21.20%), trnH-psbA (22.63% and 12.84%), matK (6.68% and 5.14%), and rbcL (2.78% and 1.94%). The highest percentages of conserved sites exhibited contrasting results, with values of 97.22%, 93.32%, 75.54%, 70.60%, and 57.60% for rbcL, matK, trnH-psbA, ITS, and ITS2, respectively. The relative % GC levels were as follows: ITS2>ITS > rbcL > matK > trnH-psbA.

Table 1.

Sequencing analysis of the DNA barcode regions from twelve Mucuna species used in this study.

DNA barcode regions
ITS ITS2 matK rbcL trnH-psbA
Length range (bp) 619–641 206–210 772–778 720 296–315
Aligned length (bp) 672 217 778 720 327
Conserve site (%) 70.6 57.60 93.32 97.22 75.54
Variable site (%) 26.1 40.55 6.68 2.78 22.63
Parsimony-informative site (%) 21.20 34.09 5.14 1.94 12.84
Average %GC content 52.3–64.4 50.0–71.5 26.2–27.9 41.8–42.2 24.1–25.9
Interspecific divergence (%) (mean/min-max) 14/0.14–22.90 15/0–35.71 3/0–4.42 1/0–1.59 11/0–22.02
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For discriminatory performance among the DNA barcode regions, the genetic divergence within and between Mucuna species in all five DNA regions was analyzed using the Kimura-two parameter (K2P) model with 10,000 bootstrap replications. In the present study, variation within all collected Mucuna species was not found, so the intraspecific divergence was zero. For interspecific divergence, ITS2 exhibited the highest mean % variation (15%), followed by ITS (14%), trnH-psbA (11%), matK (3%) and rbcL (1%) (Table 1). The relative distribution of interspecific divergence was used to calculate the percentage of K2P pairwise distance (Fig. 3). The intervals of K2P pairwise distance (%) of five DNA barcode regions among the twelve Mucuna spp. were as follows: 0.13–23.65 for ITS, 0.00–29.33 for ITS2, 0.00–4.42 for matK, 0.00–1.59 for rbcL, and 0.00–22.02 for trnH-psbA (Supplementary Tables S4–S8). These results revealed that the ITS2 region has the highest variation in interspecific distances, followed by the ITS, trnH-psbA, matK, and rbcL regions. Thus, ITS2 was the suitable DNA region for testing aphrodisiac products containing Mucuna plants.

Fig. 3.

Fig. 3

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Relative distribution of the K2P pairwise distance of DNA barcode regions, including ITS, ITS2, matK, rbcL and trnH-psbA, among twelve Mucuna species.

3.3. Phylogenetic analysis

The combined nucleotide data matrix of ITS (ITS1-5.8S-ITS2), matK, rbcL and trnH-psbA regions consisted of 2616 characters for 13 samples, including twelve Mucuna species and Pueraria candollei. The aligned matrices of ITS (ITS1-5.8S-ITS2), matK, rbcL and trnH-psbA consisted of 672, 784, 724, and 361 base pairs, respectively. The incongruence length difference (ILD) analysis suggested that only if the matK sequence of M. monosperma was discarded were all four data sets congruent, with a p value equal to 0.128 (higher than the 0.05 significance level). Therefore, the chloroplast and nuclear gene sequences were suitable enough to be combined for further phylogenetic analyses.

The best nucleotide substitution model suggested by JModelTest was General Time Reversible plus gamma distribution (GTR + G), and other parameters were set as follows: base frequencies = 0.2935, 0.2059, 0.1970; rmat = 1.3369, 2.1491, 0.8815, 2.2600, 4.5367; and gamma shape = 0.1830. Only one most parsimonious tree was found with 618 step changes, and the best ML tree found had a likelihood score of ln = −7067.519.

Three major clades were revealed from the MP and ML phylogenies (Fig. 4A and B), and they were very robust, with 100% bootstrap supporting values. Clade A was composed of M. bracteata, M. gracilipes, M. pruriens var. pruriens, and M. pruriens var. utilis. M. macrocarpa was paired with M. thailandica as clade B. Clade C had the other seven species, which were M. gigantea, M. hainanensis, M. interrupta, M. monosperma, M. revoluta, and M. warburgii. Two subclades were also recognized in clade C: the first subclade C1 had M. gigantea, M. monosperma, and M. warburgii with moderate bootstrap values, and subclade C2 had M. hainanensis, M. interrupta, and M. revoluta with high bootstrap values.

Fig. 4.

Fig. 4

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Relationship of the ML and MP phylogenies of twelve Mucuna species and Pueraria candollei. (A) The maximum likelihood tree of the combined nucleotide sequence data matrix. The best ML tree was retrieved with a score likelihood of ln = −7067.519. Numbers along branches are the 1000-replicate bootstrap supporting values of each branch. (B) The most parsimonious tree of the combined nucleotide sequence data matrix. The MP tree was found by the branch-and-bound search method with a tree length of 618. Numbers above and under the branches are the evolutionary step changes and bootstrap supporting values of each branch, respectively.

Apparently, the overall topologies of both MP and ML phylogenetic trees appeared to be very similar, except for the groupings between the three major clades. In the MP tree, clade B of M. macrocarpa and M. thailandica was grouped with clade A with 77% bootstrap support. On the other hand, clade B of the ML tree was joined with clade C instead, with the same bootstrap support (77%). Notably, although the matK sequence of M. monosperma could cause uncertainty in the partition homogeneity test of the combined data matrix, this taxon may not be a reason for this discrepancy between the MP and ML phylogenies. The position of M. monosperma was not different between the trees, as it was always clustered with M. gigantea and M. warburgii.

3.4. Testing of aphrodisiac herbal products

Ten aphrodisiac herbal products (Fig. 5A) were evaluated using HPLC for the existence of l-dopa by comparison with the reference l-dopa standard. The HPLC chromatograms (Fig. 5B) demonstrated that all ten herbal products contained l-dopa, exhibiting a peak at the same retention time as that of the l-dopa standard. A distinct peak of l-dopa at a retention time of 5.4 min was observed in all the tested products, with the variety of peak intensities indicating different amounts of l-dopa. The l-dopa content in all ten aphrodisiac herbal products ranged from 11.86 to 77.93 mg/100 mg. The highest l-dopa concentration was observed in the PP2 sample (77.93 mg/100 mg), followed by PP3, PP10, PP5, PP7, PP6, PP1, PP4, PP8, and PP9 at 56.04, 47.06, 41.22, 37.63, 31.86, 27.24, 20.34, 17.13, and 11.86 mg/100 mg, respectively. As a result, the HPLC analysis was unable to differentiate plant species that contained l-dopa at both the intrageneric and intergeneric levels. Consequently, DNA barcoding analysis based on ITS2, the DNA region that showed the highest variation in interspecific distance calculated by K2P pairwise distance, was utilized to identify the aphrodisiac herbal products. The BLASTn results revealed that all ten aphrodisiac herbal products displayed 100% BLAST homology with M. pruriens (L.) DC. Based on the nucleotide sequences of the ITS2 intergenic spacer, the phylogram of the neighbor-joining tree was constructed using the nucleotide sequences of the ITS2 region of all the aphrodisiac herbal products, twelve authentic Mucuna species, and eight leguminous plants retrieved from the GenBank database. The constructed neighbor-joining tree exhibited a clear separation in the cladogram among the twelve Mucuna species and the other Fabaceous species (Fig. 6). The results revealed that all the examined aphrodisiac herbal products belonged to the same clade as the authentic M. pruriens var. pruriens and M. pruriens var. utilis.

Fig. 5.

Fig. 5

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The aphrodisiac herbal products (PP1-10) and HPLC fingerprint. (A) Aphrodisiac herbal products collected from the e-commerce platform. (B) HPLC fingerprint of ten aphrodisiac herbal products compared with the l-dopa reference standard.

Fig. 6.

Fig. 6

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The neighbor-joining tree based on the ITS2 sequences of twelve Mucuna species, ten aphrodisiac herbal products, and some leguminous species containing l-dopa. The numbers at the nodes are bootstrap values based on 10,000 replications. The number of substitutions per site on the scale bar can be used to determine the branch lengths.

4. Discussion

4.1. HPLC analysis coupled with DNA barcoding can be used to identify Mucuna species

Traditionally, the organoleptic, morphological, and histological inspections have been recommended for identification of herbal medicine in Herbal Pharmacopoeia of many countries. Although those methods are fast and economical, they have limitations due to personal experience. The methods are also difficult to identify closely related species, highly processed materials, and herbal-mixed products [33]. Phytochemical methodologies were introduced for authentication of botanical materials and products in public Pharmacopoeias as a common regulation. Identification and authentication methods of Mucuna plants using phytochemical analysis have been published, for instance, TLC [34], HPTLC [35], and HPLC [23,36]. However, the variation in geographical location and environmental conditions can affect to phytochemical analysis [37]. Thus, DNA barcoding has been developed and used to identify herbal supplements, including for discrimination of authentic materials from counterfeited, substituted, and adulterated material in herbal drug safety monitoring [38], for example, German Chamomile [39], Saffron [40], and herbal beverage [41]. Some common Mucuna plants in India [42] and Thailand [36] namely, M. atropurpurea, M. bracteata, M. gigantea, M. interupta, M. monosperma, and M. pruriens was also carried out by DNA barcoding for plant identification purposes. In order to accurate and trustworthy results, a combination of those techniques should be applied for identifying and authenticating the botanical origin of herbal materials and products.

The consumption of M. pruriens products has led to consumer death recently in Thailand (Supplementary Fig. S1). A method for the detection of suspected Mucuna species is needed. To identify the botanical origin of food material, chemical analysis is generally a key tool to reveal detailed information on the main chemical components in herbal food products. For example, the chemical profiles of toxic tropane alkaloids have been utilized to screen the existence of some Datura and Hyoscyamus plants in Italian herbal tea and extracts [43]. Anthecotulide and senecionine have been used for discrimination of Matricaria recutita L. (German chamomile) and its toxic adulterants Anthemis cotula L., Senecio desfontainei Druce and Senecio vulgaris L [39]. For Mucuna plants, l-dopa is the main active compound found in seeds [44]. According to the results of the HPLC analysis (Fig. 2B), l-dopa was observed as a single peak in all seeds from seven Mucuna species at a retention time of 5.4 min. However, the presence of the l-dopa peak has been found in Mucuna species and other plants, viz. M. multisiana, M. andreana, M. holtonii, M. sloanei, M. urens, and M. deeringiana [45], including plants in the Fabaceous family, such as Alysicarpus rugosus (Willd.) DC., Bauhinia purpurea L., Canavalia gladiata (Jacq.) DC., Parkinsonia aculeata L., Prosopis chilensis (Molina) Stuntz, Senna floribunda (Cav.) H.S. Irwin & Barneby, Senna hirsuta (Cav.) H.S. Irwin & Barneby, and Vicia faba L [32]. Therefore, the species-specific chemical markers for the banned plants M. macrocarpa and M. pruriens should be considered. This finding is consistent with the fact that the use of non-species-specific compounds in HPLC analysis cannot differentiate plant species when the plant samples have identical chemical markers [46]. In this case, although the HPLC analysis provided ambiguous results for the identification of Mucuna species, the method could screen out some plants that lacked l-dopa from the suspected samples prior to the molecular analysis based on the DNA barcode that was applied for species identification.

DNA barcoding for species identification, which is based on the variance of DNA sequences at the species level, is an approach to support the chemical identification. In our study, a DNA barcode was applied to detect Thai FDA-banned aphrodisiac herbal products. The DNA barcode reference libraries of ITS, ITS2, matK, rbcL, and trnH-psbA from twelve species of Mucuna plants collected across Thailand were generated, characterized, and used to distinguish Mucuna species. According to the percentage of variable and parsimony-informative sites, our results revealed that the nuclear ribosomal ITS2 region had highest percentage of both parameters (40.55% and 34.09% of variable and parsimony-informative sites, respectively). Furthermore, ITS2 showed the highest percentage of mean variation (15%) in the interspecific divergence performed using the K2P model with 10,000 bootstraps. As a consequence, ITS2 is an effective and suitable DNA region for the identification of Mucuna species, which is in agreement with Rashmi's study carried out in Indian Mucuna plants [42]. Because of the practical and successful procedure for amplification and its short length with high variation among species, ITS2 has been widely used as a DNA mini-barcode to identify various plant species, including DNA-poor botanical materials [47].

4.2. Phylogenetic tree reconstruction aids the differentiation of Mucuna species

In the past decade, molecular biological methods have played an important role in phylogenetic and evolutionary studies of organisms, coupled with morphological, histological, and chemical data. For the genus Mucuna (Phaseoleae-Leguminosae-Papilionoideae), there remains a lack of precise taxonomic data to distinguish among members, at both the species and subspecies levels, because of the high variation within the genus, including several synonyms [4,48]. Basically, Mucuna Adans. was separated into two subgenera based on the main morphological differences of the fruits and seeds: M. subg. Mucuna and M. subg. Stizolobium. Based on previous phylogenetic research, phylogenetic analysis of some Indian Mucuna species using nuclear ribosomal ITS and chloroplast trnH-psbA regions exhibited two clusters of Mucuna taxa [49]. In 2016, based on the trnL-F and ITS regions, Moura and coworkers revealed three separate clades, namely, the core Mucuna clade, Stizolobium clade and Macrocarpa clade [50]. Then, the infrageneric classification of the genus Mucuna supported by biogeography, morphology, and molecular phylogeny was revised to consider the Macrocarpa clade and propose the new infrageneric taxon Mucuna subg. Macrocarpa [1]. In the consecutive year, the phylogenetic relationships of forty-six specimens of four common Mucuna species found in India were reconstructed by using DNA barcode regions, including ITS2, matK, rbcL, and trnH-psbA [42]. The tree showed two clusters representing species in the subgenera Mucuna and Stizolobium. Subsequently, phylogenetic analysis utilizing the plastid matK region was integrated with palynology to resolve the earliest diverging lineage in the genus Mucuna and to confirm the evolutionary relationships among lineages [48].

We performed phylogenetic analysis for the investigation of twelve Thai Mucuna species using maximum parsimony (MP) and maximum likelihood (ML) in the combined nucleotide data matrix of the ITS, matK, rbcL, and trnH-psbA regions. Both the MP and ML phylogenies (Fig. 4A and B) revealed three major clades, namely, clade A (M. bracteata, M. gracilipes, M. pruriens var. pruriens and M. pruriens var. utilis), clade B (M. macrocarpa and M. thailandica), and clade C (M. gigantea, M. hainanensis, M. interrupta, M. monosperma, M. revoluta, and M. warburgii), which agreed well with the previously published phylogenies of ITS along with trnL-F [1,50] and matK [48]. Based on biogeographic, morphological, molecular and palynological approaches, the revision of infrageneric taxa of the genus Mucuna led to the proposal of the subgenus Macrocarpa [1,48,50]. In this research, our phylogenetic results for twelve Mucuna species covering three subgenera correlated very well with the morphological features of the fruits (Fig. 4A and B). M. subg. Mucuna showed the main morphological characteristics of fruits, namely, oblong to linear-oblong shapes, leathery surfaces ornamented with raised lamellae and scattered bristles. Conversely, the fruiting materials of species in subgenus Stizolobium were distinct from the others, showing small fleshy fruits with a dense indumentum and a linear to linear-oblong shape (up to 10 cm long and with a maximum of five seeds). However, a newly proposed subgenus, M. subg. Macrocarpa, showed two taxa in Thailand viz. M. macrocarpa and M. thailandica. These are endemic species found only in the montane and gallery forest of Doi Inthanon, Chiang Mai. Fruits of species in this subgenus distinctively present a linear shape, woody surface with longitudinal deep wrinkles and lack ornamentation (M. macrocarpa) or scattering with red–brown bristles (M. thailandica).

4.3. Combination of HPLC and DNA barcoding for detection of the illicit aphrodisiac herbal products

Chemical identification using HPLC was undertaken to detect the existence of l-dopa in aphrodisiac products. According to the HPLC result, l-dopa was found in all the tested products. However, not only Mucuna species but also the other genera in the Fabaceae family were reported to contain l-dopa. Thus, DNA barcoding was used to address these constraints and identify the botanical origin. ITS2 is a significant and suitable DNA barcoding region for plant identification, especially in herbal plants, because it provides excellent primer universality and a high degree of interspecific variation, and simplicity of PCR amplification and sequencing [38]. Based on the nucleotide sequences of the ITS2 intergenic region, the BLASTn results of all tested products showed the best hit with M. pruriens, the prohibiting plant species. The ITS2 nucleotide sequences obtained from herbal products combined with twelve Mucuna species and l-dopa-containing leguminous plants (Alysicarpus rugosus, Bauhinia purpurea, Canavalia gladiata, Parkinsonia aculeata, Prosopis chilensis, Senna floribunda, Senna hirsuta, and Vicia faba) retrieved from the GenBank database were reconstructed using neighbor-joining (NJ) tree analysis. The NJ result demonstrated that all the aphrodisiac herbal products existed in the same clade of M. pruriens. Based on the results, the investigations showed that all the aphrodisiac herbal products originated only from M. pruriens, so their use as food health supplements was prohibited by the Thai FDA, as well as the ASEAN traditional medicine and health supplement scientific committee and European Food Safety Authority. In this study, herbal products derived from M. macrocarpa were not found. These findings indicated that M. pruriens products are in high demand and are extensively used. Therefore, the products are still traded in Thai herbal markets, including on e-commerce platforms. Another reason why the product is widely sold because that M. pruriens is a common weed and can be found naturally across Thailand [3]. The plant is also easy to cultivate; moreover, the species has high seed productivity [51]. There are reports in which DNA barcoding and HPLC methods was a successfully implemented to identify herbal products, for instance, Labisia pumila (Blume) Fern.-Vill [52], Daphne giraldii Nitsche [53] and Saussurea involucrata (Kar. Et. Kir.) Sch. Bip [54]. Hence, the usage of binary approaches of DNA barcoding and chemical analysis could accurately confirm the botanical identities of these plants in food safety and pharmacovigilance applications [55]. Accessible digital databases providing essential information on herbal products, such as morphological, chemical, and molecular information, should be considered and generated for use by all researchers, traders, and customers [56].

4.4. The importance of DNA barcoding and herbal pharmacovigilance in food safety

As the use of herbal products continues to escalate rapidly across the world, toxicity and adverse events, such as mild to serious side effects, hypersensitivity, subchronic and chronic toxicity and death related to herb usage and misidentification, are also increasing. There were two case reports linked with dietary supplements containing extracts of Garcinia cambogia, Gymnema sylvestre, Camelia sinensis, and chromium polynicotinate for weight loss-induced acute hepatitis in the USA [57]. In Thailand, one fatal case related to herbal product use was reported. A young woman who consumed an unregistered product of M. pruriens seed extract in capsule dosage form suffered an allergic reaction and died shortly after thereafter [22]. In addition, the unexpected toxicities of herbal products caused by quality issues, including adulterated or contaminated herb materials and inferior quality, incorrect or misidentified herbs, incorrect processing methods, and the addition of modern medicines or synthetic drugs to achieve the expected effects, have contributed to serious adverse events [58]. Therefore, awareness of the quality and fidelity of herbal products has been raised at several levels, ranging from health authorities to the general public, to develop methods for monitoring the safety of marketed herbal products [59]. In the past decade, the WHO has enhanced the support and provision of technical, educational, and other services to strengthen the pharmacovigilance system in various countries [60]. However, pharmacovigilance for herbal products is associated with several challenges: ambiguous nomenclature, problems of substitution and adulteration, lack of monitoring, and necessity of standardization [61]. To address the limitations of herbal pharmacovigilance related to the aforementioned challenges, the identification and authentication of herbal products is essential.

5. Conclusion

Currently, the consumption of herbal products containing toxic plants is one of the causes of death. To identify the banned aphrodisiac plants M. macrocarpa and M. pruriens, the dual approaches of HPLC analysis and DNA barcoding were applied. HPLC analysis can detect the existence of l-dopa in all the Mucuna seeds while DNA barcoding can differentiate all Mucuna species. The coupled approaches revealed that the prohibited Mucuna species are still sold in the market. Thus, we urge the FDA to raise awareness among consumers and manufacturers and cease the sale of all products containing M. macrocarpa and M. pruriens available in the herbal market and e-commerce platforms. This finding will influence future research to develop immunochromatographic assays or DNA-chromatographic detection strips for fast and simple diagnostic applications in food safety.

Author contribution statement

Aekkhaluck Intharuksa: Contributed reagents, materials, analysis tools or data; Performed the experiments; Analyzed and interpreted data; Wrote the paper.

Jessada Denduangboripant, Kannika Thongkhao: Analyzed and interpreted data; Wrote the paper.

Sunee Chansakaow: Conceived and designed experiment; Contributed reagents, materials, analysis tools or data.

Suchada Sukrong: Conceived and designed experiment; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This work was supported by a new researcher grant from the Thailand Research Fund and Chiang Mai University (grant number MRG6080203).

Data available statement

Data included in article/supp. material/referenced in article.

Declaration of interest’s statement

The authors declare no competing interests.

Acknowledgements

The authors are grateful to the Faculty of Pharmaceutical Sciences, Chulalongkorn University and Faculty of Pharmacy, Chiang Mai University, for providing facilities. The authors would like to thank the Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment for permitting the collection of plant materials in the conservation areas. Furthermore, the authors would like to acknowledge Mr. Wittaya Pongamornkul and Ms. Wannaree Charoensup for their support in identifying the plant materials.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2023.e14130.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (4.5MB, docx)

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