Abstract Senegalia rugata (Lam.) Britton & Rose has been used in the treatment of traditional ailments for a long time in Thailand. However, the specifications of S. rugata remain limited, which is crucial for quality control. The aims of this study are to authenticate and evaluate the quality of S. rugata by employing DNA barcoding and pharmacognostic characterization of pods. The molecular identification using six DNA nucleotides sequences was studied. The chemical determination, microscopic and physico-chemical properties of pods from six different sources were also investigated while the saponin content was quantitively detected by spectroscopy using quillaja saponin as a reference standard. The result of nucleotide sequence analysis and intraspecific variation of the six DNA barcode regions of all specimens showed that they are the same species and presented the different length of nucleotide sequences among each DNA region namely ITS (530-532 bp), psbA-trnH (416-418 bp), and trnL-F (1,025-1,030 bp). However, the sequences of matK, rbcL, and ycf1 from all samples were identical in length at 864, 723, and 913 bp, respectively. Intraspecific polymorphism and gaps occurred in ITS, matK, psbA-trnH, rbcL, and trnL-F, while no nucleotide variation found in ycf1 regions. Thin layer chromatographic chromatograms from both ethanolic and aqueous extracts showed the same chemical patterns while total saponin content quantified as quillaja saponin equivalent of the aqueous extract was significantly higher than the ethanolic extract. The DNA barcoding, microscopic characters and physico-chemical properties were firstly reported which could be used to identify and control the quality of S. rugata.

Keywords: Senegalia rugata, Acacia concinna, Molecular identification, Microscopic, Quillaja saponin

Funding: This research project is supported by Research Fund for Fiscal Year 2021, the Faculty of Pharmacy, Chiang Mai University.

Citation:  Rungsimakan, S., Charoensup, W., Intharuksa, A., Yanaso, S., and Phrutivorapongkul, A. 2026. DNA barcoding coupled with pharmacognostic profile of Senegalia rugata (Lam.) Britton & Rose for authentication and quality control. Natural and Life Sciences Communications. 25(4): e2026071.

Graphical Abstract:

INTRODUCTION

Senegalia rugata (Lam.) Britton & Rose belongs to family Fabaceae. Its synonym, Acacia concinna (Willd.) DC., is commonly known throughout Asia (Flora of Thailand, 1985; Maslin et al., 2019; World Flora Online, 2024).  The vernacular names include Purple-pod Senegalia (Maslin et al., 2019), Som khon, Som poi (Northern Thailand) (Flora of Thailand, 1985), Teng jin he huan (China PR) (Flora of China, 2008), Saptalaa, Shitalaa, Saatalaa, Shrivalli, Kantvalli (Ayurvedic), Shikaakaai, Kharunb Nabti (Unani), Seekai, Sigakai (Siddha/Tamil) (Khare, 2007). This plant is woody climber with branchlets spiny and bipinnate leaves (Figure 1). Leaflets shows 10–35 pairs per pinna, opposite, sessile, base asymmetrically truncate. Mostly axillary inflorescence, white or yellowish flowers in heads with pink bud, floral bracts 0.5–1 mm not projecting beyond the flowers in bud are common. Pod is 10–15 by 1.7–2.7 cm, oblong, often with sinuate margins, thick, fleshy, very wrinkled when dried, glabrous and venation inconspicuous. Seeds are 6.5–11 by 4.5–8 mm elliptic to orbicular, often of irregular shape (Flora of Thailand, 1985; Maslin et al., 2019; World Flora Online, 2024).

Figure 1. Senegalia rugata (Lam.) Britton & Rose. Liana habit showing leaves and flowers (1), Leaves showing pairs of pinnate (2), Inflorescence showing flowers in heads with pink bud (3), Flower showing hairy calyx (4) and Dried pods and seeds (5).

In Thailand, the pods are used as traditional medicine for anti-dandruff, laxative, expectorant, and external uses for hair detergent and wound dressing. Leaves have been traditionally used for expectorant and external uses for cleansing and an ingredient in a herbal massage ball (DTAM, 2015). A Thai herbal preparation containing pods of S. rugata called “Ya Fai Ha Kong” is listed in the Thai Herbal Preparation Pharmacopoeia and Thai National list of essential herbal medicine for treatment of gynecological symptoms (DTAM, 2022; Thai government gazette, 2023). Other Thai herbal preparations containing pods and leaves for treatment various traditional ailments are published in the National Thai traditional medicine formulary 2021 edition (DTAM, 2021). Dried pods have also been used ethnobotanically by being steeped in scented water for ablution during the water festival and new year celebration (Flora of Thailand, 1985). In India, an infusion of leaves is given in malarial fever. Decoction of pods and seeds is used to remove dandruff (known as Shikaakaai) and extensively used as a detergent. An ointment is also used for skin diseases. Bark extract is used in leprosy (Khare, 2007).

Phytochemical studies of pods reported chemical constituents, i.e. saponins, triterpenoids, monoterpenes and others. Saponin fraction from the pods gave kinmoonosides A-C which showed significant cytotoxicity against human HT-1080 fibrosarcoma cells (Tezuka et al., 2000) and spinasteryl glucoside and its dihydro derivative (Kiuchi et al., 1997; Gafur et al., 1997). Triterpenoidal prosapogenols, namely concinnosides A-E, glycosides and their aglycone, namely, acaciaside, julibroside A1, julibroside A3, albiziasaponin C, acacic acid lactone, monoterpenes and their glycosides, namely menthiafolic acid, (2E)-6-hydroxy-2-hydroxymethyl-6-methyl-2,7-octadienoic acid, 4-O-[(2E)-6-hydroxyl-2-hydroxymethyl-6-methyl-2,7-octadienoyl]-D-quinovopyranose were also reported (Gafur et al., 1997; Kiuchi et al., 1997; Tezuka et al., 2000). Saponins possess extensive bioactivities including cytotoxic, expectorant, anti-inflammatory, vasoprotective, hypocholesterolemic, immunomodulatory, hypoglycaemic, molluscicidal, antifungal, antiparasitic effects and many others (Podolak et al., 2010). S. rugata pod extracts showed good in vitro antioxidant activity on DPPH and ABTS assays with IC₅₀ of 0.83 mg/mL and 0.14 mg/mL, respectively. Cytotoxic effect on human peripheral blood mononuclear cells (PBMCs) using MTT assay showed comparable percentage of viability of hydroethanolic extract-treated PBMCs to ascorbic acid at each individual concentration (Poomanee et al., 2015). Antimicrobial activity against Klebsiella pneumoniae, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus was also reported by agar cup diffusion method by using S. rugata pods extract (Todkar et al., 2010). The significant antidermatophytic activity was reported for the extracts prepared with ethanol, ethyl acetate and hexane against the dermatophytes, namely Trichophyton rubrum, T. mentagrophytes, T. violaecum, Microsporum nanum and Epidermophyton floccosum, with the MIC value of 62.5 μg/mL (Natarajan and Natarajan, 2009).

Senegalia Raf. (Synnonym Acacia) is a genus of 217 species (233 taxa) that occurs in the tropics and subtropics worldwide. It is represented by 97 species in the Americas (with Brazil being the centre of species-richness), 62 species in Africa (plus 11 species in Madagascar and Mascarene Islands), 56 species in Asia (i.e. Arabian Peninsula to China and Southeast Asia), and 2 species in Australia (Maslin et al., 2019).  Though certain botanical appearances of S. rugata are recognized, the similarity of the common characteristics in this genus could lead to the wrong identification. Additionally, the assurance of quality, safety, and efficacy represents the ultimate goal in the utilization of medicinal plants (DMSC, 2021). These attributes are established through the standardisation and specification of medicinal plants and their products, including authentication by conventional pharmacognostic methods or modern molecular techniques (e.g., DNA barcoding), together with quality evaluation based on physicochemical parameters and chemical identification. The aims of this study are to authenticate S. rugata by employing the combination of DNA barcoding of six DNA barcode regions (ITS, matK, rbcL, psbA-trnH, trnL-F, and ycf1) and pharmacognostic characterization, including morphological and microscopical analyses, and to evaluate the quality of S. rugata pods by means of the physico-chemical method and chemical identification using thin layer chromatography (TLC). The saponin content of the pods are also investigated quantitatively and reported as quillaja saponin equivalent unit.

MATERIALS AND METHODS

Plant material

Six S. rugata samples were collected throughout Northern Thailand and kept as reference samples at the Faculty of Pharmacy, Chiang Mai University. All samples were identified by Ms. Wannaree Charoensup, a botanist of Faculty of Pharmacy, Chiang Mai University. Pod part was dried at 50°C, ground and pressed through a fine sieve (mesh no. 60) for pharmacognostic studies and saponin content analysis. The seeds were sown in a pot during monsoon season (August 2020). At the seedling stage, the first true leaf was collected, frozen in liquid nitrogen and ground into a fine powder for DNA analysis. 

DNA barcoding analysis

Genomic DNA extraction, polymerase chain amplification, and sequencing

S. rugata samples and accession number of DNA barcode are shown in Table 1. The genomic DNA was isolated using a DNeasy Plant Mini Kit (Qiagen, German) according to the manufacturer’s instruction with minor modifications. The DNA template about 100–120 ng was amplified by polymerase chain reaction (PCR) in the nuclear internal transcribed spacer (ITS) and five plastid regions: matK, psbA-trnH, rbcL, trnH-L, and ycf1. A 25-µL PCR reaction mixture contained 2 x PCR buffer for KOD FX Neo (12.5 µL), each of dNTPs (0.4 mM), each of primers shown in Table 2 (0.15 µM) and KOD FX Neo DNA polymerase (Toyobo, Japan) (0.5 unit). The PCR cycles of reaction were comprised of initial denaturation at 94°C, 2 min, followed by 35 cycles of denaturation at 94°C, 15 sec, annealing at 52°C, 30 sec, initial extension at 68°C, 45 sec, and final extension at 68°C, 5 min. The PCR products were separated by size in 1.8% agarose gel electrophoresis stained with RedSafe™ nucleic acid staining solution (iNtRON Biotechnology, Korea) and visualized under UV light radiation in Gel Doc™ EZ Imager (Bio-Rad, USA). The amplified products were purified using a MEGAquick-spin^TM Plus Total Fragment DNA Purification Kit (Intron Biotechnology, Korea) and then directly sequenced using ABI PRISM 3730 XL sequencer (Applied Biosystems, USA).

Table 1.  S. rugata samples and accession number of DNA barcode.

Table 2. Primers used in this study.

Sequence alignment and analysis

The bidirectional nucleotide sequences from six regions were edited and corrected using BioEdit software (Hall, 1999) and aligned using MUSCLE tools (Edgar, 2004) in Molecular Evolutionary Genetics Analysis (MEGA) version X software (Kumar et al., 2008) with manual adjustment. The consensus sequences of all samples were deposited to GenBank database of National Center for Biological Information (NCBI), and the accession numbers are shown in Table 2. Intraspecific genetic distance among S. rugata sample was calculated using the Kimura 2-parameter (K2P) model of evolution with 10,000 bootstrap replications (Kimura, 1980).

Microscopic characterization

Microscopic analysis was carried out using transverse sections of the entire pod and powdered dried pod samples, in accordance with the Thai Herbal Pharmacopoeia (DMSC, 2021) to observe the cells, tissues, and contents of each sample was performed by placing either cross section of pod part or pod’s powder and 2–3 drops of the reagent used, such as water, iodine water, and picric acid, on a glass slide using a needle tip moistened with the used reagent. The cover slip was closed gently to avoid air bubbles (any air bubbles present could be removed by passing the slide carefully over the small fame of a micro burner). An upright microscope, Nikon ECLIPSE E200, was used for the observations and combined with a camera (CANNON EOS 880D) for photomicrography. Microscopic observations were prepared at least 5 slides for each sample and performed under objectives with a magnification of 4x, 10x, and 40x.

Thin layer chromatography (TLC)

One g of powder of each sample was extracted with 20 mL of 80% ethanol via mechanical shaker (Daihan Scientific SHO-1D, Korea co. ltd) for 30 min. Then the sample extracts were filtered and evaporated using a rotary evaporator (Buchi 300, Switzerland). The water extracts obtained by using DI water instead of 80% ethanol. All extracts were kept in a refrigerator at 4°C until ready for use. For TLC, the sample extracts were dissolved in methanol to give concentrations of 5 mg/mL. Each sample was spotted (8 μL) in the form of bands 9 mm of bandwidth with an automatic TLC sampler (CAMAG^® Linomat 5) on an activated aluminum plate of precoated silica gel 60 GF254 (Merck, Germany): plate size 20×10 cm; saturated TLC tank for 1 h and then developed using a mobile phase system; chloroform: methanol: water (65: 40: 1) with 8 cm of developing distance. The TLC plate was subsequently dried and detected under UV 254 nm, UV 366 nm, sprayed with anisaldehyde TS and observed under white light and UV 366 nm using CAMAG^® TLC Visualizer 2 REF QS.

Physico-chemical properties

Various parameters, i.e. foreign matter, water content (loss on drying), total ash, acid-insoluble ash, water-soluble extractive and ethanol-soluble extractive were performed by using methods described in Thai Herbal Pharmacopoeia (DMSC, 2021). All values were obtained from analyses performed in triplicate for each sample.

Determination of total saponin content

The total saponin content of S. rugata was determined using a modified vanillin-sulfuric acid spectrophotometric method. Briefly, 1 g of dried S. rugata pod powder, passed through a 60-mesh sieve, was extracted in 20 mL of either deionized (DI) water or 80% ethanol using a shaker for 30 min. The extracts were then filtered through Whatman No. 1 filter paper and diluted 10-fold with DI water before analysis. Quillaja saponin (Sigma-Aldrich) was used as the reference standard. For the assay, 25 µL of the sample or standard was reacted with 25 µL of 8% vanillin in ethanol and 200 µL of 70% sulfuric acid in a 96-well microplate. The mixture was incubated at 80°C for 10 min, followed by cooling in an ice bath for 2 min. Absorbance was subsequently measured at 480 nm using a microplate reader (Biochrom^® EZ Read 2000). All experiments, including blank determinations, were performed in triplicate. The calibration curve was established using standard quillaja saponin at concentrations of 10, 100, 150, 250, and 300 μg/mL. The total saponin content was calculated from a quillaja saponin calibration curve and expressed as mg quillaja saponin equivalent/g dried herbal powder (mg QSE/g). The statistical analyses were performed using SPSS Statistics 17.0. Statistical differences between solvents were analysed using a paired t-test, while variations among different sample sources were evaluated via one-way ANOVA. The statistical differences were considered if P < 0.05.

RESULTS

Nucleotide sequence analysis and intraspecific variation of the six DNA barcode regions

Six specimens of S. rugata collected from Thailand were analysed for nucleotide sequences across six DNA barcode regions: ITS, matK, psbA-trnH, rbcL, trnL-F, and ycf1. These regions were used as molecular references for the authentication of their crude drugs. All regions were successfully amplified by PCR. The PCR products were verified using 1.8% agarose gel electrophoresis (e.g., rbcL and matK; Supplementary Figure S1) and subsequently sequenced using Sanger sequencing. The aligned nucleotide sequences for each locus are presented in Supplementary Information S2–S7. The length of the nucleotide sequences varied among the DNA regions, with ITS ranging from 530 to 532 bp, psbA-trnH from 416 to 418 bp, and trnL-F from 1,025 to 1,030 bp. In contrast, the sequences of matK, rbcL, and ycf1 were identical in length across all samples, measuring 864 bp, 723 bp, and 913 bp, respectively (Table 3). Intraspecific polymorphisms and gaps were observed in the ITS, matK, psbA-trnH, rbcL, and trnL-F regions, whereas no nucleotide variation was detected in the ycf1 region, as summarized in Table 3. Notably, all samples exhibited overlapping peaks in the DNA sequence electropherograms of ITS and trnL-F region. Specifically, a G base insertion was observed at nucleotide position 70 in the ITS region, and an A base insertion was found at nucleotide position 346 in the trnL-F region, resulting in overlapping peaks in all samples. An analysis of intraspecific divergence among the S. rugata specimens, using the K2P model of evolution with 10,000 bootstrap replications, revealed zero divergence across all six DNA barcode loci. However, the genetic distances for ITS, matK, and psbA-trnH ranged from 0 to 0.0019, 0.0023, and 0.0024, respectively. Five variable sites were identified in the ITS, matK, psbA-trnH, rbcL, and trnL-F regions. Based on the sequencing and genetic distance analyses, the nucleotide lengths of matK, rbcL, and ycf1 were consistent across all samples, measuring 864 bp, 723 bp, and 913 bp, respectively.

Table 3. Intraspecific polymorphism and length of nucleotide sequences in the six DNA regions of S. rugata samples.

Note: Asterisk (*) indicates the same nucleotide of top sequence; hyphen (-) indicates nucleotide deletion; R denotes A and G; W denoted A and T

Microscopic characterization

Histology of the pericarp, seed and cotyledon was investigated by transverse sectioning to study the arrangement and characteristic of the cells and tissues including cell inclusion as shown in Figures 2–6. A reddish-brown powder of pods with a characteristic odour and a slight sour taste was also microscopically observed. The prominent characters include exocarp showed epidermis and sclereids; mucilaginous cell and vascular bundle in mesocarp; elongated sclereids of endocarp; malpighian cells of palisade layer and sclerified parenchyma containing brown substances in seed coat; numerous starch grains and aleurone mass, and vascular bundle in seed part (Figure 7).

Figure 2. Transverse section of pericarp stained with fast green solution; 1 exocarp, 2 mesocarp, 3 endocarp, 4 sclereid, 5 mucilage cell, 6 vascular bundle, 7 parenchyma and 8 elongate sclereid.

Figure 3. Transverse section of pericarp stained with fast green solution; sclereid of exocarp (1), mucilage cell of mesocarp (2), parenchyma of mesocarp (3) and elongate sclereid of endocarp (4).

Figure 4. Transverse section of seed coat; 1 exotesta (palisade layer), 2 mesotesta, 3 cuticle, 4 light line, 5 malpighian cell of palisade layer, 6 lumen of malpighian cell and 7 sclerified parenchyma.

Figure 5. Transverse section of cotyledon stained with iodine solution (a) and picric acid (b); 1 abaxial and adaxial epidermis, 2 starch grains, 3 cotyledon parenchyma and 4 vascular bundle.

Figure 6. Transverse section of cotyledon stained with picric acid; 1 aleurone layer, 2 starch grains, 3 parenchyma containing starch grains and aleurone mass and 4 aleurone mass.

Figure 7. Powder of pod; outermost of exocarp in surface view (1), sclereid of exocarp (2), elongate sclereids of endocarp (3), elongate parenchyma (4), palisade layer with light line of testa (5), mucilage cells (6), starch grains (st) and aleurone layer (ar) (7), vessels (8) and thick-walled cells of tegmen (9).

Thin layer chromatography (TLC)

Chemical compounds of pod were separated by TLC from sample extracts of 80% ethanol and water as shown in Figure 8. TLC technique was successfully performed by using a mixture of chloroform: methanol: water (65: 40: 1) as the developing solvent and detecting under a UV wavelength at 254 nm, 366 nm, anisaldehyde TS and 366 nm after anisaldehyde TS derivatization. TLC chromatograms (Figure 8) showed a similar chemical pattern between the 80% ethanol and water extracts.

Figure 8. TLC chromatograms of pod from 80% ethanol (tracks 1-6) and water (tracks 7-12) extractions; mobile phase-chloroform: methanol: water (65: 40: 1); under UV 254 nm (a), under UV 366 nm (b), Anisaldehyde TS (c) and Anisaldehyde TS and under UV 366 nm (d).

Physico-chemical properties

The evaluation of the physico-chemical values, including foreign matter, water content, total ash, acid-insoluble ash and extractive values are the general requirements for the quality control of medicinal plant. These values of SR1 to SR6 used in this study are close to each other as shown in Table 4.

Table 4. Physico-chemical properties of pods.

Note: *NMT-Not more than, NLT-Not less than

Determination of total saponin content

The total saponin content in S. rugata pods, extracted with DI water and 80% ethanol, were determined using vanillin-sulfuric acid assay, with quillaja saponin as the standard compound. The results were expressed in mg QSE/g, based on the standard curve equation: y = 0.0017x + 0.0049, R² = 0.9923; where y is the average absorbance (n=3) at 480 nm and x is the concentration of standard quillaja saponin solution (µg/mL), as demonstrated in Figure 9.

Figure 9. Standard calibration curve of quillaja saponin (Error bars represent SD (n = 3)).

The total saponin content of six samples (SR1–SR6) from different sources are presented in Figure 10. The results revealed that the total saponins of S. rugata pods extracted with DI water and 80% ethanol were found to be 11.54–27.78 and 7.42–21.31 mg QSE/g, respectively. The results of the aqueous extract were significantly higher than those of the ethanolic extract, as analysed by a paired t-test (P-value < 0.05). SR1 exhibited the highest total saponin whilst SR6 was the lowest. One-way ANOVA statistical analysis revealed that the total saponin of these samples differed significantly when comparing the various raw material sources.

Figure 10. Total saponins of S. rugata pods (SR1–SR6), extracted with DI water and 80% ethanol; values within each column followed by different letters (a–i) indicate statistically significant differences (P < 0.05), as determined by one-way ANOVA.

DISCUSSION

Although the number of studies involving molecular identification, species and genetic diversity, and evolutionary or phylogenetic analyses using DNA barcoding has increased by a geometric average of 15.4 studies per year (Priyono et al., 2023), a review of the GenBank database reveals that nucleotide sequence data for S. rugata remain limited. As of September 9, 2024, only 57 nucleotide sequence samples were available for various gene loci (GenBank, September 9, 2024). This limited dataset highlights the need for further analysis and the development of comprehensive DNA barcode data to facilitate the accurate identification of S. rugata crude drugs, particularly for pharmacological and medicinal purposes. In this study, six DNA barcode regions (ITS, matK, rbcL, psbA-trnH, trnL-F, and ycf1) were employed to authenticate S. rugata samples. The core loci matK and rbcL are recommended by the CBOL Plant Working Group for their universality, while ITS and psbA–trnH are widely used as supplementary markers due to their higher interspecific variability and improved species-level resolution (Kress et al., 2005; CBOL Plant Working Group, 2009; China Plant BOL Group, 2011). Additionally, ycf1 and trnL-F were included as alternative markers with demonstrated high discriminatory power and reliable performance in phylogenetic and species identification studies (Taberlet et al., 1991; Dong et al., 2015). We successfully amplified and sequenced all S. rugata samples across all gene loci, ensuring comprehensive coverage of the selected barcode regions. The intraspecific divergence analysis among all S. rugata specimens, using the K2P model of evolution with 10,000 bootstrap replications, revealed zero divergence across all six DNA barcode loci. However, slight genetic distances were observed for the ITS, matK, and psbA-trnH regions, ranging from 0 to 0.0019, 0.0023, and 0.0024, respectively. These findings demonstrate that while the sequences are highly conserved, there is still minimal variation in specific loci, which could be relevant for differentiating closely related species. Our results align with previous studies, such as the work of Abdel-Hamid et al. (2021) and Ismail et al. (2020), who utilized rbcL and matK loci as effective DNA barcoding markers to distinguish between various Senegalia species collected from different regions of Saudi Arabia and Pakistan. Additionally, the matK gene, which encodes the maturase K enzyme in the chloroplast, has been reported as a suitable candidate for DNA barcoding within the Fabaceae family, helping to clarify phylogenetic relationships within this group (Abdelsalam et al., 2022).  Moreover, the use of other plastid regions, particularly rpl32-trnL, has been suggested for barcoding in Senegalia species, as demonstrated by Nevill et al. (2013).  Their study reported that chloroplast regions such as rpl32-trnL, psbA-trnH, trnL-F, and trnK had mixed success in resolving taxonomic differences in the studied taxa. They proposed that multi-locus data sets, which include multiple DNA regions, provide better resolution than single-locus data sets for species identification and phylogenetic analysis. The results of this study highlight the utility of DNA barcoding, particularly through multi-locus approaches, in accurately identifying S. rugata and potentially other medicinal species. In future studies, expanding the nucleotide sequence dataset of S. rugata will contribute to the establishment of a more comprehensive and robust reference database for species authentication. In addition, the incorporation of phylogenetic analyses and sequence similarity approaches will enable comparative evaluation with closely related taxa and improve species-level resolution. Furthermore, the development of rapid and reliable molecular identification techniques, such as Bar-HRM, LAMP, and ddPCR, will enhance the practical application of these findings for accurate discrimination of S. rugata in herbal materials.

The macroscopic characters of all samples were not different.  They had purplish brown or pinkish brown wrinkled pericarps, 30–180 mm long, 8–23 mm wide, somewhat constricted between seeds and seeds 6–16 per pod. Their powders were brown and mucilaginous, odour acidic and taste sour. Pharmacognostic studies of S. rugata pods including morphological and microscopical analyses, together with the physico-chemical, TLC and total saponin content evaluations, were carried out. The histological structure of the pod in transverse section showed an exocarp, mesocarp, endocarp, seed coat and seed (Figures 2–6). Microscopical characterization of powder presented the specific cells and tissues from pericarp, namely sclereid, elongate parenchyma, mucilage cells, elongate sclereids of endocarp and vessels. Malpighian cells of palisade layer of exotesta, starch grains and aleurone grains from seed were extensively observed (Figure 7). For physico-chemical parameters, trace amount of acid-insoluble ash was obtained which might be fewer inorganic compounds in pods. The water-soluble extractive content was 45% which was more than the ethanol-soluble extractive. These physico-chemical parameters indicate the soluble compounds particularly saponin though TLC chromatograms of 80% ethanol and water extracts were comparable. Physico-chemical specifications, i.e. foreign matter NMT 0.5 %(w/w), water content (loss on drying) NMT 11.0% (w/w), total ash NMT 4.5% (w/w), acid insoluble ash NMT 0.1% (w/w), water-soluble extractive NMT 38.0% (w/w) and ethanol-soluble extractive NLT 32.0% (w/w), could be used quantitatively for quality control of pods.  Previous report showed the physico-chemical values of a Shikakai sample collected in India (Khanpara et al., 2012). Certain results, i.e. loss on drying (10.00% (w/w)) and water-soluble extractive (43.25 %(w/w)) were comparable as the methods were possibly similar. However, alcohol-soluble extractive in this study was carried out by using methanol which was not the common solvent listed in Pharmacopoeias. Additionally, this report presented the ash value and acid insoluble ash analyses at 0.60% (w/w) and 16.66% (w/w), respectively which could not compare to our study.

The saponin content was quantified as quillaja saponin equivalent, and statistical analyses were conducted using SPSS Statistics 17.0, including paired t-tests and one-way ANOVA, to ensure a reliable evaluation of the active components in the pods of S. rugata. The saponin content in the aqueous extracts of six samples (SR1–SR6) ranged from 11.54 to 27.78 mg QSE/g, which was significantly higher than that observed in the 80% ethanol extracts (7.42–21.31 mg QSE/g). A previous study employing quantitative microscopy reported that the saponin content in S. rugata pods was 8.04% (Khanpara et al., 2012). Furthermore, spectrophotometric analysis indicated a total saponin content of 11.79% (w/w) in dried pod powder, expressed as quillaja saponin equivalent (Anamika et al., 2017). In addition, total saponin content extracted with 80% methanol and water was reported as 1.15 and 0.25 mg/g extract, respectively, expressed as diosgenin equivalent (Wisetkomolmat et al., 2020).  Saponins from S. rugata pods consist of a complex derivatives of triterpenes, monoterpenes and sugar moieties, which have been shown to inhibit pancreatic lipase activity and reduce lipid accumulation in 3T3-L1 adipocytes (Zhuoyue et al., 2021). The biotechnological applications of S. rugata saponins have also been widely explored. For instance, silk fibroin derived from silkworms contains sericin, a glue-like protein commonly used in biomedical applications such as wound dressing and drug delivery. Saponins isolated from S. rugata demonstrated effective degumming properties, enabling the extraction of silk fibroin from cocoons without the use of toxic chemicals or generating harmful effluents (Dan et al., 2022). Moreover, plant-based saponins from S. rugata pods have potential applications in textile processing. They function as biosurfactants in cotton scouring and in routine fabric washing, offering a sustainable alternative to conventional alkalis and enzymatic treatments (Thakker, 2020). The surfactant properties of these saponins have also attracted attention due to their natural origin, biodegradability, and eco-friendly characteristics, which make them suitable substitutes for synthetic chemicals in various industries. For example, saponins from S. rugata have demonstrated the ability to interact with cationic dyes such as Rhodamine B and undergo self-degradation under aerobic conditions at ambient temperature, highlighting their potential as environmentally friendly surfactants (Begum et al., 2024). In addition, aqueous extracts of S. rugata pods containing saponins have been utilized as natural surfactants, reducing agents, and stabilizers in the green synthesis of palladium nanoparticles. These nanoparticles exhibited excellent catalytic activity in Suzuki-Miyaura coupling reactions and could be reused for at least five cycles while maintaining high product yields (Gaikwad et al., 2019). Furthermore, optimized concentrations of saponins from S. rugata fruits have been shown to act as effective dispersants and stabilizers during the pipeline transport of iron ore particles (Gupta et al., 2024). Overall, S. rugata represents a promising natural source of saponins with significant potential for further research and development in health-related and biotechnological applications whenever its authentication and quality evaluation should be approved.

The authentication of S. rugata pods distributed throughout Thailand achieved through the integration of conventional pharmacognostic methods, including macroscopic and microscopic analyses, and modern techniques such as DNA barcoding. This demonstrates a valuable approach that can be applied to the authentication of other medicinal plants or the identification of plant materials sold in herbal markets. To ensure the quality, safety, and efficacy of medicinal plants, comprehensive evaluation of physicochemical parameters and chemical identification using simple chromatographic techniques, such as TLC, can be effectively implemented and scaled for industrial applications. These approaches collectively contribute to the ultimate goal of ensuring the quality, safety, and efficacy in the utilization of medicinal plants within the country.

CONCLUSION

This study is the first to report the integration of DNA barcoding techniques for analyzing nucleotide sequence variation across multiple barcode regions with pharmacognostic evaluations (morphological and microscopic analyses) as a robust method for authenticating S. rugata. TLC chromatograms of six S. Rugata pods are comparable and this chromatographic technique could be used for primarily chemical identification. Physico-chemical specifications and total saponin content using rapid spectrophotometry will aid in quantifying and control the pods’ quality. High saponin content in the pods could also be further developed as an ingredient in various health products such as wound dressing and biodegradable detergent.

SUPPORTING INFORMATION

The nucleotide sequences of six regions, including ITS, matK, psbA-trnH, rbcL, trnL-F and ycf1, in Senegalia rugata (Lam.) Britton & Rose are available as Supporting Information.

ACKNOWLEDGEMENTS

We are grateful to the Faculty of Pharmacy, Chiang Mai University for financial (Fiscal Year 2021) and equipment supports and thankful for technician of Laboratory of Pharmacognosy, Faculty of Pharmacy, Chiang Mai University to provide research facility.

AUTHOR CONTRIBUTIONS

Supattra Rungsimakan: Writing – Original Draft (Lead), Investigation (Lead), Visualization (Equal), Writing – Review & Editing (Equal); Wannaree Charoensup:  Writing – Original Draft (Lead), Visualization (Equal); Aekkhaluck Intharuksa: Formal Analysis (Equal), Investigation (Lead), Validation (Equal), Data Curation (Equal), Supervision (Equal), Writing – Review & Editing (Equal); Suthira Yanaso: Formal Analysis (Equal), Investigation (Lead), Validation (Equal), Data Curation (Equal), Supervision (Equal), Writing – Review & Editing (Equal); Ampai Phrutivorapongkul: Conceptualization (Lead), Data Curation (Lead), Funding Acquisition (Lead), Supervision (Lead), Investigation (Equal), Validation (Equal), Visualization (Equal), Writing – Review & Editing (Equal).

CONFLICT OF INTEREST

No potential conflict of interest was reported by the author (s).

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Supplementary information

Figure S1. Representative of gel electrophoresis of rbcL and matK regions in S. rugata samples. 1.8% Agarose gel electrophoresis showing PCR amplification of two chloroplast DNA barcode regions (rbcL and matK) from six S. rugata samples (SR1–SR6). Clear and distinct bands of the expected size were observed for all samples, indicating successful amplification of both loci. The lane labeled “–ve” represents the negative control, showing no amplification, while “M” denotes the DNA ladder used as a molecular size marker.

S2. Nucleotide Sequences of the ITS Region in Senegalia rugata (Lam.) Britton & Rose.

S3. Nucleotide Sequences of the matK Region in Senegalia rugata (Lam.) Britton & Rose.

S4. Nucleotide Sequences of the psbA-trnH Region in Senegalia rugata (Lam.) Britton & Rose.

S5. Nucleotide Sequences of the rbcL Region in Senegalia rugata (Lam.) Britton & Rose.

S6. Nucleotide Sequences of the trnL-F Region in Senegalia rugata (Lam.) Britton & Rose.

S7. Nucleotide Sequences of the ycf1 Region in Senegalia rugata (Lam.) Britton & Rose.