DNA barcoding and High-Performance Liquid Chromatography as golden standards of authentication in Blumea balsamifera and Ehretia microphylla products
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Abstract. Lola AMA, Binag SDA, Alfeche NKG, Alejandro GJD. 2025. DNA barcoding and High-Performance Liquid Chromatography as golden standards of authentication in Blumea balsamifera and Ehretia microphylla products. Biodiversitas 26: 1258-1270. In the Philippines, the Department of Health (DOH) has included Blumea balsamifera (sambong) and Ehretia microphylla (tsaang-gubat) among the top ten medicinal plants in the country. Sambong is known for treating urinary stones, while tsaang-gubat is used for helping increase the motility of intestines. Despite their known healing effects, there remains a lack of strict implementation for the approval of these products to be commercially available. This study aimed to employ DNA barcoding and High-Performance Liquid Chromatography (HPLC) by creating a Standard Reference Material (SRM) for both methods. Eight blindly selected sambong herbal medicinal products (HMPs), and six blindly selected tsaang-gubat HMPs are authenticated using the stated methods. DNA barcoding reveals that samples BB4 and EMP1 are cases of substitution for the products containing Oryza glaberrima and Cymbopogon citratus, respectively. HPLC analysis also reveals that all sambong derived HMPs contain quercetin, except for samples BB2, BB4, and BMP3, whereas for the tsaang-gubat herbal products, only sample EMP1 does not contain rosmarinic acid as its active compound. Dissidence in the results indicates that both protocols should be used in coordination as standard authentication tools to promote accuracy of the results and to provide safety to all the consumers. Furthermore, DNA barcoding authenticates the actual plant material used while HPLC checks the presence of active compounds.
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References
Abubakar BM, Salleh FM, Omar MSS, Wagiran A. 2017. DNA barcoding and chromatography fingerprints for the authentication of botanicals in herbal medicinal products. Evid?Based Complement Alternat Med 2017: 1352948. DOI: 10.1155/2017/1352948.
Abubakar BM, Salleh FM, Shamsir Omar MS, Wagiran A. 2018. Assessing product adulteration of Eurycoma longifolia (tongkat ali) herbal medicinal product using DNA barcoding and HPLC analysis. Pharm Biol 56 (1): 368-377. DOI: 10.1080/13880209.2018.1479869.
Alfeche NK, Binag SD, Medecilo MM, Alejandro GJ. 2019. Standard reference material (SRM) DNA barcode library approach for authenticating Antidesma bunius (L.) Spreng. (bignay) derived herbal medicinal products. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 36 (12): 1777-1786. DOI: 10.1080/19440049.2019.1670868.
Anantha Narayana DB, Johnson ST. 2019. DNA barcoding in authentication of herbal raw materials, extracts and dietary supplements: A perspective. Plant Biotechnol Rep 13 (3): 201-210. DOI: 10.1007/s11816-019-00538-z.
Buddhachat K, Osathanunkul M, Madesis P, Chomdej S, Ongchai S. 2015. Authenticity analyses of Phyllanthus amarus using barcoding coupled with HRM analysis to control its quality for medicinal plant product. Gene 573 (1): 84-90. DOI: 10.1016/j.gene.2015.07.046.
Cafino EJV, Lirazan MB, Marfori EC. 2016. A simple HPLC method for the analysis of [6]-gingerol produced by multiple shoot culture of ginger (Zingiber officinale). Intl J Pharmacogn Phytochem Res 8 (1): 38-42.
Catublas HAL. 2016. Knowledge, attitudes and practices in the use of herbal medicine: The case of urban and rural mothers in the Philippines. Mahidol Univ J Pharm Sci 43 (1): 1-16. DOI: 10.14456/mujps.2016.1.
Chaowuttikul C, Palanuvej C, Ruangrungsi N. 2020. Quantification of chlorogenic acid, rosmarinic acid, and caffeic acid contents in selected Thai medicinal plants using RP-HPLC-DAD. Braz J Pharm Sci 56: e17547. DOI: 10.1590/s2175-97902019000317547.
Custers D, Van Praag N, Courselle P, Apers S, Deconinck E. 2017. Chromatographic fingerprinting as a strategy to identify regulated plants in illegal herbal supplements. Talanta 164: 490-502. DOI: 10.1016/j.talanta.2016.12.008.
de Guzman CC. 2013. Country status report on medicinal and aromatic plants in the Philippines. In: Paroda R, Dasgupta S, Mal B, Ghosh SP, Pareek SK (eds). Expert Consultation on Promotion of Medicinal and Aromatic Plants in the Asia-Pacific Region. Asia-Pacific Association of Agricultural Research Institutions (APAARI), Bangkok.
Fay MF, Swensen SM, Chase MW. 1997. Taxonomic affinities of Medusagyne oppositifolia (Medusagynaceae). Kew Bull 52: 111-120. DOI: 10.2307/4117844.
FDA [Food and Drug Administration Philippines]. 2018. Manufacture, sale and distribution of traditional and alternative medicines. https://ww2.fda.gov.ph/index.php/issuances-2/pharml-1/pharml-fda-circular/161090-fda-circular-no-2014-015.
Frigerio J, Agostinetto G, Mezzasalma V, De Mattia F, Labra M, Bruno A. 2021. DNA-based herbal teas’ authentication: An ITS2 and psbA-trnH multi-marker DNA metabarcoding approach. Plants 10 (10): 2120. DOI: 10.3390/plants10102120.
Galimberti A, Casiraghi M, Bruni I, Guzzetti L, Cortis P, Berterame NM, Labra M. 2019. From DNA barcoding to personalized nutrition: The evolution of food traceability. Curr Opin Food Sci 28: 41-48. DOI: 10.1016/j.cofs.2019.07.008.
Ghorbani S, Sonboli A, Ebrahimi SN, Mirjalili MH. 2020. Molecular authentication and phytochemical assessment of Ruscus hyrcanus Woron. (Asparagaceae) based on trnH-psbA barcoding and HPLC-PDA analysis. Biocatal Agric Biotechnol 25: 101585. DOI: 10.1016/j.bcab.2020.101585.
Hassan B. 2012. HPLC uses and importance in the pharmaceutical and industrial field. Pharm Anal Acta 3: 9. DOI: 10.4172/2153-2435.1000E133.
Hebert PD, Cywinska A, Ball SL, deDewaard JR. 2003. Biological identifications through DNA barcodes. Proc R Soc Lond B Biol Sci B: Biol Sci 270 (1512): 313-321. DOI: 10.1098/rspb.2002.2218.
Hollingsworth PM, Graham SW, Little DP. 2011. Choosing and using a plant DNA barcode. PloS One 6 (5): e19254. DOI: 10.1371/journal.pone.0019254.
Ichim MC. 2019. The DNA-based authentication of commercial herbal products reveals their globally widespread adulteration. Front Pharmacol 10: 1227. DOI: 10.3389/fphar.2019.01227.
Jirakitticharoen S, Wisuitiprot W, Jitareerat P, Wongs-Aree C. 2022. Phenolics, antioxidant and antibacterial activities of immature and mature Blumea balsamifera leaf extracts eluted with different solvents. J Trop Med 2022: 7794227. DOI: 10.1155/2022/7794227.
Kool A, De Boer HJ, Kruger A, Rydberg A, Abbad A, Bjork L, Martin G. 2012. Molecular identification of commercialized medicinal plants in southern Morocco. PLoS One 7 (6): e39459. DOI: 10.1371/journal.pone.0039459.
Kumar S, Stetcher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33 (7): 1870-1874. DOI: 10.1093/molbev/msw054.
Kuzmina ML, Johnson KL, Barron HR, Hebert PD. 2012. Identification of the vascular plants of Churchill, Manitoba, using a DNA barcode library. BMC Ecol 12: 25. DOI: 10.1186/1472-6785-12-25.
Legaspi CLB, Bagaoisan DMA. 2020. Ehretia microphylla tablet formulation for biliary and gastrointestinal colic: A review of its phytochemical constituents, pharmacologic activities and clinical researches. Acta Med Philipp 54 (1): 80-85. DOI: 10.47895/amp.v54i1.1108.
Liang YZ, Xie P, Chan K. 2004. Quality control of herbal medicines. J Chromatogr B Analyt Technol Biomed Life Sci 812 (1-2): 53-70. DOI: 10.1016/j.jchromb.2004.08.041.
Lv YN, Yang CY, Shi LC, Zhang ZL, Xu AS, Zhang LX, Li XL, Li HT. 2020. Identification of medicinal plants within the Apocynaceae family using ITS2 and psbA-trnH barcodes. Chin J Nat Med 18 (8): 594-605. DOI: 10.1016/S1875-5364(20)30071-6.
Mageswari S, Karpagam S. 2015. Evaluation of antimicrobial studies on root of Carmona retusa (Vahl.) Masam. Intl J Curr Pharm Res 7 (3): 60-63.
Makino T, Furuta Y, Wakushima H, Fujii H, Saito KI, Kano Y. 2003. Anti-allergic effect of Perilla fructescens and its active constituents. Phytother Res 17: 240-243. DOI: 10.1002/ptr.1115.
Maramba-Lazarte CC, Cavinta LL, Sara, MCL. 2020. Antibacterial activity of guyabano, ulasimang bato, sambong, and tsaang gubat leaf extracts against common drug-resistant bacteria. Acta Med Philipp 54 (1): 17-21. DOI: 10.47895/amp.v54i1.1087.
Michel CI, Meyer RS, Taveras Y, Molina J. 2016. The nuclear internal transcribed spacer (ITS2) as a practical plant DNA barcode for herbal medicines. J Appl Res Med Aromat Plants 3 (3): 94-100. DOI: 10.1016/j.jarmap.2016.02.002.
Mishra P, Kumar A, Nagireddy A, Mani DN, Shukla AK, Tiwari R, Sundaresan V. 2016. DNA barcoding: An efficient tool to overcome authentication challenges in the herbal market. Plant Biotechnol J 14 (1): 8-21. DOI: 10.1111/pbi.12419.
Monroy-Garcia IN, Carranza-Torres IE, Carranza-Rosales P, Oyón-ardoiz M, García-Estévez I, Ayala-zavala JF, Viveros-valdez E. 2021. Phenolic profiles and biological activities of extracts from edible wild fruits Ehretia tinifolia and Sideroxylon lanuginosum. Foods 10 (11): 2710. DOI: 10.3390/foods10112710.
Montealegre CM, De Leon RL. 2016. Blumea balsamifera (sambong) tea as a therapeutic drink for calcium oxalate stones. MATEC Web Conf 62: 02002. DOI: 10.1051/matecconf/20166202002.
Nessa F, Ismail Z, Karupiah S, Mohamed N. 2005. RP-HPLC method for the quantitative analysis of naturally occurring flavonoids in leaves of Blumea balsamifera DC. J Chromatogr Sci 43 (8): 416-420. DOI: 10.1093/chromsci/43.8.416.
Newmaster SG, Grguric M, Shanmughanandhan D, Ramalingam S, Ragupathy S. 2013. DNA barcoding detects contamination and substitution in North American herbal products. BMC Med 11 (1): 222. DOI: 10.1186/s12916-024-03504-x.
Olivar J, Alaba J, Atienza J, Tan J, Umali IV M, Alejandro GJ. 2016. Establishment of a Standard Reference Material (SRM) herbal DNA barcode library of Vitex negundo L. (lagundi) for quality control measures. Food Addit Contam: Part A 33 (5): 741-748. DOI: 10.1080/19440049.2016.1166525.
Orman E, Bekoe SO, Jato J, Spiegler V, Asare-Nkansah S, Agyare C, Hensel A, Bekoe EO. 2022. Quality assessment of African herbal medicine: A systematic review and the way forward. Fitoterapia 162: 105287. DOI: 10.1016/j.fitote.2022.105287.
Palhares RM, Goncalves M, dos Santos B, Pereira G, das Graças Lins Brandão M, Oliveira G. 2015. Medicinal plants recommended by the world health organization: DNA barcode identification associated with chemical analyses guarantees their quality. PLoS One 10 (5): e0127866. DOI: 10.1371/journal.pone.0127866.
Pedales R, Damatac A, Limbo C, Marquez C, Navarro A, Molina J. 2016. DNA barcoding of Philippine herbal medicinal products. J AOAC Intl 99 (6): 1479-1489. DOI: 10.5740/jaoacint.16-0185.
Penecilla GL, Magno CP. 2011. Antibacterial activity of extracts of twelve common medicinal plants from the Philippines. J Med Plants Res 5 (16): 3975-3981. DOI: 10.5897/JMPR.9000122.
Rasonabe ZMP, Cruz JD, Tiausas CG, Areza F. 2023. Chemical composition and antimicrobial activity of the extracts and essential oil of Blumea balsamifera from the Philippines. Intl J Herb Med 11 (1): 6-14. DOI: 10.22271/flora.2023.v11.i1a.847.
Republic Act No. 8423. 1997. Traditional and Alternative Medicine Act (TAMA). The LAWPHIL Project. http://www.lawphil.net/statutes/ repacts/ra1997/ra_8423_1997.html
Sahoo N, Manchikanti P, Dey S. 2010. Herbal drugs: Standards and regulation. Fitoterapia 81: 462-471. DOI: 10.1016/j.fitote.2010.02.001.
Seethapathy GS, Balasubramani SP, Venkatasubramanian P. 2014. nrDNA ITS sequence based SCAR marker to authenticate Aconitum heterophyllum and Cyperus rotundus in Ayurvedic raw drug source andprepared herbal products. Food Chem 145: 1015-1020. DOI: 10.1016/j.foodchem.2013.09.027.
Semagn K. 2014. Leaf tissue sampling and DNA extraction protocols. Methods Mol Biol 1115: 53-67. DOI: 10.1007/978-1-62703-767-9_3.
Shukla A, Kaur A. 2018. A systematic review of traditional uses bioactive phytoconstituents of genus Ehretia. Asian J Pharm Clin Res 11 (6): 88-100. DOI: 10.22159/ajpcr.2018.v11i6.25178.
Srirama R, Santhosh Kumar JU, Seethapathy GS, Newmaster SG, Ragupathy S, Ganeshaiah KN, Shaanker RU, Ravikanth G. 2017. Species adulteration in the herbal trade: causes, consequences and mitigation. Drug Saf 40: 651-661. DOI: 10.1007/s40264-017-0527-0.
Tarmizi AAA, Wagiran A, Mohd Salleh F, Chua LS, Abdullah FI, Hasham R, Binte Mostafiz S. 2021. Integrated approach for species identification and quality analysis for Labisia pumila using DNA barcoding and HPLC. Plants 10: 717. DOI: 10.3390/plants10040717.
Techen N, Parveen I, Pan Z, Khan IA. 2014. DNA barcoding of medicinal plant material for identification. Curr Opin Biotechnol 25: 103-110. DOI: 10.1016/j.copbio.2013.09.010.
Tolentino RD, Tomas VCB, Travezonda JC, Magnaye BP. 2019. Herbal medicine utilization among Batangueños. Asian Pac J Educ Arts Sci 6 (1): 9-22.
Toralba JV, Quiming NS, Palacpac JS. 2015. RP-HPLC analysis of quercetin in the extract of sambong (Blumea balsamifera (L.) DC.) leaves. Sci Diliman 27 (1): 48-63.
Tupas GD, Gido LJFJ. 2021. Perspective on herbal medicine in the Philippines, economic demands, quality control, and regulation. In: Egbuna C, Mishra AP, Goyal MR (eds). Preparation of Phytopharmaceuticals for the Management of Disorders. Academic Press, London. DOI: 10.1016/B978-0-12-820284-5.00009-5.
Umali FAC, Chua AH. 2017. Ehretia microphylla (Tsaang gubat) versus Loratadine as treatment for allergic rhinitis: A randomized controlled trial. Philipp J Otolaryngol Head Neck Surg 32 (2): 6-10. DOI: 10.32412/pjohns.v32i2.57.
Upton R, David B, Gafner S, Glasl S. 2020. Botanical ingredient identification and quality assessment: Strengths and limitations of analytical techniques. Phytochem Rev 19: 1157-1177. DOI: 10.1007/s11101-019-09625-z.
Vassou SL, Kusuma G, Parani M. 2015. DNA barcoding for species identification from dried and powdered parts: A case study with authentication of the raw drug market samples of Sida cordifolia. Gene 559 (1): 86-93. DOI: 10.1016/j.gene.2015.01.025.
Wang H, Provan GJ, Helliwell K. 2003. Determination of rosmarinic acid and caffeic acid in aromatic herbs by HPLC. Food Chem 87 (2): 307-311. DOI: 10.1016/j.foodchem.2003.12.029.
Xu H, Li P, Ren G, Wang Y, Jiang D, Liu C. 2021. Authentication of three source spices of Arnebiae radix using DNA barcoding and HPLC. Front Pharmacol 12: 677014. DOI: 10.3389/fphar.2021.677014.
Yang J, Dong LL, Wei GF, Hu HY, Zhu GW, Zhang J, Chen SL. 2018. Identification and quality analysis of Panax notoginseng and Panax vietnamensis var. fuscidicus through integrated DNA barcoding and HPLC. Chin Herb Med 10: 177-183. DOI: 10.1016/j.chmed.2018.03.008.
Zarsuelo MAM, Zordilla ZD, Anacio DB, 2018. Review of regulatory policies on and benefits of herbal medicine in the Philippines. Acta Med Philipp 52 (5): 473-479. DOI: 10.47895/amp.v52i5.334.
Zhang Z, Zhang Y, Zhang Z, Yao H, Liu H, Ben GZ, Liao Y. 2016. Comparative analysis of DNA barcoding and HPLC fingerprint to trace species of Phellodendri Cortex, an important traditional Chinese medicine from multiple sources. Biol Pharm Bull 39 (8): 1325-1330. DOI: 10.1248/bpb.b16-00210.