DNA Barcoding for the Identification of Phyllanthus Taxa Used Medicinally in Brazil

Abstract

Plants of the genus Phyllanthus, principally Phyllanthus amarus, Phyllanthus urinaria, Phyllanthus niruri, and Phyllanthus tenellus, are used in Brazilian folk medicine to treat kidney stones as well as other ailments, where the latter two species are listed in the Brazilian Pharmacopeia as quebra-pedra (stone-breaker). However, only P. niruri has been shown to be effective in a clinical setting. Nuclear ribosomal internal transcribed spacer (ITS1 – 5.8S rRNA-ITS2), internal transcribed spacer 2, and chloroplasts rbcL, matK, psbA-trnH, trnL, and trnL-trnF were screened for their potential as DNA barcodes for the identification of 48 Phyllanthus taxa in Brazilian medicinal plant germplasm banks and in “living pharmacies”. The markers were also tested for their ability to validate four commercial herbal teas labelled as quebra-pedra. Using the criterion of high clade posterior probability in Bayesian phylogenetic analysis, the internal transcribed spacer, internal transcribed spacer 2, and chloroplast matK, psbA-trnH, trnL, and trnL-trnF markers all reliably differentiated the four Phyllanthus species, with the internal transcribed spacer and matK possessing the additional advantage that the genus is well represented for these markers in the Genbank database. However, in the case of rbcL, posterior probability for some clades was low and while P. amarus and P. tenellus formed monophyletic groups, P. niruri and P. urinaria accessions could not be reliably distinguished with this marker. Packaged dried quebra-pedra herb from three Brazilian commercial suppliers comprised P. tenellus, but one sample was also found to be mixed with alfalfa (Medicago sativa). An herb marketed as quebra-pedra from a fourth supplier was found to be composed of a mixture of Desmodium barbatum and P. niruri.

Key words

Phyllanthus - Phyllanthaceae - DNA barcoding - urolithiasis - folk medicine

• References

• 1 Agência Nacional de Vigilância Sanitária (ANVISA). Formulário de Fitoterápicos da Farmacopéia Brasileira. Available at: http://www.anvisa.gov.br/hotsite/farmacopeiabrasileira/conteudo/Formulario_de_Fitoterapicos_da_Farmacopeia_Brasileira.pdf Accessed March 27, 2018
• 2 Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS). Available at: http://portalarquivos.saude.gov.br/images/pdf/2014/maio/07/renisus.pdf Accessed March 27, 2018
• 3 Matos FJA. O projeto Farmácias – Vivas e a fitoterapia no nordeste do Brasil. Rev Cienc Agrovet 2006; 5 Número Especial 1 (Plantas Medicinais): 24–32
• 4 Barbosa K. Há indícios de que chá de quebra-pedra seja eficaz para litíase renal. Arq Bras Fitomed Cientif 2006; 6: 86-88
• 5 Barros ME. Efeito do Phyllanthus niruri (quebra-pedra) sobre a cristalização de oxalato de cálcio in vitro. [PhD Thesis]. São Paulo: Escola Paulista de Medicina, UNIFESP; 2001
• 6 Dawson CH, Tomson CRV. Kidney stone disease: pathophysiology, investigation and medical treatment. Clin Med 2012; 12: 467-471
• 7 Lorenzi H, Matos FJA. Plantas Medicinais no Brasil: nativas e exóticas cultivadas. Nova Odessa, São Paulo: Instituto Plantarum; 2002: 512
• 8 Calixto JB, Santos AR, Cechinel Filho V, Yunes RA. A review of the plants of the genus Phyllanthus: their chemistry, pharmacology, and therapeutic potential. Med Res Rev 1998; 18: 225-258
• 9 Silva MJ, Sales MF. O gênero Phyllanthus L. (Phyllantheae – Euphorbiaceae Juss.) no bioma Caatinga do estado de Pernambuco – Brasil. Rodriguesia 2004; 55: 101-126
• 10 Zappi DC. BFG – THE BRAZIL FLORA GROUP. Growing knowledge: an overview of Seed Plant diversity in Brazil. Rodriguésia 2015; 66: 1085-1113
• 11 Silva MJ, Sales MF. Phyllanthus L. (Phyllanthaceae) em Pernambuco, Brasil. Acta Bot Bras 2007; 21: 79-98
• 12 Marques LC. Phyllanthus niruri (Quebra-Pedra) no Tratamento de Urolitíase: Proposta de Documentação para Registro Simplificado como Fitoterápico. Rev Fitos 2010; 5: 20-33
• 13 Boim MA, Heilberg IP, Schor N. Phyllanthus niruri as a promising alternative treatment for nephrolithiasis. Int Braz J Urol 2010; 36: 657-664
• 14 Ignácio SRN, Ferreira JLP, Almeida MB, Kubelka CF. Nitric oxide production by murine peritoneal macrophages in vitro and in vivo treated with Phyllanthus tenellus extracts. J Ethnopharm 2001; 74: 181-187
• 15 Santos ARS, Cechinel Filho V, Viana AM, Moreno FN, Campos MM, Yunes RA, Calixto JB. Analgesic effects of callus culture extracts from selected species of Phyllanthus in mice. J Pharm Pharmacol 1994; 46: 755-759
• 16 Asare GA, Addo P, Bugyei K, Gyan B, Adjei S, Otu-Nyarko LS, Wiredu EK, Nyarko A. Acute toxicity studies of aqueous leaf extract of Phyllanthus niruri . Interdiscip Toxicol 2011; 4: 206-210
• 17 Kushwaha SK, Dashora A, Dashora N, Patel JR, Kori ML. Acute oral toxicity studies of the standardized methanolic extract of Phyllanthus amarus Schum & Thonn. J Pharm Res 2013; 6: 720-724
• 18 Silva TCL, Veras Filho J, Araújo J, Albuquerque UP, Lima V, Amorim ELC. Acute toxicity study of stone-breaker (Phyllanthus tenellus Roxb.). Rev Ciênc Farm Básica Apl 2012; 33: 205-210
• 19 Martins ER, Lima LR, Cordeiro I. Phyllanthus (Phyllanthaceae) no estado do Rio de Janeiro. Rodriguésia 2014; 65: 405-424
• 20 Newmaster SG, Grguric M, Shanmughanandhan D, Ramalingam S, Ragupathy S. DNA barcoding detects contamination and substitution in North American herbal products. BMC Med 2013; 11: 222
• 21 Hollingsworth PM, Graham SW, Little DP. Choosing and using a plant DNA barcode. PLoS One 2011; 6: e19254
• 22 Kathriarachchi K, Samuel R, Hoffmann P, Mlinarec J, Wurdack KJ, Ralimanana H, Stuessy TF, Chase MW. Phylogenetics of tribe Phyllantheae (Phyllanthaceae; Euphorbiaceae sensu lato) based on nrITS and plastid matK DNA sequence data. Am J Bot 2006; 93: 637-655
• 23 China Plant BOL Group, Li DZ, Gao LM, Li HT, Wang H, Ge XJ, Liu JQ, Chen ZD, Zhou SL, Chen SL, Yang JB, Fu CX, Zeng CX, Yan HF, Zhu YJ, Sun YS, Chen SY, Zhao L, Wang K, Yang T, Duan GW. Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants. Proc Natl Acad Sci U S A 2011; 108: 19641-19646
• 24 Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 2010; 5: e8613
• 25 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215: 403-410
• 26 Aita AM, Matsuura HN, Machado CA, Ritter MR. Espécies medicinais comercializadas como “quebra-pedras” em Porto Alegre, Rio Grande do Sul, Brasil. Rev Bras Farmacogn 2009; 19: 471-477
• 27 Sarin B, Verma N, Martín JP, Mohanty A. An overview of important ethnomedicinal herbs of phyllanthus species: present status and future prospects. Sci World J 2014; 2014: 839172
• 28 Srirama R, Senthilkumar U, Sreejayan N, Ravikantha G, Gurumurthyd BR, Shivannae MB, Sanjappab M, Ganeshaiaha KN, Shaanker RU. Assessing species admixtures in raw drug trade of Phyllanthus, a hepato-protective plant using molecular tools. J Ethnopharmacol 2010; 130: 208-215
• 29 Palhares RM, Gonçalves Drummond M, Dos Santos Alves Figueiredo Brasil B. Pereira Cosenza G, das Graças Lins Brandão M, Oliveira G. Medicinal plants recommended by the world health organization: DNA barcode identification associated with chemical analyses guarantees their quality. PLoS One 2015; 10: e0127866
• 30 Kool A, de Boer HJ, Krüger Å, Rydberg A, Abbad A, Björk L, Martin G. Molecular identification of commercialized medicinal plants in Southern Morocco. PLoS One 2012; 7: e39459
• 31 De Boer HJ, Ouarghidi A, Martin G, Abbad A, Kool A. DNA barcoding reveals limited accuracy of identifications based on folk taxonomy. PLoS One 2014; 9: e84291
• 32 Ghorbani A, Saeedi Y, de Boer HJ. Unidentifiable by morphology: DNA barcoding of plant material in local markets in Iran. PLoS One 2017; 12: e0175722
• 33 Palhares RM, Drummond M, Brasil BS, Krettli AU, Oliveira GC, Brandão MG. The use of an integrated molecular-, chemical- and biological-based approach for promoting the better use and conservation of medicinal species: a case study of Brazilian quinas. J Ethnopharmacol 2014; 155: 815-822
• 34 Brasil. Ministério da Saúde. Agência Nacional de Vigilância Sanitária. Resolução da Diretoria Colegiada n. 18, de 3 de abril de 2013. Dispõe sobre as boas práticas de processamento e armazenamento de plantas medicinais, preparação e dispensação de produtos magistrais e oficinais de plantas medicinais e fitoterápicos em farmácias vivas no âmbito do Sistema Único de Saúde (SUS). Diário Oficial [da] União da República Federativa do Brasil, Brasília, DF, 5 April 2013. Available at: http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2013/rdc0018_03_04_2013.html Accessed June 1, 2018
• 35 Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 1987; 19: 11-15
• 36 Wen J, Zimmer EA. Phylogeny and biogeography of Panax L. (the ginseng genus, Araliaceae): inferences from its sequences of nuclear ribosomal DNA. Mol Phylogenet Evol 1996; 6: 167-177
• 37 Taberlet P, Gielly L, Pautou G, Bouvet J. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 1991; 17: 1105-1109
• 38 Tate JA, Simpson BB. Paraphyly of Tarasa (Malvaceae) and diverse origins of the polyploid species. Syst Bot 2003; 28: 723-737
• 39 Sang T, Crawford DJ, Stuessy TF. Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae). Am J Bot 1997; 84: 1120-1136
• 40 Kress WJ, Erickson DL, Jones FA, Swenson NG, Perez R, Sanjur O, Bermingham E. Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proc Natl Acad Sci U S A 2009; 106: 18621-18626
• 41 Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 1999; 41: 95-98
• 42 Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 2013; 30: 772-780
• 43 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 2012; 61: 539-542
• 44 Huelsenbeck JP, Larget B, Alfaro ME. Bayesian phylogenetic model selection using reversible jump Markov chain Monte Carlo. Mol Biol Evol 2004; 21: 1123-1133