Phylogeny of Medicinal Phyllanthus Species in China Based on Nuclear ITS and Chloroplast atpB-rbcL Sequences and Multiplex PCR Detection Assay Analysis

Abstract

Plants of the genus Phyllanthus are commonly used in India, China and Korea for medicinal purposes. Although they are widely cultivated and marketed, there has been uncertainty about the efficacy of different species. A prerequisite of Good Agricultural Practice (GAP) is the authentication of relevant species, and this is now made unequivocal by applying DNA sequence tools. In this study the phylogenetic relationships among 18 Phyllanthus species found in China were investigated by DNA sequence analyses of the nuclear internal transcribed spacers (ITS1 and ITS2) along with the combined chloroplast atpB and rbcL sequences. Cladistic analysis indicated that this genus is paraphyletic and strongly supports the notion that two problematic and confusing species, P. niruri and P. amarus, are two individual, albeit closely related, species. We have also developed an ITS rDNA-based multiplex PCR assay to differentiate three medicinally important species, P. amarus, P. niruri and P. urinaria.

Abbreviations

atpB:ATP synthase subunit B

cpDNA:chloroplast DNA

ITS:18S-26S internal transcribed spacer region

MP:maximum parsimony

NJ:neighbor-joining

nrDNA:nuclear DNA

rbcL:ribulose-1,5-bisphosphate carboxylase large subunit

Phyllanthaceae - Phyllanthus - internal transcribed spacer (ITS) region - atpB gene - rbcL gene - molecular phylogeny - multiplex PCR

References

• 1 Unander D W, Webster G L, Blumberg B S. Records of usage or assays in Phyllanthus (Euphorbiaceae) I. subgenera Isocladus, Kirganelia, Cicca and Emblica .  J Ethnopharmacol. 1990;  30 233-64
• 2 Li P T. Family Euphorbiaceae.  In: Flora of Reipublicae Popularis Sinicae. Volume 44, Part 2 Beijing; Science Press 1994: pp 78-116
• 3 Bandyopadhyay S K, Pakrashi S C, Pakrashi A. The role of antioxidant activity of Phyllanthus emblica fruits on prevention from indomethacin induced gastric ulcer.  J Ethnopharmacol. 2000;  70 171-6
• 4 Shen Z Q, Dong Z J, Peng H, Liu J K. Modulation of PAI-1 and tPA activity and thrombolytic effects of corilagin.  Planta Med. 2003;  69 1109-12
• 5 Thyagarajan S P, Subramanian S, Thirunalasundari T, Venkateswaran P S, Blumberg B S. Effect of Phyllanthus amarus on chronic carriers of hepatitis B virus.  Lancet. 1988;  2 764-6
• 6 Lee C D, Ott M, Thyagarajan S P, Shafritz D A, Burk R D, Gupta S. Phyllanthus amarus down-regulates hepatitis B virus mRNA transcription and replication.  Eur J Clin Invest. 1996;  26 1069-76
• 7 Notka F, Meier G R, Wagner R. Inhibition of wild-type human immunodeficiency virus and reverse transcriptase inhibitor-resistant variants by Phyllanthus amarus .  Antiviral Res. 2003;  58 175-86
• 8 Unander D W, Webster G L, Blumberg B S. Uses and bioassays in Phyllanthus (Euphorbiaceae): a compilation II. The subgenus Phyllanthus .  J Ethnopharmacol. 1991;  34 97-133
• 9 Huang S T, Yang R C, Yang L J, Lee P N, Pang J HS. Phyllanthus urinaria triggers the apoptosis and Bcl-2 down-regulation in Lewis lung carcinoma cells.  Life Sci. 2003;  72 1705-16
• 10 Giridharan P, Somasundaram S T, Perumal K, Vishwakarma R A, Karthikeyan N P, Velmurugan R. et al . Novel substituted methylenedioxy lignan suppresses proliferation of cancer cells by inhibiting telomerase and activation of c-myc and caspases leading to apoptosis.  Br J Cancer. 2002;  87 98-105
• 11 Wang F, Chien N, Zhang Y, Yang H. Pollen flora of China. 2nd edition Beijing; Science Press 1997: pp 178-89
• 12 Ma X Q, Duan J A, Zhu D Y, Dong T TX, Tsim K WK. Species identification of Huangqi (Radix Astragali) by DNA sequence of its 5S rRNA spacer domain.  Phytochemistry. 2000;  54 363-8
• 13 Lau D TW, Shau P C, Wang J, But P PH. Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA.  Planta Med. 2001;  67 456-60
• 14 Kuo H C, Su Y L, Yang H L, Chen T Y. Identification of Chinese medicinal fungus Cordyceps sinensis by PCR-single-stranded conformation polymorphism and phylogenetic relationship.  J Agric Food Chem. 2005;  53 3963-8
• 15 APG (The Angiosperm Phylogeny Group). An update of the Angiosperm phylogeny group classification for the orders and families of flowering plants: APG II. Bot J Linn Soc 2003 141: 399-436
• 16 Wurdack K J, Hoffmann P, Samuel R, de Brujin A van der, Bank M, Chase M W. Molecular phylogenetic analysis of Phyllanthaceae (Phyllanthoideae pro parte, Euphorbiaceae sensu lato) using plastid rbcL DNA sequences.  Am J Bot. 2004;  91 1882-900
• 17 Kathriarachchi H, Hoffmann P, Samuel R, Wurdack K J, Chase M W. Molecular phylogenetics of Phyllanthaceae inferred from five genes (plastid atpB, matK, 3'ndhF, rbcL, and nuclear PHYC).  Mol Phylogenet Evol. 2005;  36 112-34
• 18 White T J, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Sninsky J, White TJ, editors PCR protocols: a guide to methods and application. San Diego; Academic Press 1990: pp 315-22
• 19 Swofford D L. PAUP*: phylogenetic analysis using parsimony (*and other methods). Massachusetts; Sinauer Associates, Inc. 1998
• 20 Webster G L. A monographic study of the West Indian species of Phyllanthus .  J Arnold Arbor. 1957;  38 295-373