Food Chemistry 141 (2013) 2522–2525

Contents lists available at SciVerse ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Short communication

Application of novel loop-mediated isothermal amplification (LAMP) forrapid authentication of the herbal tea ingredient Hedyotis diffusa Willd.

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.05.085

⇑ Corresponding author at: School of Life Sciences, The Chinese University ofHong Kong, Shatin, N.T., Hong Kong. Tel.: +852 39431363; fax: +852 26037246.

E-mail addresses: liming@cuhk.edu.hk (M. Li), wongylsally@cuhk.edu.hk (Y.-L.Wong), aijiang0@hotmail.com (L.-L. Jiang), klwong@cuhk.edu.hk (K.-L. Wong),alicewonginss@gmail.com (Y.-T. Wong), claralau@cuhk.edu.hk (C.B.-S. Lau),pcshaw@cuhk.edu.hk (P.-C. Shaw).

1 These authors contributed equally to this work.

Ming Li a,1, Yuk-Lau Wong a,1, Li-Li Jiang a, Ka-Lok Wong a, Yuen-Ting Wong b, Clara Bik-San Lau a,Pang-Chui Shaw a,b,⇑a State Key Laboratory of Phytochemistry and Plant Resources in West China (CUHK), Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kongb School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong

a r t i c l e i n f o a b s t r a c t

Article history:Received 19 October 2012Received in revised form 1 February 2013Accepted 16 May 2013Available online 24 May 2013

Keywords:BaihuasheshecaoHedyotis corymbosaHedyotis diffusaInternal transcribed spacer (ITS)Loop-mediated isothermal amplification(LAMP)Molecular authentication

Hedyotis diffusa Willd. (Baihuasheshecao) is an ingredient of herbal teas commonly consumed in the Ori-ent and tropical Asia for cancer treatment and health maintenance. In the market, this ingredient is fre-quently adulterated by the related species Hedyotis corymbosa (L.) Lam. The objective of this study is todevelop a novel loop-mediated isothermal amplification (LAMP) technique to differentiate H. diffusa fromits adulterant H. corymbosa. A set of four internal control primers (F3, FIP, BIP and B3) were designedbased on six loci in the internal transcribed spacer (ITS) for LAMP of both H. diffusa and H. corymbosa.Two specific primers (S_F3 and S_FIP) were designed for specific LAMP detection of H. diffusa only. Ourdata showed that LAMP was successful for both H. diffusa and H. corymbosa in internal control. In contrast,only H. diffusa was detected in specific LAMP using the specific primers S_F3 and S_FIP. This study showedthat LAMP was useful to differentiate H. diffusa from its adulterant H. corymbosa. This study is significantfor the verification of the authenticity for better quality control of this common herbal tea ingredient. Thestrategy of including an internal control assures the quality of the concerned DNA region for LAMP.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Hedyotis diffusa Willd. (Rubiaceae, common name: Baihuashe-shecao) is an ingredient of popular herbal teas in the Orient andtropical Asia (Perry, 1980). It has been commonly consumed forhealth maintenance and also as a dietary medicine for the treat-ment of prostate cancer (Liang, 2004). Scientific studies alsoshowed that H. diffusa possessed anti-cancer properties (Gupta,Zhang, Yi, & Shao, 2004; Willimott, Barker, Jones, & Opara, 2007),and its effects were related to the stimulation of the immune sys-tem, activation of caspase and burst of superoxide (Shan, Zhang,Du, & Li, 2001; Yadav & Lee, 2006). Along with the commercializa-tion of this herb, adulteration by a related species, Hedyotis cor-ymbosa (L.) Lam, has been more frequent in the market (Zhaoet al., 2005). The chemical components and therapeutic value ofthese two herbs are quite different. For example, two anti-prostatetumor chemicals, 6-O-(E)-p-coumaroyl scandoside methyl esterand 10(S)-hydroxylpheophytin a, were found only in H. diffusa

but undetectable in H. corymbosa (Li et al., 2010). Although thesetwo Hedyotis species are not closely related sister groups fromthe phylogenetic point of view (Guo, Simmons, But, Shaw, & Wang,2011), they look very much alike even in fresh. They differ only bythe shape of stems, number of flowers and size of pedicels (Ko,1999), and these characters were difficult to observe when thematerials are dried, cut or pulverized. Thin layer chromatography,high performance liquid chromatography, and DNA sequencinghave been developed to differentiate H. diffusa from H. corymbosa(Lau et al., 2012; Li et al., 2010; Yu et al., 2012). These methods,however, may be interfered by chemical variation among samplesor time consuming. A rapid molecular authentication technique isneeded for better quality assurance of this herbal tea ingredient.Loop-mediated isothermal amplification (LAMP) is a cutting-edgemolecular technique which enables amplification of DNA to a largeamount under constant operation temperature (Nagamine, Hase, &Notomi, 2002; Notomi et al., 2000). This technique has been ap-plied for the identification of Escherichia coli in food, detection ofgenetically modified food, and authentication of Chinese medicinalmaterials (Chaudhary, Hemant, Mohsin, & Ahmad, 2012; Chen,Guo, Wang, Kai, & Yang, 2011; Liu et al., 2009; Sasaki, Komatsu,& Nagumo, 2008; Wang, Jiang, & Ge, 2012). The objective of thisstudy is to develop LAMP technique to distinguish H. diffusa fromits adulterant H. corymbosa. Internal control was developed to en-sure the quality of the concerned DNA region for specific LAMP.

M. Li et al. / Food Chemistry 141 (2013) 2522–2525 2523

2. Materials and methods

2.1. Samples studied

Three authentic samples of the herbal materials of H. diffusawere collected from Jiangxi (Hd-1), Jiangsu (Hd-2) and Guangxi(Hd-3). Another three samples of the adulterants H. corymbosa werecollected from Guangxi (Hc-1) and Hong Kong (Hc-2 and Hc-3).These materials were identified by Shenzhen Institute For DrugControl, Shenzhen, China. The samples were stored in the Instituteof Chinese Medicine, The Chinese University of Hong Kong.

2.2. DNA extraction and quantification

Approximately 5 mg of herbal materials were used for DNAextraction using modified cetyltrimethylammonium bromide(CTAB) method as described previously (Li et al., 2012). DNA wasquantified by absorbance at 260 nm using NanoDrop (Thermo Sci-entific, US) and stored at �20 �C.

2.3. Primer design

The DNA sequences of ITS region of H. diffusa (EF570985,EF570986, and EF570988) and H. corymbosa (EF570974 and

Fig. 1. Primer design based on internal transcribed spacer (ITS) for internal control andGenBank and aligned. The internal control primers (F3, FIP, B3 and BIP) were designed frspecific forward primers (S_F3 and S_FIP) were designed from three loci (S_F3, S_F2 andin LAMP. (B) The structures of LAMP primers are shown and the polymorphic sites are i

EF570975) were retrieved from GenBank, National Center forBiotechnology Information. Multiple sequence alignment and iden-tification of polymorphic sites were performed using the sequenceanalysis software Bioedit 7.0 (Hall, 1999). Four common primerswere designed from six loci in ITS region, including the forwardouter primer F3 (50-TTG TCG AAT CCT GCA AA-30), forward innerprimers FIP (50-TTG AGT AGT CCT TGG CGC TTA CCG CGA ACACGT TTT-30), backward inner primer BIP (50-ACC AAT ACG ACTCTC GGC AAC GTC GCA TTT CGC TAC GTT CTT-30) and backward out-er primer B3 (50-ACG GGA TTC TGC AAT TCA CA-30), using thePrimerExplorerV4 (http://primerexplorer.jp) and OligoAnalyzer3.1 (Integrated DNA technologies, US). Specific forward outer pri-mer S_F3 (50-TAA AAA CCA GCG GGC TG-30) and specific forward in-ner primer S_FIP (50-GGA CGA TCC GTT TGA ACA GCC GTC TGC CAGCA-30) were also designed for LAMP specific to H. diffusa (Fig. 1).

2.4. Loop-mediated isothermal amplification (LAMP)

LAMP reaction was carried out using an Isothermal Mastermixamplification kit (OptiGene, UK) accordingly to the manufacturer’sinstruction. In brief, a 25 ll reaction mixture containing 0.2 lM ofeach outer primer, 2 lM of each inner primer, 1 ll DNA templateand 1� reagent mix was subjected to isothermal amplification at

specific LAMP. (A) ITS sequences of H. diffusa and H. corymbosa were retrieved fromom six loci (F3, F2, F1c, B1c, B2 and B3) as indicated in empty boxes. Two H. diffusaS_F1c) as shown in grey boxes. Arrows represent the direction of 50–30 amplificationndicated in grey boxes.

2524 M. Li et al. / Food Chemistry 141 (2013) 2522–2525

65 �C for 60 min followed by inactivation at 98 �C for 5 min. Prim-ers F3, FIP, BIP and B3 were used for internal control. Primers S_F3,S_FIP, BIP and B3 were used for specific LAMP. LAMP was real-timemonitored using the portable Genie II LAMP detector (OptiGene,UK). The LAMP products were visualized by 1% agarose gel electro-phoresis stained with ethidium bromide.

3. Results and discussion

H. diffusa is an herbal tea ingredient for daily consumption tomaintain health. This anti-tumor herb has been adulterated by H.corymbosa which showed different chemical constituents andmedicinal values (Li et al., 2010). In this study, we have appliedLAMP for rapid differentiation of these two Hedyotis species. Thedesign of LAMP primers was based on the ITS region, which is asupplementary DNA barcode commonly used in the molecularidentification of herbal materials including H. diffusa (Li et al.,2010, 2012). Four internal control primers (F3, FIP, BIP and B3)were designed from six loci in the ITS region (Fig. 1). These primerscontain minimum amount of polymorphic sites so that both H. dif-fusa and H. corymbosa were detected by LAMP started from 30 to40 min and saturated from 40 to 60 min (Fig. 2). In contrast, twoforward primers specific to H. diffusa (S_F3 and S_FIP) were de-signed based on the ITS region and many sites polymorphic to H.corymbosa were included at the 30 ends (Fig. 1). The use of thesetwo specific primers together with the two backward primers(BIP and B3) led to specific LAMP of H. diffusa started from 20 to30 min and saturated from 30 to 50 min (Fig. 2). LAMP is a highlysensitive amplification technique which could amplify targets with

Fig. 2. Differentiation of H. diffusa from H. corymbosa using LAMP. (A) Internal control (lefdiffusa and H. corymbosa were detected using the internal control primers, while only Happlied as the negative control (neg). (B) End-point detection of internal control and sprepresents 100 bp DNA ladder, the size of 800 bp is indicated for reference. ‘Hd’ represe

as low as six copies of DNA template (Notomi et al., 2000). Our datasuggested that LAMP was detectable with 30 ng to 3 pg of DNAtemplate for both internal control and specific LAMP (Fig. 3).

In the molecular authentication of herbal materials and otherprocessed samples such as food products and Chinese medicinalmaterials, poor DNA quantity, integrity and the presence of inhib-itors are the major concerns of successful identification (Li, Cao,But, & Shaw, 2011). For example, LAMP was applied to distinguishthe herbal material Ginseng Radix (Panax ginseng C. A. Mey.,Araliaceae) from Panacis Japonici Rhizoma (Parribacus japonicusC. A. Mey., Araliaceae) and Glycyrrhizae Radix (Glycyrrhiza uralensisFisch. ex DC., Fabaceae) (Sasaki et al., 2008). Although DNA purityand quantity were considered, the problem of DNA degradation insamples of Panacis Japonici Rhizoma and Glycyrrhizae Radix wasnot addressed and this may lead to false negative identificationresults. In fact, it is quite difficult to determine the degree ofDNA integrity in small amount of DNA. Alternatively, the introduc-tion of internal control can serve as an indication of sufficient DNAtemplate, intact DNA region of concern and the absence of inhibi-tors. The specific primers, either forward, backward or both, mustbe designed in the region flanked by the outer primers of theinternal control (F3 and B3) so that DNA integrity of the concernedregion in specific LAMP could be assured (Fig. 2).

Correct use of ingredient is important for quality assurance offood and drinks. In this study, we have successfully applied LAMPtechnique for the identification of the herbal tea ingredient H. dif-fusa and differentiated it from its adulterant H. corymbosa. Internalcontrol was developed to avoid false identification conclusiondrawn by negative LAMP results. This study provided a novel

t panel) and specific LAMP (right panel) was real-time monitored by Genie II. Both H.. diffusa was detected using the specific primers. LAMP without DNA template wasecific LAMP by 1% agarose gel electrophoresis stained with ethidium bromide. ‘M’nts H. diffusa and ‘Hc’ represents H. corymbosa.

Fig. 3. Detection limit of LAMP using internal control primers and specific primers. DNA templates of H. diffusa (Hd-1) (30 ng) (100) were 10-fold serial-diluted (10�1 to 10�6)and LAMP was (A) real-time monitored by Genie II (left panel: internal control; right panel: specific LAMP) and (B) end-point detected by DNA gel electrophoresis (left panel:internal control; right panel: specific LAMP). LAMP without DNA template was applied as the negative control (neg). ‘M’ represents 100 bp DNA ladder, the size of 800 bp isindicated for reference.

M. Li et al. / Food Chemistry 141 (2013) 2522–2525 2525

molecular technique for rapid identification of H. diffusa and thestrategy employed may be extended to other medicinal materials.

Acknowledgements

We thank Shenzhen Institute for Drug Control for identifyingthe herbal samples. This project was supported by the Innovationand Technology Fund (ITS/162/11), Innovation and TechnologyCommission, Hong Kong SAR.

References

Chaudhary, A. A., Hemant Mohsin, M., & Ahmad, A. (2012). Application of loop-mediated isothermal amplification (LAMP)-based technology for authenticationof Catharanthus roseus (L.) G. Don. Protoplasma, 249(2), 417–422.

Chen, L., Guo, J., Wang, Q., Kai, G., & Yang, L. (2011). Development of the visual loop-mediated isothermal amplification assays for seven genetically modified maizeevents and their application in practical samples analysis. Journal of Agriculturaland Food Chemistry, 59(11), 5914–5918.

Guo, X., Simmons, M. P., But, P. P. H., Shaw, P. C., & Wang, R. J. (2011). Application ofDNA barcodes in Hedyotis L. (Spermacoceae, Rubiaceae). Journal of Systematicsand Evolution, 49(3), 203–212.

Gupta, S., Zhang, D., Yi, J., & Shao, J. (2004). Anticancer activities of Oldenlandiadiffusa. Journal of Herbal Pharmacotherapy, 4(1), 21–33.

Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor andanalysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41,95–98.

Ko, W. C. (1999). Hedyotis. In X. R. Luo (Ed.). Flora republicae popularis sinicae (Vol.71, pp. 32–77). Beijing: Science Press.

Lau, C. B., Cheng, L., Cheng, B. W., Yue, G. G., Wong, E. C., Lau, C. P., et al. (2012).Development of a simple chromatographic method for distinguishing betweentwo easily confused species, Hedyotis diffusa and Hedyotis corymbosa. NaturalProduct Research, 26(15), 1446–1450.

Li, M., Cao, H., But, P. P. H., & Shaw, P. C. (2011). Identification of herbal medicinalmaterials using DNA barcodes. Journal of Systematics and Evolution, 49(3),271–283.

Li, M., Jiang, R. W., Hon, P. M., Cheng, L., Li, L. L., Zhou, J. R., et al. (2010).Authentication of the anti-tumor herb Baihuasheshecao with bioactive markercompounds and molecular sequences. Food Chemistry, 119(3), 1239–1245.

Li, M., Wong, K. L., Chan, W. H., Li, J. X., But, P. P. H., Cao, H., et al. (2012).Establishment of DNA barcodes for the identification of the botanical sources ofthe Chinese ‘cooling’ beverage. Food Control, 25, 758–766.

Liang, X. Y. (2004). A case of prostate cancer treated with Chinese medicines.Academic Periodical of Changchun College of Traditional Chinese Medicine, 20(2),29.

Liu, M., Luo, Y., Tao, R., He, R., Jiang, K., Wang, B., et al. (2009). Sensitive and rapiddetection of genetic modified soybean (Roundup Ready) by loop-mediatedisothermal amplification. Bioscience, Biotechnology, and Biochemistry, 73(11),2365–2369.

Nagamine, K., Hase, T., & Notomi, T. (2002). Accelerated reaction by loop-mediatedisothermal amplification using loop primers. Molecular and Cellular Probes,16(3), 223–229.

Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N.,et al. (2000). Loop-mediated isothermal amplification of DNA. Nucleic AcidsResearch, 28(12), E63.

Perry, L. M. (1980). Medicinal plants of east and southeast Asia: Attributed propertiesand uses. Cambridge: MIT Press.

Sasaki, Y., Komatsu, K., & Nagumo, S. (2008). Rapid detection of Panax ginseng byloop-mediated isothermal amplification and its application to authentication ofGinseng. Biological and Pharmaceutical Bulletin, 31(9), 1806–1808.

Shan, B. E., Zhang, J. R., Du, X. N., & Li, K. X. (2001). Immunommodulatory activityand anti-tumor activity of Oldenlandia diffusa in vitro. Chinese Journal ofIntegrated Traditional and Western Medicine, 21, 370–374.

Wang, F., Jiang, L., & Ge, B. (2012). Loop-mediated isothermal amplification assaysfor detecting shiga toxin-producing Escherichia coli in ground beef and humanstools. Journal of Clinical Microbiology, 50(1), 91–97.

Willimott, S., Barker, J., Jones, L. A., & Opara, E. I. (2007). Apoptotic effect ofOldenlandia diffusa on the leukaemic cell line HL60 and human lymphocytes.Journal of Ethnopharmacology, 114, 290–299.

Yadav, S. K., & Lee, S. C. (2006). Evidence for Oldenlandia diffusa-evoked cancer cellapoptosis through superoxide burst and caspase activation. Journal of ChineseIntegrative Medicine, 4, 485–489.

Yu, J., Li, M., Guo, X., Wang, R. J., Chiu, S. W., Shaw, P. C., et al. (2012). Value of FINSdepends on choice of DNA locus as shown in Baihuasheshecao. Journal of Foodand Drug Analysis, 20(4), 879–886.

Zhao, Z. Z., Huang, W. H., Wu, J. L., Yu, T., Huang, R. D., Tsang, L. Y. I., et al. (2005).Easily confused chinese medicines in Hong Kong. Hong Kong: Chinese MedicineMerchants Association.