Identi fication of three kinds of Plumeria flowers by DNA barcoding and HPLC specific chromatogram

Leilei Zhao,Xiaoxue Yu,Jie Shen,Xinjun Xu,*

aSchool of Pharmaceutical Sciences,Sun Yat-sen University,Guangzhou 510006,China

bGuangdong Technology Research Center for Advanced Chinese Medicine,Guangzhou,China

1.Introduction

Plumeriais a kind of deciduous tree belonging to Apocynaceae.It has a significant medicinal value and can be used for the treatment of various ailments,such as sore throat,heatstroke,bellyache,cough,and dysentery[1].The main chemical components ofPlumeriaare iridoids,triterpenes, flavonoids,essential oil and so on.Mansour et al.[2]found that the iridoids inPlumeriahave a toxic effect on a series human tumor cells.The extracts of the flowers can significantly inhibit streptococcus faecalis,bacillus,and corynebacterium pyogenes[3].Gupta et al.[4]indicated that the flowers extracts ofPlumeriahad the antipyretic-analgesic effect.As a medicine with dual-purpose of drug and food,the flowers ofPlumeriaare also popular among people in making herbal tea to relieve the summer heat.

There are many varieties ofPlumeria,such asPlumeria rubraL.,Plumeria rubravar.alba,andPlumeria rubra'Acutifolia'.Some researches have indicated that the chemical components vary from differentPlumeriaflowers,some of which may have good bioactivities[5–8].However,it is difficult to identifyPlumeriaflowers because they are very alike after dried and processed.Thus,authenticity assurance is crucial for their quality control.We intended to use DNA barcoding technique and HPLC specific chromatograms to identify three kinds ofPlumeria.

DNA barcoding is a technique for identifying biological specimens using short DNA sequences from either nuclear or organelle genomes[9].This technique has three advantages as follows.Firstly,the results have good repeatability compared to other molecular identification methods.Secondly,DNA barcoding technique is highly universal.The traditional taxonomic method requires that the leaves, flowers,fruits and other organs of plants must exist.However,DNA barcoding technique is not limited by growth stage,organ,tissue difference or the external environment.And a small number of samples are enough for the identification.Lastly,it can establish a unified database and identification platform to realize digital species identification[10].DNA barcoding technique has been applied to identification of animals,gymnosperms,angiosperms,fungi and so on[11].

The chloroplast DNA(cpDNA)has been commonly used for DNA barcoding studies in plants.The cpDNA contains variable DNA regions,among which the most commonly used cpDNA intergenic spacer ispsbA-trnH,which has shown high variability and can be used to elucidate genetic relationships at the intraspecific level[12,13].In this pioneering study,we selectedpsbA-trnH to identify three varietas ofPlumeria,at the same time,the HPLC specific chromatogram that is commonly used for identification was also used for distinguishing these three varieties ofPlumeriato make comparison.We hope that our established method will be helpful for the future quality control ofPlumeriaflowers.

2.Experimental

2.1.Materials and reagents

Twelve samples ofPlumeriaflowers were collected from three districts in Guangzhou,Guangdong,China(Table 1).They were authenticated by Associate Professor Lin Jiang,Sun Yat-Sen University,China.Methanol was of analytical grade and manufactured by Tianjin Zhiyuan Chemical Reagent Factory(Tianjin,China).TAE buffer,agarose,PVP-40 andTaqPCR Master Mix(2×,blue dye)were purchased from Sangon Biotech(Shanghai,China).DNA secure Plant Kit was purchased from Tiangen Biotech(Beijing,China).Goldview(MYM Biological Technology Co.,Ltd.,USA)was used for agarose examination.Acetonitrile was of HPLC grade manufactured by SK Chemicals(Korea).Ultrapure water was obtained from a Milli-QRG purification unit(Millipore,Bedford,MA,USA).

2.2.Apparatus

An electronic balance(KERN ABT 220-5DM,0.1 mg,Germany),an ultrasonic machine(SB25-12DTD,Xinzhi Biotechnical Ltd.,Ningbo,China)and an eppendorf centrifuge 5417R(Eppendorf AG,Hamburg,Germany)were used for sample preparation and DNA extraction.PCR amplification was performed on the K960 thermal cycler(Hangzhou Jingge Scientific Instrument Co.,Ltd,Hangzhou,China).HPLC analysis was performed on the Shimadzu LC-15C high performance liquid chromatograph(Shimadzu,Japan)with a Dikma Diamonsil C18column(250 mm × 4.6 mm,5 μm;Dikma,Beijing,China)and a guard column(15 mm × 4.6 mm,5μm;Dikma,Beijing,China).

2.3.Sample pre-treatment for DNA extraction and total DNA extraction

The fresh flowers were washed up and wiped with 75%alcohol aqueous.About 100mg of each sample was grinded with 1%(m/m)PVP-40.Then,DNA secure Plant Kit was used for total DNA extraction.The process followed the instruction of the Kit.

2.4.PCR amplification and DNA sequencing

DNA barcodes were amplified by polymerase chain reaction using universal primers(fwd∶5′-GTTATGCATGAACGTAATGCTC-3′and rev∶5′-CGCGCATGGATTCACAATCC-3′).Each 25 μL reaction mixture contained 12.5μLTaqPCR Master Mix,1μL Genomic DNA,1μL of each 10μM primer,1μL MgCl2solution and 8.5 μL ddH2O The PCR conditions for amplification were 1 cycle 94°C for 5 min;40 cycles of 94 °C 30 s,56 °C 30 s,72 °C 45 s;and 1 cycle 72 °C for 10 min,and hold 4°C.To detect amplified products successfully,PCR products were examined on 2% agarose gels stained with Goldview and visualized under ultraviolet light.

2.5.Sequence alignment and analysis

All the amplified products were sent to Sangon Guangzhou for sequencing.The sequences were analyzed by MEGA 5.0.

2.6.Preparation of sample solution for HPLC analysis

Test solutions were prepared by extracting 0.5 g dried and pulverized herbs with 10 mL 70%methanol aqueous under ultrasonic condition at room temperature for 30 min.After cooling,the extracted solution was added with 70%methanol aqueous to the original weight.The extracts were filtered through a 0.45 μm filter before used for HPLC analysis.

2.7.HPLC conditions

Chromatographic separation was carried out on a Diamonsil C18column along with a guard column.The separation was conducted at 35°C with a flow rate of 0.7 mL/min.0.5%acetic acid aqueous solution(A)and acetonitrile(B)were used as the mobile phase in gradient elution mode.The elution gradient was set as follows∶0–10 min,10%(B);10–30 min,10%→15%(B);30–70min,15%→20%(B);70–85 min,20%→65%(B);85–90 min,65%→90%(B);90–95 min,90%(B).The detection wavelength was 240nm.The injection volume was 20μL.

2.8.Chemometric analysis

Principal components analysis(PCA)and hierarchical clustering analysis(HCA)were conducted on the SAS 9.0 software to demonstrate the variability among the 12 samples.

3.Results and discussion

3.1.DNA barcode result analysis

Authenticity assurance is crucial for quality control of natural products.It is essential to develop different approaches to authenticate the natural products as each approach has advantages that complementary to one another[14].A desirable DNA barcodeshould process high interspecific divergences and low intraspecific variations.The Consortium for the Barcode of Life(CBOL)suggested comparing theinterspeciesdistance(dinter)and intraspecies distance(dintra)to estimate the identification effectiveness of the selected barcode.The interspecific divergences of an ideal ‘barcoding gap’should be significantly larger than intraspecific divergences.Ifdinter/dintrais smaller than 1,it may not be a suitable DNA barcode[10].

Table 1 Information of the collected sample.

Table 2 Properties of the psbA-trnH region of the 12 samples.

The results of DNA barcoding showed a good differentiation.ThepsbA-trnH of all samples were successfully amplified from total DNA and sequenced.Properties of thepsbA-trnH region are summarized in Table 2.The genetic distance was calculated by MEGA 5.0,based on Kimura-2-parameter model.The intraspecies distances ofPlumeria rubraL.andPlumeria rubra'Acutifolia'were 0.001 and 0.004,respectively.There was no intraspecies distance forPlumeria rubravar.alba.The interspecies distance was 0.007 betweenPlumeria rubraL.andPlumeria rubravar.alba,0.034 betweenPlumeria rubraL.andPlumeria rubra'Acutifolia',0.033 betweenPlumeria rubravar.albaandPlumeria rubra'Acutifolia'.Results of eachdinter/dintrawere larger than 1,which indicated that thepsbA-trnH region was suitable for the identification of three varietas ofPlumeria.The neighbor joining tree was built by MEGA 5.0 by repeated 1000 times bootstrap(Fig.1).The neighbor joining tree showed that the samples ofPlumeria rubraL.,Plumeria rubravar.albaandPlumeria rubra'Acutifolia'can be clustered into three groups,respectively.Therefore,thepsbA-trnH region was an appropriate DNA barcode for identifying these three varietas ofPlumeria.

Fig.1.The neighbor joining tree of Plumeria rubra L.(R1-R4),Plumeria rubra var.alba(P1-P4)and Plumeria rubra(W1-W4)'Acutifolia'based on the psbA-trnH sequence with K2P model.

Fig.2.The liquid chromatograms of the three Plumeria(A∶Plumeria rubra L.,B∶Plumeria rubra var.alba,C∶Plumeria rubra'Acutifolia').

3.2.HPLC analysis

Fig.2 shows the HPLC specific chromatograms of samples R1(Plumeria rubraL.),P1(Plumeria rubravar.alba)and W1(Plumeria rubra'Acutifolia'),their HPLC chromatograms were so similar that it was difficult to separate the three varietas visually.

3.3.Principal component analysis(PCA)

Fig.3.Score plot(A)and loading plot(B)of PCA.

Fig.4.The dendrogram of 12 batches of Plumeria by HCA.R1-R4∶Plumeria rubra L.,P1-P4∶Plumeria rubra var.alba,and W1-W4∶Plumeria rubra'Acutifolia'.

PCA,a multivariate analysis technique,could visualize similarities or differences within multivariate data[15].It was employed to analyze the differences among these 12 samples.The peak areas of 6 characteristic peaks were set as variables,while 12 samples were set as observations.PC1 explained 41.2%of the total variance in the data set while PC2 explained 32.4%.The cumulative proportion of PCA as well as the loading diagram is shown in Fig.3.According to the loading diagram,PC1 showed a strong correlation with peak 5 and peak 6.PC2 showed a strong correlation with peak 2 and peak 4.PC3 showed a strong correlation with peak1 and peak 5.The distinguished results of HPLC specific chromatograms combined with PCA were not as accurate as that of DNA barcoding.

3.4.Hierarchical cluster analysis(HCA)

HCA,one of the most commonly used unsupervised pattern recognition methods,is a useful multivariate statistic technique to assign a data set into groups by creating a cluster tree or dendrogram according to similarity[15].In order to assess the resemblance and differences of these samples,HCA ofPlumeriasamples was performed based on the peak area of the 6 characteristic chromatographic peaks by SAS 9.0 software.The Ward's method was applied as the amalgamation rule and the squared Euclidean distance was selected to measure the resemblance and classify the 12 samples.The result is shown in Fig.4.Samples R1–R4(Plumeria rubraL.)were categorized into one cluster,W1–W4(Plumeria rubra'Acutifolia')were categorized into another cluster.However,samples P1–P4(Plumeria rubravar.alba)cannot cluster together.Samples R1–R4 and W1–W4 were collected from the same district,but samples P1–P4 were from three different districts.Therefore,we speculated that the environment affected the chemical component which affected the result of PCA and HCA.

4.Conclusion

This study has shown that DNA barcoding combined with chemometrics analysis can distinguishPlumeria rubraL.,Plumeria rubra'Acutifolia'andPlumeria rubravar.alba.while the HPLC method can not identify these three varietas because the specific chromatograms were similar.DNA barcoding technique is hopeful for automation as it is more stable,accurate and not affected by growth stage,tissue difference or external environment.This technique is an effective supplement for traditional authentication methods,especially when different species are mixed together.This work also provides an experimental reference for identification of natural medicines by DNA barcoding technique.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the Industry-University-Research Cooperation Program from Science and Technology Department of Guangdong Province(No.∶2013B090600058);the National Key Research and Development Program of China(2017YFC170116).

[1]Chinese Herbal Medicines Guangxi Standard,1st volume,The Guangxi Zhuang Autonomous Region Health Department,Guangxi Science and Technology Press,Nanning,1990∶59.

[2]A.K.Mansour,M.M.Eid,N.S.A.M.Khalil,Synthesis and reactions of some new heterocyclic carbohydrazides and related compounds as potential anticancer agents,Molecules 8(2003)744–755.

[3]P.A.Egwaikhide,S.O.Okeniyi,C.E.Gimba,Screening for anti-microbial activity and phytochemical constituents of some Nigerian medicinal plants,J.Med.Plants Res.3(2009)1088–1091.

[4]M.Gupta,U.K.Mazumder,P.Gomathi,Evaluation of antipyretic and antinociceptive activities ofPlumeriaacuminata leaves,J.Med.Sci.7(2007)452–456.

[5]Z.Zahee,A.G.Konale,K.A.Patel,et al.,Comparative phytochemical screening of flowers ofPlumeria albaandPulmeria rubra,Asian J.Pharm.Clin.Res.3(2010)88–89.

[6]N.Tohar,M.A.Mohd,I.Jantan,et al.,A comparative study of the essential oils of the genusPlumeriaLinn.from Malaysia,Flavour Fragr.J.6(2006)304–309.

[7]N.Tohar,K.Awang,M.A.Mohd,et al.,Chemical composition of the essential oils of four Plumeria specials grown on Peninsular Malayasia,J.Essent.Oil Res.6(2006)613–617.

[8]Y.Q.Liu,H.W.Wang,S.Wei,et al.,Chemical composition and antimicrobial activity of the essential oils extracted by microwave-assisted hydrodistillation from the flowers of two Plumeria species,Anal.Lett.45(2012)2389–2397.

[9]N.Techen,I.Parveen,Z.Q.Pan,et al.,DNA barcoding of medicinal plant material for identification,Curr.Opin.Biotechnol.25(2014)103–110.

[10]S.L.Chen,DNA Barcoding Molecular Markers in Chinese Medicinal Materials,People's Medical Publishing House,Beijing,2012∶18-19.

[11]S.L.Chen,Y.Hui,J.P.Han,et al.,Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species,PLoS One 5(2010)e8613.

[12]H.Azum,J.G.García-Franco,V.Rico-Gray,et al.,Molecular phylogeny of the Magnoliaceae∶the biogeography of tropical and temperate disjunctions,Am.J.Bot.88(2001)2275–2285.

[13]M.B.Hamilton,J.M.Braverman,D.F.Soria-Hernanz,Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in new world species of the Lecythidaceae,Mol.Biol.Evol.20(2003)1710–1721.

[14]P.C.Shaw,J.Wang,P.P.H.But,Authentication of Chinese Medicinal Materials by DNA Technology,World Scientific,Singapore,2002∶1–23.

[15]Z.S.Xie,S.Z.Lam,J.W.Wu,et al.,Chemical fingerprint and simultaneous determination of flavonoids in Flos Sophorae Immaturus by HPLC-DAD and HPLC-DAD-ESI-MS/MS combined with chemometrics analysis,Anal.Methods 6(2014)4328–4335.