Review: DNA Barcoding and Chromatography Fingerprints for the Authentication...

Review ArticleReview DNA Barcoding and Chromatography Fingerprints forthe Authentication of Botanicals in Herbal Medicinal Products

Bashir Mohammed Abubakar12 FaezahMohd Salleh1

Mohd Shahir Shamsir Omar3 and AlinaWagiran1

1Department of Biotechnology amp Medical Engineering Faculty of Biosciences and Medical EngineeringUTM 81310 Skudai Johor Malaysia2Department of Biological Sciences Bauchi State University Gadau PMB 065 Bauchi Nigeria3Department of Biosciences amp Health Sciences Faculty of Biosciences and Medical Engineering UTM 81310 Skudai Johor Malaysia

Correspondence should be addressed to Alina Wagiran alinafbbutmmy

Received 8 November 2016 Revised 11 February 2017 Accepted 16 March 2017 Published 27 April 2017

Academic Editor Victor Kuete

Copyright copy 2017 Bashir Mohammed Abubakar et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

In the last two decades there has been a tremendous increase in the global use of herbal medicinal products (HMPs) due to theirclaimed health benefits This has led to increase in their demand and consequently also resulted in massive adulteration This isdue to the fact that most of the traditional methods cannot identify closely related species in a process product form Thereforethe urgent need for simple and rapid identification methods resulted in the discovery of a novel technique DNA barcoding is aprocess that uses short DNA sequence from the standard genome for species identification This technique is reliable and is notaffected by external factors such as climates age or plant part The difficulties in isolation of DNA of high quality in addition toother factors are among the challenges encountered using the DNA barcoding in the authentication of HMP These limitationsindicated that using DNA barcoding alone may ineffectively authenticate the HMPTherefore the combination of DNA barcodingwith chromatographic fingerprint a popular and generally accepted technique for the assessment and quality control of HMP willoffer an efficient solution to effectively evaluate the authenticity and quality consistency of HMP Detailed and quality informationabout the main composition of the HMPs will help to ascertain their efficacy and safety as these are very important for qualitycontrol

1 Introduction

In the last two decades there was a tremendous increasein the global use of herbal medicinal products (HMPs) [1]These general increases in the consumption of HMP are dueto their claimed health benefits easy availability perceivedeffectiveness and safety [2 3] HMP generally referred to anymedicinal product that exclusively contained active ingredi-ent herbal substance (part of the plant) or herbal extract [4]In the context of this paper HMPs or herbal medicinesencompass both simple preparation andmanufactured prod-ucts containing one or more herbal active ingredients

It is estimated that 56 billion people approximately 80of the world population rely on HMPs for their primaryhealth care [5ndash9] More than 90 of Africans as well as 70

and 40 of Indian and Chinese population respectivelycontinue to rely on HMP for general health care [10] Despitethe great advances achieved in the field of modern medicineplants still play a significant role in health care At least 25of drugs in modern pharmacopeia are directly or indirectlyderived from plants [11 12] (Palhares et al 2015a) and morethan 60 of antitumour drugs are generally derived fromnatural product [13] The international trade of HMPs isbecoming a lucrative business due to their high demand theirglobal market is estimated at US$83 billion (Palhares et al2015a) and it is expected to reach US$107 billion by 2017 [3]

The increase in usage of these HMPs is not only restrictedto developing countries however developed countries suchas USA Italy France and Germany have produced guide-lines for the registration of herbal medicine [11] The WHO

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 1352948 28 pageshttpsdoiorg10115520171352948

2 Evidence-Based Complementary and Alternative Medicine

reported that the annual sales of herbal medicine in Germanyfor the year 2002 have reached US$2432 billion [14] Nahinet al [15] also reported that the market of HMPs in USAhas expanded tremendously from US$29 billion to US$48billion from 1995 to 2008

So also the average import of these HMPs in USA in2004ndash2008 is more than US$220 million and the highestexporters for that very period were China and India valuedat around US$348 and US$93 million respectively [16] Aftera period of 3 years the sales of herbal medicine in Chinaincreased tremendously as reported by National Bureau ofStatistics [14] They reported that the sale value in herbalindustries has reached US$68 billion (RMB418 billion) withan annual growth rate of 379 So also the export annualrate of the herbal medicine from China to US in the sameyears has reached up to 663 [17]

Despite the extensive usage of HMP for a long period oftime issues related to their assessment and quality controlare always a major problem because active phytocompoundshave inherent risk just like all the active chemical compounds[18] The problems encountered are mostly attributed tothe poor quality of raw material or finished products andmay result in different types of adverse effects Basically thequality of the herbal products can be categorised into externaland internal categories [19] The external issue affectingthe quality includes contamination cultivation processingadulteration and misidentification while internal issues arebasically caused by the presence of bioactive chemicals in theherbal products

Both the external and internal qualities of the raw mate-rial are very important due to the fact that both safety andefficacy of the herbal products depend on them Accordingto the WHO adulteration of the herbal product is notonly a fraud but a threat to the consumerrsquos health as thismight result in adverse effects and eventually death [20]False identification of HMPs has resulted in several toxiceffects to consumers A dramatic example of such incidentoccurs in an 18-month-old baby who was diagnosed withvenoocculusive disease after regularly consuming a herbaltea containing high amount of pyrrolizidine alkaloid [21]Other cases include adulteration of herbal medicine teawith a neurotoxin compound from Illicium anisatum [22]Aconitum carmichaelii and Aconitum kusnezoffii have longbeen used to relief pain conditions such as rheumatismarthritis bruises and fractures [23] Most of the toxicity ofthese HMPs is primarily derived as a result of the presenceof diterpene alkaloids particularly aconitine others includemesaconitine and hypaconitine [24] In traditional Chinesemedicine (TCM) practices aconitine has been effectivelyused to treat various ailments despite their toxic effects Sucheffects were related to that causing ventricular tachycardia[25] bradycardia and hypotension [26]

The proportion of adulterated HMPs sold in developingcountries can account for up to 10 but may increase up to50 when purchased through unregulated channels such asonline transaction [27] The popular demand in such marketespecially in developing countries may pose further a seriousproblemwhere drug regulation is weak Reports have showedthatmore than 80of theHMPs sold inAfrica are counterfeit

or adulterated [28] All these incidences keep compoundingand increasing frequently due to lack of proper identificationmethods for primary plant source [27]

In general HMPs are classified into two products mono-herbal and polyherbal products [29] Monoherbal productscontained only one herb or herbal component for exampleEurycoma longifolia capsule which consists of its extract onlyThe polyherbal normally can be found either in the formof capsule tablets or tea which consists of more than one(two or more) herb component which can work in synergy todeliver their therapeutic effects Liuwei Dihuang pill (LDP)a well known TCM widely used for the treatment of kidneyyin deficiency contains 6 different Chinese medicinal herbs[30] Hwangryunhaedok-tang (HHT) a traditional herbalmedicine that is used for the treatment of inflammation feverhypertension and gastritis in China and Japan is composedof four herbal substances [31] Other examples of polyherbalproducts are shown in Table 1

Due to the widespread use of medicinal plants forthe past decades tandem with the assurance of its qualitysafety and efficacy of medicinal plant the WHO has pub-lished monographs [32ndash35] for selected medicinal plants forreferences Each volume of these monographs consists ofdifferent species of plant with their synonyms and vernac-ular names commonly used parts geographical locationknown active compounds dosage forms potential adverseeffects contraindication recognised medicinal benefits andhow to correctly and effectively use them Some countrieshave also established a series of monographs for their localusage such as Chinese pharmacopeia [36] United Statespharmacopeia [37] Ayurvedic Pharmacopoeia of India [38]Japanese pharmacopeia [39] and other countries The mainpurpose of developing all these monographs is to provideaccurate scientific information on the safety efficacy andquality assurance of the widely used medicinal plants and tohelp other countries to develop their own

Certain steps are of enormous importance in order toguarantee the quality of HMPs and these include (1) correctidentification of the plant species and (2) correct identifi-cation and analyses of the purity of the pharmacologicallyactive constituent (biomarkers with their required minimumconcentration) [40] In this aspect the identification of theexact and correct raw material used for the production ofHMPs is always challenging Correct identification of thesource of raw materials of medicinal herbal plants used isthe prerequisite and is of utmost criticalness to ensure theirquality safety and therapeutic efficacy [41 42] Generallythere are different types of identification methods most arecommonly based on botanical and morphological analysesby taxonomist expert thus identification by common peoplemay lead to incorrect specieThis is also true in the sense thatmost of the HMP consists of dried or unidentifiable part andthis makes the species identification process difficult even tohighly trained and experienced professional taxonomist [27]

The identification of herbal medicinal plant for qualitycontrol and standardization of raw materials should com-ply with the requirement specified in pharmacopeia theseinclude organoleptic evaluation (identification of sensestouch smell sight and taste) morphological characteristics

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Examples of polyherbal HMPs

Polyherbal product Medicinal value Composition References

Xue-Fu-Zhu-Yu decoction

Treatment ofatherosclerosis and

coronary heart diseaselowering the level ofcholesterol in serum

Paeonia lactiflora Ligusticum chuanxiongCitrus aurantium Carthamus tinctoriusPrunus persicaand Bupleurum falcatum

Zhang and Cheng [65]

Ayurved SirirajPrasachandaeng Antipyretic drug

Myristica fragrans Citrus aurantifoliaBouea macrophylla Knema globulariaCaesalpinia sappan Conioselinumunivitatum Dracaena loureiri

Kaempferia galanga Mesua ferreaJasminum sambac and Mammea

siamensis Nelumbo nucifera

Akarasereenont et al [66]

Ge-Gen Decoction

Treatment of common coldfever and influenzaIt is also used for thetreatment of different

diseases such as cervicalspondylosis and primary

dysmenorrhea

Pueraria Radix (Gegen) Ephedrae Herba(Mahuang)

Cinnamomi Ramulus (Guizhi)Glycyrrhizae Radix et Rhizoma Preparata(prepared Gancao) Paeoniae Radix AlbaPreparata (prepared Shaoyao) Zingiberis

Rhizoma Recens (Shengjiang) andJujubae Fructus (Dazao)

Yan et al [67]

Danggui-Shaoyao-San(DSS)

Treatment ofgynaecological disordersuch as amenorrhea and

dysmenorrhea

Radix Paeoniae alba Radix Angelicasinensis Rhizoma Chuanxiong Poria

cocos Rhizoma Atractylodismacrocephalae and Rhizoma Alismatis

Chen et al [68]

Shrishadi To treat infectiousrespiratory disorders

Albizia lebbeck Cyprus rotan andSolanum xanthocarpum Kajaria et al [69]

Hongdoushan capsule Treatment of ovarian andbreast cancers

Glycyrrhiza uralensis Fisch PanaxGinseng Taxus chinensis var mairei Zhu et al [70]

such as microscopic and macroscopic (shape colour andtexture) and chemical profiling such as HPLC TLC andGC [43] First of all if the identity of the plant part isverified the process then proceeds with chemical analysisusing the chromatographic methods such as HPLC so as toidentify or to determine the profile of the active compounds(activebiomarkers) of the product and to quantify them(Palhares et al 2015a) [43]

However these methods have their own limitations asthe former methods primarily depend on human expertise[27] the latter may be affected by several factors such asage growth and storage condition [42] These methodscannot identify closely related species which shared similar oridenticalmorphological characteristics and chemical profilesThis indicates that chemical profiling techniques can onlydetect an indirect fraud as it lacks the temerity to revealthe identity of the adulterated species However this showsthat neither of thesemethods can distinguish between closelyrelated species therefore there is an urgent need to introducean accurate simple and rapid identification method forproper inspection of HMPs

In addition to other identificationmethodsDNAbarcod-ing can serve as a powerful tool to overcome some of theproblems encountered DNA barcoding is a process whichinvolves the use of short DNA sequence from the standardpart of the genome for species identification This uniquetechnique was first coined by Canadian zoologist Herbert in

2003 he used the 51015840 end of cytochrome C oxidase subunit ofmitochondria to identify closely related allies of lepidopterans[44] Contrary to chemical profiling DNA barcoding canprovide a reliable and consistent result because it is not influ-enced by sample age environmental factors physiologicalcondition growth condition harvesting period cultivationarea plant part and storage condition [42 45]

The species-level discrimination of DNA barcoding hasmade this technique widely accepted as a regulatory toolfor evaluating the authenticity of different types of HMPs[20 46ndash48] The authentication of HMPs is made possibleusing DNA barcoding because DNA is more reliable is stablein contrast to other macromolecules (eg RNA) as it isnot affected by internal or external factors and is found inall tissues [45] The stability of DNA makes it possible toidentify species in a dried or process product form against themorphological features of the plant onwhich the current Lin-naean taxonomic system is based on [20] Moreover despitethe fact that DNA barcoding serves as an efficient methodfor identifying and authenticating HMPs it is also character-ised by some limitations [49 50]

For these reasons it can be observed that none of themethods are 100 fit for authentication and evaluation ofthe quality of the HMP This review tends to highlight theneed to combine both DNA barcoding and chromatographicfingerprint in order to effectively validate the authenticity andevaluate the quality of the HMPs Detailed knowledge about


themain composition of theHMPswill help to ascertain theirefficacy and safety for quality purpose control

2 High Performance LiquidChromatography (HPLC)

Chromatography is a technique used for the separation ofa mixture into their constituent component This techniquewas first coined by a Russian botanist Mikail Tswett in1903 when he separated plant pigments such as chlorophylland xanthophylls by passing their solution through a glasscolumn packedwith finely divided calcium carbonate [51] Asthe plant pigments are coloured the separation was namedusing two Greek words ldquoChromerdquo and ldquographeinrdquo meaningldquocolourrdquo and ldquoto writerdquo respectively After the invention thevalue of his workwas not appreciated for a few years but aftera few decades Tswett discovery was reconsidered leading toan explosive emergence of various modalities of chromatog-raphy The complex mixtures are separated due to the differ-ence in their time taken for each component to travel througha system that contains an immiscible bed of material or sta-tionary phase andmobile phase [52]Themobile phase trans-ports analyte while the stationary phase is immobile Thereare different types of chromatography and these are classifiedbased on the physical state of the mobile phase such asgas chromatography (GC) for gas mobile phase while liquidmobile phase is known as liquid chromatography (LC) [53]

Due to the versatile application of these techniqueschromatography fingerprints are now widely used in theanalysis of complex compounds such as HMPs for qualitycontrol including authentication processes and efficacy andsafety evaluation [54] Chromatography fingerprint in herbalmedicine is defined as the study of the chromatographicpattern of pharmacological active compounds present in theherbal medicine under study [55] and has been accepted byWHO since 1991 [56 57]

However among the different types of chromatographyfingerprint that exist the high performance liquid chro-matography (HPLC) is undoubtedly one of the most popularand widely used chromatography fingerprints for the analysisof herbal medicines [58ndash62] Since 1991 the WHO hasaccepted this technology as a strategy for identification andquantification of herbal medicine [56 57] QualitativelyHPLC provides information about the presence of com-pounds and quantitatively the actual amount of that com-pound is present in the herbal extract or finished HMP Thequalitative and quantitative identification of the unknowncompounds is done by comparing their HPLC retentiontimes with unknown and UV spectra [63]

High reproducibility sensitivity selectivity and the abilityto analyse a number of constituents in HMP are among thegreat advantages of usingHPLC techniques [18 64]Howeverfor effective HPLC operation and to achieve the desiredobjective certain important factors have to be taken intoconsideration starting from sample preparation to the finalanalysis

21 Sample Preparation Sample preparation is a crucialaspect in any analytical process as it occupies around two-thirds of the time of the whole process and accounts foralmost one-third of the errors generated [158 159] It istherefore a very important part of chemicalbiological anal-ysis as it assists in removing potential interference and theanalyte becomes detectable [160] Sample preparation is alsoimportant in HPLC analysis as it helps to remove analytesfrom its original matrix into one that is homogenous andreproducible for proper analysis [161] Complete dissolutionof the sample in the eluent subsequently followed by filtrationusing a microfilter (usually with 045120583mM) are the funda-mental steps before HPLC analysis This is very importantbecause it will help to produce a solution that will be suitablefor injection into the column during HPLC analysis Theoverall outcome of any HPLC analysis is solely dependenton the quality of the sample preparation used There aredifferent methods of sample preparation that are necessaryto modify the sample amenable for HPLC analysis so as toimprove the result of the analysis This can involve a numberof operations that may be necessary to modify the samplesand this may also have various purposes for example samplefractionation cleanup and concentration of the analytes

Before embarking on the normal operation of samplepreparation of HMP for HPLC analysis there exists a routinesample manipulation which is weighing the sample It is veryimportant to know the appropriate amount of sample thatwill be taken into the analysis as this may affect the analysisresult as much as the final output of the chromatographicmeasurement Other basic operation techniques to be usedfor the HPLC analysis depend largely on the nature of thesamplematrix to be analysed either as solid semisolid liquidor gas [159] For example simpler sample preparation isemployed if the sample matrix is soluble in the mobile phaseHowever in a situation where the sample matrix is notsuitable in themobile phase as in the case of powder or herbsa suitable extraction procedure should be applied It is veryimportant that the chosen extraction method should containlow molecular constituent of herbal products and this ismostly achieved by the use of methanol and ethanol Some ofthe basic extraction protocols or operations in sample prepa-ration of HMP for HPLC analysis include homogenisation ofthe sample matrix dissolution and filtration

211 Sample HomogenisationSize Reduction This is one ofthe most used steps in the preparation of HMPs for HPLCanalysis due to their physical nature of the solid samplematrix This is usually achieved by reducing the samplematrix size into a fine powder using pestle andmortar [162] orany other laboratory mills or blender The ground material isthen transferred into a volumetric flask for further extraction

212 Extraction Technique Extractions are mostly perfor-med at room temperature due to the fact that most analyteshave aqueous solubility or a mixture of aqueous and organicmedia [163] Extraction can be done in one step with singlesolvent or in multiple numbers of steps with numeroussolvent depending on the nature of the analytes Informationabout the nature of the analytes (at least class of the analytes


such as organic inorganic ionic character or functionalgroup of the organic compound) to be measured is of vitalimportance This is due to the fact that the solvent to beused for the extraction process must be able to solubilize theanalytes andmust be compatiblewith theHPLCmobile phasefor the final analyses Other qualities of solvent include highpurity compatibility with the detector and low viscosity tokeep the system back pressure low [164] Furthermore thedissolved oxygen present in the solvent may lead to systeminstability but can be removed by degassing step

Dissolution of the analytes can enhance the extractionprocess and this can be achieved by agitation of the wholeHMPs (eg capsule or grounded powder) by the use of ultra-sound machine Ultrasonic extraction process is the mostused extraction processes for HMPs [30 165ndash167] and thishasmore benefits than other techniques [168] Fast extractionrates better solvent penetration greater yield extractionmaintaining low temperature increased mass transfer andless dependency on solvent are all the features that makesonication more advantageous than other types of technique[169ndash171]

Other methods used include mixing shaking inver-sion and vortexing [71] Sonication process facilitates thetransfer of analytes from the sample matrix to the solventand this is generally achieved in a short period of time[172] Methanol is the most commonly used solvent in thesonication process ofHMP extraction [70 86 173 174] Othersample preparation techniques include solvent immersionsolvent partitioning refluxing solid-phase microextractionsupercritical-fluid extraction pressurized-liquid extractionmicrowave-assisted extraction solid-phase extraction andsurfactant-mediated extraction

The extracted analytes of the HMP are further filteredusing microfilter (normally 045 120583m) before being subjectedto HPLC analysis as reported by several studies [175 176] andthis is a crucial step because it may remove any particulatewhich may block the column or interfere with the HPLCanalysis

22 Mode of Separation Mobile and Stationary Phase HPLCis one of the most popular methods for the analysis ofany crude mixtures such as HMPs and this requires eitherreverse-phase or normal-phase chromatographic mode [164177] The mobile phase is the liquid phase of HPLC whichcarries the analytes and continues flowing through the sta-tionary phase the phase where the analytes are separated byadsorption The use of any mode (mobile phase) depends onthe compatibility of the stationary phase with the nature ofthe extract or mixture to be analysed So also the mode to beused also depends on the chemical nature of the crude extract

It is an easy method which generally requires eitherreverse-phase or normal phase whose choice of usagedepends on the compatibility of the stationary phase with themixture or extract to be analysedThemethod to be used alsodepends on the chemical nature of the crude extract Amongthe two the reverse-phase column is the most commonlyused method for the separation of HMP when compared tothe normal phase and is alwaysmore likely to produce a satis-factory final result [178]

221 Normal-Phase HPLC (NP-HPLC) The normal-phaseHPLC (NP-HPLC) which was formally known as adsorptionHPLC is not the most popular form of HPLC analysis foranalysis of HMPs This method is best used for analysis ofpolar analytes as it utilizes a polar stationary phase whichusually consists of silica compared to a nonpolar mobilephase for example n-hexane This is due to the fact that thepolar compounds will strongly bind or be adsorbed to thepolar silica in the column making the nonpolar compoundquickly pass through the stationary phase Increasing thepolarity of solvent in the mobile phase will decrease theelution (retention) time of the analytes On the other handmore hydrophobic solvent will decrease the retention timeThe popularity of using NP-HPLC decreased in 1970 with thedevelopment of reverse phase (RP-HPLC) due to the lack ofreproducibility and retention times [179]

The eluents that are mostly used in NP-HPLC mostlyconsist of a mixture of different types of hydrocarbons suchas aliphatic (eg n-hexane and n-heptane) halogenated(eg chloroform) more polar oxygenated (eg ethyl acetatediethyl acetate and acetone) or hydroxylated solvents (egisopropanol and methanol) [164]

222 Reverse-Phase HPLC (RP-HPLC) Reverse-phaseHPLC (RP-HPLC) is the most commonly used HPLC modethat is used for the analysis of HMP [18 61] As the nameimplies it is just the reversemode of NP-HPLCThe selectionof stationary phase is one of the most important factors forfingerprint development [180] Commonly used RP-HPLCstationary phase for herbal fingerprint is the silica which ismodified or treated with nonpolar RMe

2SiCl R is a straight

chain alkyl group where the long chain (eg C18H37) is more

hydrolytically stable than short chain group (eg C8H17)

[177 181] Typically 2ndash5120583mparticle size is used as this is veryimportant for maintaining their efficiency backpressure isdrastically increased if small particle size is used [29 180]

In contrast with NP-HPLC RP-HPLC has a broad rangeof selectivity coupled with a high degree of reproducibility[177] The retention time of RP-HPLC is longer for less polarmolecules as such the polarmolecules elutemore readily Soalso most of the eluents used in the RP-HPLC are composedof water mixed with miscible organic solvents which aremostly acetonitrile methanol or tetrahydrofuran [164] PHalso plays a vital role as it changes the hydrophobicity of theanalytes For this reason buffer acids or bases are also addedin order to control the PH The effects of buffer and acidsdiffer tremendously by application but generally improvechromatography result [179]

23 Isocratic Flow and Gradient Elution The term isocraticmeans constant composition [179] It is a type of separationanalysis in which the mobile phase composition is constantthroughout the HPLC analysis as such any equilibration ofthe column is not required Isocratic flow elution is alwayssuitable for separation of mixture with few compoundsIsocratic flow with commonly used reverse-phase column isalso used in the analysis of HMP [71 173 182 183] In terms ofgradient elution it is the reverse case of isocratic analysis asthe mobile composition changes overtime Gradient elution


with reverse-phase is the most commonly used in the HPLCanalysis of multiple constituent present in herbal preparationor medicinal plants [184ndash189]

24 Detectors Adetector which consists of a sensor and elec-tronics is a device used with HPLC to detect or monitorthe presence of sample components in a medium whileeluting the column After the columns have performed theiractual role of separation the next task is to identify thepresence of the separated component in the sample and thisfunction is achieved using detectors This situation makesthe detector the secondmost important component of HPLCanalysis as it will be meaningless to separate componentsin a sample without identifying them The detection ofchemicals using HPLC analysis is possible as a result ofa specific physiochemical property of the sample which isdifferent from that of themobile phaseThe detector providesan electric signal which is proportional to the quantity orconcentration of the componentwithin the detector at a givenmomentThe signals which are produced in the formof peaksare then further processed by passing them into an integratorwhich will be used to quantify the amount of the componentspresent The use of peak areas in the chromatogram is themost common way of quantification [190]

The criteria for a specific choice for the selection ofdetectors depend on some factors which include sensitivityof the detector for a specific property difference in propertiesfrom that of the mobile phase and separation conditionused for the analysis The detection of molecules after elutingthem from the chromatographic column can be achievedusing varieties of principles and techniques and this can beclassified as those that can detect all sample componentsparticular group of compound or only one compound [191]Thefirst groupwhich can response to all samples componentsare called universal and a good example of such detector is theUVVis detectorThe second are known as selective while thelast are specific

The various detection techniques used for the HPLCanalysis of HMP include the Ultraviolet-Visible (UV-Vis)or photodiode-array the evaporative light scattering (ELS)and the spectrometry (MS) as well as other types of otherdetection techniques However as detectors are very crucialin HPLC analysis they are expected to possess some qualitieswhich include sensitivity linearity in a wide range of samplereproducibility in the response stability to changes in flowand environmental parameters and capability of detectionin a small volume of sample [190] In general the mostfrequently used detectors for the analysis of HMP are theUV-Vis [71 80] and ELSD [75 192ndash194]

UVVis is a universal detector used in HPLC system andwas first described by Horvath and Lipsky in 1966 [195]This type of detector is capable of absorbing electromagneticradiation in UVVisible wavelength range of 190ndash600 nm Itis extremely important as it gives room for analysis of nat-ural products containing chromophores such as flavonoidscoumarin or isoflavin The UVVIS detectors are easy tooperate particularly suitable for less skilled operators Thediode-array detector (DAD is also known as photodiode-array PDA) is an advanced form of UVVis that is frequently

used for the analysis of HMP coupled with an HPLC toform hyphenated technique HPLC-PDA also known as LC-PDA [72 79 196] The PDA provides more information onsample composition than any other detectors due to the factthat it can collect data at one or more wavelength acrossa chromatogram in one or more analytes in a single run[64] However the inherent limitation of using PDAUVVISdetector is that it can only absorb compounds that haveabsorbing chromophores those that do not have them willtherefore have little or no effects on theUVdetector [61 197]PDA is one of the most commonly used detectors utilized forthe screening of vitamin drugs natural products and herbalmedicines [198] probably due to its high sensitivity to manysolutes simplicity and low cost [29]

The ELSD is also a universal type of detector used for theHMP analysis As a universal detector it has the potential tobe used for any type of sample including those that do nothave good light absorbance in the UV The work of this typeof detector begins with nebulization of the solution elutedfrom the column to form aerosol that is further convertedto larger droplets so as to enhance detection [199] Theonly requirement for using this type of detector techniqueis that the component of the sample should be less volatilethan the component of the mobile phase [195] ELSD hasalso played a vital role in the analysis of compounds withlittle or weak chromophores such as saponins [57] aglycone[200] terpenes [83] glycosidic form [193] and other formsof alkaloids [201] which may be difficult to detect using UVdetector However nonlinearity of ELSD as reported by someresearchers (Verhelst and Vandereecken [202] Carbognani[203]) is among the drawbacks or limitations of using thistype of detector in the analysis of complex mixture ofcompounds

Due to some limitations ofHPLC-UV-Vis orHPLC-PDAnowadays HPLC coupled with mass spectrometry (MS) hasbecome a powerful tool for the qualitative and quantitativeanalysis of complex mixture such as HMP or natural productextract [204]This is due to the fact that hyphenatedMS withHPLC as a detector has high sensitivity and selectivity forthe analysis of natural product HMPThe term ldquohyphenatedrdquorefers to coupling of an independent analytic instrument toprovide detection In HPLC-LC also known as LC-MS thesamples separated from the column can be observed in theform of spectral data [196] Atmospheric-pressure ionization(API) interfaces which include electrospray ionization (ESI)and atmospheric-pressure chemical ionization (APCI) areused to overcome the problem of high mobile phase volumeduring the HPLC analysis and it is the most successfulinterface used in HPLC-MS configuration [63 205] The useof interfaces together with MS in the analysis of HMP hasbecome a powerful tool due to its high sensitivity low level ofsample consumption and rapid analysis time [206] Most ofthese ionization techniques used in this detection technique(HPLC-MS) are generally soft ionization techniques and thisdoes not typically produce many fragments

The use of LC-MS in the analysis of HMP has nowattracted a lot of attention compared to any type of detectionmethod due its high sensitivity and selectivity As mentionedearlier the introductions of the various types of interface


more especially ESI and APCI with respect to the analysisof HMP have also made this type of detection techniqueextremely important This is due to the fact that withthe presence of these interfaces various analysers such asquadrupole (Q) ion trap (IT) and time of flight (TOF) canalso be used and all these conferred a better advantage in thequalitative and quantitative analysis of the HMP Howeverupon the various advantages of using this type of hyphenatedtechnique in the analysis of HMP the quality of responsestrongly depends on the nature of the compound to be usedthe type of solvent and buffer to be used in the mobile phasethe type of interface to be used and so on All these factorsare problems associated with the use of this type of detectiontechnique

Other HPLC hyphenated techniques such as NMR spec-troscopy are available for the analysis of HMP The HPLC-NMR or LC-NMR which has been in existence for the pasttwo decades helps in providing useful information aboutthe structure elucidation of natural product [196] Howeverupon the various advantages of using this type of detectiontechnique in the analysis of natural product LC-NMR hasnot become a widely accepted hyphenated technique forHPLC analysis and this could be attributed as a result of itslow sensitivity The summary of the different types of thedetection technique used in the analysis of the HMP is shownin Table 2

The qualitative and quantitative analysis of the HMP arevery significant in the authentication of HMPs as they serveas the prerequisite for disclosing their overall benefits and risk[206] The developments of the modern analytical techniqueof HPLC coupled with other detectors have been extensivelyused for the qualitative and quantitative analysis of HMPHPLC coupled to mass spectrometry is also becoming verypopular in the qualitative and quantitative analysis of HMPmore especially in the presence of the various types of softionization technique The development of the different typesof mass analysers such as Quadruple (Q) time of flight(TOF) and Ion trap (IT) also makes the qualitative andquantitative analysis of complex HMP applicable

The qualitative analyses ofHMP are generally used for theidentification of unknown compounds by comparing themwith standard ones [104] However despite the presence ofvarious powerful analytical tools the confirmation of chem-ical component contained in the HMP is still a challengeThis is due to the fact that most of the components inHMP receipts are limited in target compound and the iden-tification techniques rely on the authentic standard [206]HPLC analysis has been widely applied for the qualitativeanalysis of HMPs such as Brucea javanica [93] ScutellariabaicalensisGeorgi [103]Dalbergia odorifera [104] and Panaxnotoginseng [102]

After the qualitative analysis next is the quantitative ana-lysis and this is very crucial as it is the prerequisite for thequality control of theHMP A number of quantitative analysismethods develop using HPLC are simple stable and durable[207] The coupling of the HPLC with different types ofdetectors such as the UV ELSD and MS is frequently usedin the quantitative analysis of HMP The UV detectors arethe most frequently used detectors in the quantification of

HMPs despite the fact that they have a poor response incompounds with few or no chromatophores Many kindsof components present in HMP were quantified using thisdetection technique which includes Yiqing capsule [208] andQingfu Guanjieshu (QFGJS) capsule [167] However theseobstacles are usually overcome through the use of ELSDwhich can detect and quantify the nonchromophoric com-pounds present in theHMP Some of theHMPs quantitativelyanalysed using the ELSD include Flos Lonicerae [209] andPanax notoginseng (Burk) F Chen [210] The couplings ofHPLC with MS are mostly used in quantitative analysis ofHMP due to their high rate of sensitivity and selectivityWhen developing HPLC-MS method for the quantificationof complex material soft ionization technique such as theESI and API detection mode and the condition of themobile phase should be optimized [207]The development ofvarious types of mass analysers as earlier stated can make thequantitative analysis of HMP practically feasible The HPLC-MS has been widely applied in the quantitative analysis ofseveralHMPs such asRadixAngelicaeDahuricae [105]Yuccagloriosa L [106] Shenqi Fuzheng injection [107] and Panaxnotoginseng (Sanqi) [102]

Apart from the quantitative and qualitative analysis of theHMP some semiapproaches have been proposed [206] Thisapproach gives room for the simultaneous determination of amultiple active compound based on the presence of only oneor several standards For example Gao et al [108] selectedrhizome and used a single standard emodin as both internaland external standard to simultaneously determine other sixanthraquinones in rhubarb The result showed that therewas no significant difference between the results obtainedusing the external standard The result shows that this alter-native method can be used without standard Simultaneousabsolute or semiquantification of multiple components hasbeen widely applied for the quantitative analysis of variousHMPs such as Brucea javanica [110] Triticum durum [111]and Fritillaria [109] The examples of some selected qualita-tive quantitative and semiquantitative analysis of HMP areshown in Table 3

25 Data Analysis Most of the data analyses from HMP arecarried out using PC-based software such as the Chromeleonfor Dionex which normally comes with theHPLC system andcan be used for quality control and authentication purposesIn case of the chromatogram the data may be composedof different components such as peak area peak heightand retention time while for spectroscopic (MS or NMR)techniques the data are obtained in the form of absorbanceor transmittance absorbance These data are analysed for thequantification and identification of separated component ofthe HMP These data are analysed and obtained from HPLCfingerprint analysis of the HMP

Other than the above technique chemometrics andprincipal component analysis (PCA) are also techniques usedto verify and distinguish the similarity or dissimilar structureof obtained data in the quality control and authentication oftheHMPThe chemometrics analysis is composed of group ofmethods which include basic statistics mathematics signalprocessing detection and other methods in order to retrieve


Table 2 Example of some detectors used for the detection of HMPs

Types of HMPs Medicinal value Column Detector Chromatographiccondition References

Valerian Neuralgia epilepsy andrelieving digestive

VP-ODS C18 column(46 times 250mm 5 120583m) HPLC-UV

aAcetonitrile08mLmin isocratically

d10 120583LGhafari et al [71]

VidangaAnthelmintic

carminative andstimulant

Chromatopak Peerlessbasic C18 column

(46 times 250mm 5120583m)HPLC-PDA

aMeOH-phosphateb14mLmin

isocratically c25∘Cd20 120583L

Sudani et al [72]

Radix Isatidis (Isatisindigotica)

Influenza epidemichepatitis and epidemic

encephalitis B

ODS-3 Inertsil column(25 cm times 46mm id

5 120583m)HPLC-DAD

aWater-acetonitrileb1mLmin gradient

c35∘C d10 120583LZou et al [73]

Buyang Huanwudecoction

Promoting blood flowtreatment of

cerebrovascular diseases

Zorbax SB-C18 column(46 times 50mm 18120583m)

HPLC-DAD-TOFMS

a03 formic acid water-acetonitrile

b08mLmin gradientc25∘C d1 120583L

Liu et al [74]

GinsengAntidiabetic

anti-inflammatory andantitumour activities

C18 column (250mm times46mm id 5 120583m) HPLC-ELSD

aAcetonitrile-water-acetic acid (10 85 5

vvv) andAcetonitrile-water

(80 20 vv)b15mLmin gradient

c40∘C d10 120583L

Sun et al [75]

Ge-Gen decoction Treatment of commoncold fever and influenza

Agela Venusil MP-C18column

(46 times 250mm id5 120583m)

HPLC-Q-TOF-MSMS

a01 formicacid-water-acetonitrileb10mLmin gradient

c30∘C d10 120583L

Yan et al [67]

Radix Paeoniae RubraAnti-inflammatoryincreasing coronary

blood flow

Waters Symmetry C18column (250mm times46mm id 5 120583m)

HPLC-PDAaAcetonitrile-phosphoric

acid b08mLmingradient c25∘C d20120583L

Xu et al [76]

Cimicifuga racemosaAntipyretic and

anti-inflammatoryactivities

C-18 column(5 120583m 120 A 46mm times

250 mm)HPLCndashPDAndashELSD

aAcetonitrile-waterb16mLmin gradient

c43∘C d20 120583LLi et al [77]

Renshen (Panaxginseng)

Treatment ofcardiovascular diseases

cancer

A CSH C18 column(21 times 100mm 17 120583m) HPLCQTOF-MSE

CH3CN-H

2O

b03mLmin gradientc25∘C d2120583L

Qiu et al [78]

Glycyrrhiza glabra LAnti-inflammatory

antiviral antiallergy andantiulcer properties

C18-HL (150 times 100mm10 120583mHichrom Ltd) HPLC-PDA

01 TFA in water-01of TFA in MeOH

300mLmin gradientc25ndash28∘C d100 120583L

Basar et al [79]

Sanqi (Panaxnotoginseng)

Hemostatic andcardiovascularproperties

Waters Symmetry C18column

(250mm times 46mm id5 120583m)

HPLC-UVaWater-acetonitrile


Lau et al [80]



cancer

WatersODS C18 column(150 times 21mm id

5 120583m)

HPLC-APCIMS

aAcetonitrile-water(33 67 vv)b02mLmin

isocratically c35∘C

Ma et al [81]

Evodia rutaecarpa Anti-inflammatory andantibacterial effects

Zorbax SB-C18 column(250mm times 46mm id

5 120583m)HPLC-DAD


c25∘C d10 120583LZhao et al [82]

Fritillaria pallidiflora Treatment of cough Kromasil C18 column(200 times 46mm id) HPLC-ELSD

aAcetonitrile-water-diethylamine

(70 30 01 vv)1mLmin isocratically

c88∘C d20 120583L

Li et al [83]


Table 2 Continued


Hongdoushan capsule Treatment of ovarianand breast cancers

Shimadzu C18 column(46 times 250mm 5120583m) HPLC-PDA


c25∘C d5 120583LZhu et al [70]

Panax quinquefoliumImproving

cardiovascular activityinsomnia

Waters Spherisorb S3ODS2 column(150 sdot 20mm)

HPLC-APCI-MS

aAcetonitrile water(24 76 vv)b02mLmin


Ma et al [84]

Coptidis rhizome(Huanglian)

Suppressing feverdispelling dampness andantimicrobial properties

Kromasil C18 analyticalcolumn

(250mm times 46mm5 120583m)

HPLC-ELSD

aAcetonitrile water(30 70 vv) pH wasadjusted to 60 with

02moLLtrichloroacetic acidb28 Lmin c115∘C

d20 120583L

Kong et al [85]

Menoprogen Treatment of menopauseKromasil ODS C18

column (46 times 250mm5 120583m)

HPLC-PDA01 phosphoric acid-

01 acetonitrileb10 Lmin c25∘C d10 120583L

Wang et al [86]

Fructus AurantiiImmaturus

Dispersing painfulabdominal mass

Sepax C18 column(5 120583m 250mm times

46mm)HPLC-DAD

aAcetonitrile-methanol-polyphosphoric


Xu et al [87]

Tong-Xie-Yao-Fang Diarrhoea-predominantirritable bowel syndrome

Kromasil C18 column(250mm times 6mm

5 120583m)

HPLC-DAD-ESI-M

aAcetonitrile-01formic acid and waterb06mLmin gradient

c35∘C d5120583L

Yan et al [88]

Tulsi (Ocimumsanctum)

Carcinogenschemotherapeutic agents

C18 column(250 times 46mm2 5mm

Waters USA)HPLC-PDA


c40∘CChanda et al [89]



Diamonsil C18 column(5 120583m 250mm times

46mm)HPLC-UVndashvis

aAcetonitrile-05 ofphosphate acid


Zhang et al [90]

Eurycoma longifolia JErectile dysfunctionlibido and male

infertility

C18 column(21mm times 50mm

27 120583m)LC-MSMS

a01 formic acid inwater-01 formic acidin acetonitrilewater


c40∘C d2120583L

Han et al [91]

Antike capsule Antineoplastic property Zorbax SB-C18 column(46 times 250mm 5 120583m) HPLC-PDA

MeCN and 01HOAcndash05 KH

2PO4

aqueous solution(adjusted to pH = 24

with H3PO4)


Duan et al [92]

Brucea javanicaTreatment of canceramebic dysentery and

malaria

Agilent EclipseXDB-C18 column(250mm times 46mm

5 120583m)

HPLC-QTOFMSWater-acetonitrileb1mLmin gradient

c25∘CTan et al [93]

Fufang ZhenzhuTiaozhi Dyslipidemia

Dionex Acclaim C18column (250mm times46mm 5 120583m)

HPLC-DAD

aAcetonitrile-potassiumdihydrogen phosphatesolution (40 60 vv) PHadjusted to 3 with 17 gLsodium dodecyl sulfateand phosphoric acid

b10minmLisocratically c30∘C

d20 120583L

Chen et al [94]


Table 2 Continued


Black cohosh(Cimicifuga racemosa)

Antipyretic andanti-inflammatory

activities

C-18 column(150 times 46mm 5 120583m) HPLC-ELSD

aWater-acetonitrile-alcohol b10mLmingradient c40∘C d10 120583L

Ganzera et al [95]

Psoralea corylifolia Osteoporosisosteomalacia

Zorbax SB-C18 column(30 times 100mm 35 120583m)

HPLC-DAD-TOFMS-QITMS

a01 aqueous formicacid-acetonitrile


Tan et al [96]

Cimicifuga foetida LAntimicrobialtranquilizer and

hypotensive treatments

Hypersil UK ODS2column (200 times 40mm

id 5 120583m)HPLC-ELSD

aMethanol-waterb08mLmin gradient

c30∘C d10 120583LKong et al [97]

Xue-Fu-Zhu-Yudecoction

Atherosclerosis andcoronary heart disease

ZorbaxSB-C18 column

(46 times 250mm 5120583m)HPLC-ESI-MS

a(CH3COOHH

2O)-

(CH3OH) b05mLmingradient c30∘C

Zhang and Cheng[65]

Curculiginis Rhizoma

Treatment of paininflammation andimmune and hepatic

disorders

Altima C18 ODScolumn (46 times 250mm

5 120583m)HPLC-DAD

aAcetonitrile with 002TFA-water with 05TFA b10mLmin

gradient c30∘C d10 120583L

Bian et al [98]

Danggui-Shaoyao-San

Dysmenorrheaamenorrhea

Alltima C18 column(250mm times 46mm id

5 120583m)

HPLC-DAD-ESI-MS

aAcetonitrile-water-formic acid b1mLmin

gradient c30∘CChen et al [68]

Ojeok-san Primary dysmenorrhealclastogenicity

Gemini C18 column(46 times 250mm 5120583m) HPLC-DAD

a01 formic acid inwater-acetonitrile


Kim et al [99]

Gui-Zhi-Jia-Shao-Yao-Tang

Effect against pepticulcer by alleviating

gastrospasm

Zorbax SB-C18 RapidResolution HT column(46mmtimes 50mm18120583m Agilent)

LC-MS and NMR



Wang et al [100]


Cardiovascular diseasescancer

Capcell Pak C18column (250mm times46mm id 5 120583m)

HPLC-ESI-MS

a02 acetic acid inwater-acetonitrile


Zhang et al [101]




5 120583m)

HPLC-QTOFM


containing a01 formicacid b08mLmingradient c25∘C

Tan et al [102]

aMobile phase bflow rate ctemperature dinjection volumeHPLC-UV Ultraviolet-Visible detector HPLC-DAD liquid chromatography-diode-array detector HPLC-PDA liquid chromatography photodiode-array HPLC-ELSD liquid chromatography evaporative light scattering detector LC-MS liquid chromatography-mass spectrometric detector NMRnuclearmagnetic resonanceHPLC-DAD-ESI-MS liquid chromatography-diode-array detector-electrospray ionization-mass spectrometry HPLC-APCI-MSliquid chromatographyndashatmospheric-pressure chemical ionization-mass spectrometry HPLC- DAD-TOFMS liquid chromatography-diode-array detector-time of flight-mass spectrometry LC-MS and NMR liquid chromatography-mass spectrometry-nuclear magnetic resonance HPLC-QTOFMS liquidchromatography-quadrupole time of flight-mass spectrometry HPLC-ESI-MS liquid chromatography-electrospray ionization-mass spectrometry HPLC-DAD-TOFMS-QITMS liquid chromatography-diode-array detector-time of flight-mass spectrometry-quadrupole ion trap mass spectrometry LC-MSMSliquid chromatography-mass spectrometrymass spectrometry HPLC-PDA-ELSD Liquid chromatography-photodiode-array-evaporative light scatteringdetector

more information from the chromatographic data [29] Thechemometrics analysing technique was first coined by SvanteWold in 1971 and is a simple application that integrates math-ematics and statistical technique to providemore informationfrom the various spectroscopy and chemical measurementdata [211] However with the massive progress observed inboth electronics and computational technique chemometricshas become one of the most important and frequently usedtools for the analysis of HMP as it helps to provide usefulinformation from the original statistics

The chemometrics techniques are usually classified intotwo categories pattern recognition methods (unsupervisedand supervised) and multivariate calibration for qualitativeand quantitative evaluation respectively [212] The principalcomponent analysis (PCA) and clustering analysis (CA) arethe most commonly unsupervised patterns of recognitiontechnique used in the analysis of HMPThe PCA portrays theoriginal measurement by discovering the dominant factorswhile excluding the relevant interference factors thereby


Table 3 Examples of some selected qualitative quantitative and semiquantitative analysis of HMPs

Types of HMPs Medicinal value Type of analysis Method of analysis Reference


malariaQualitative analysis HPLC-QTOFMS Tan et al [93]

Scutellaria baicalensisGeorgi (Huang-Qin)

Treatment of feversulcers cancers and

inflammationQualitative analysis HPLC-DAD-MS Horvath et al [103]

Dalbergia odoriferaTreatment of blood

disorders ischemia andswelling

Qualitative analysis HPLC-DADESI-MSMS Liu et al [104]



Qualitative analysis HPLC-QTOFMS Tan et al [102]

Radix AngelicaeDahuricae

Anticancer andantibacterial properties Quantitative analysis HPLC-ESI-MSMS Zheng et al [105]

Yucca gloriosa LHigh antioxidant and

antiproliferativeactivities

Quantitative analysis LC-ESI-MS Skhirtladze et al[106]

Shenqi Fuzhenginjection (SFI)

Goodimmunoenhancementand anticancer activity

Quantitative analysis SPE-HPLC-UVELSD Wang and Qu [107]



Quantitative analysis HPLC-QTOFMS Tan et al [102]

Rhubarb (Rhei rhizome) Cathartic and laxativeproperties

Semiquantitativeanalysis HPLC-DAD Gao et al [108]

Fritillaria Treatment of chroniccough lungs cancer

Semiquantitativeanalysis LCESI-TOF-MS Zhou et al [109]


malaria

Semiquantitativeanalysis HPLC-ELSD Tan et al [110]

Triticum durum

Antioxidantanti-inflammatoryantimicrobial and

anticancer

Semiquantitativeanalysis LCESI-MS Cavaliere et al [111]

HPLC-DAD liquid chromatography-diode-array detector HPLC-DAD-MS liquid chromatography-diode-array detector-mass spectrometry HPLC-DADESI-MSMS liquid chromatography-diode-array detectorelectrospray ionization-mass spectrometrymass spectrometry HPLCndashESI-MSMS liq-uid chromatography-electrospray ionization-mass spectrometrymass spectrometry HPLC-DAD-TOFMS liquid chromatography-diode-array detector-time-of-flight-mass spectrometry HPLC-DAD-ESI-MS liquid chromatography-diode-array detector-electrospray ionization-mass spectrometry HPLC-QTOFMS liquid chromatography-quadrupole time-of-flight- mass spectrometry LC-MSMS liquid chromatography-mass spectrometry-mass spectrom-etry LCESI-TOF-MS liquid chromatographyelectrospray ionization time of flight mass spectrometry SPE-HPLC-UVELSD solid-phase extraction highperformance liquid chromatography and ultravioletevaporative light scattering detection

allowing a more accurate estimate [211] The PCA techniqueis also the most commonly applied fingerprint used forhandling multivariate data without prior knowledge aboutthe sample under investigation [29]

In terms of the cluster analysis the objects are groupedbased on their chemical characteristics that is similar objectscluster (group) more together (forming a branch structurecalled dendrograms) to each other than those of the othergroup The clustering technique is generally divided into twosubtypes which include the hierarchical clustering analysis(HCA) and nonhierarchical one The hierarchical clusteringanalysis (HCA) is themost popular clustering technique usedin the quality evaluation of HMP [211] The flexibility toalter the similaritymeasurement criterion applied the linkage

method to suit different applications and all the results arealways on the original scale of the data which are themain advantages of the HCA [212] The combination ofPCA and CA has been widely used in identification andauthentication of medicinal plants due to the fact that theformer can directly reflect the differences between sampleswhereas the latter can classify objects based on their quan-titative characteristics [212]

Peng et al [113] analysed the fingerprint of Artemisiaselengensis Turcz using HPLC-PAD The samples were anal-ysed using PCAHCA and SAThe results from eachmethodused produce different properties of the data matrix fromthe different Artemisia selengensis Turcz sample type Thestudy provides important information about the matching


and discrimination of the fingerprint Guo et al [118] devel-oped HPLC-DAD to simultaneously determine 10 triter-penoid acids from the roots of Ziziphus jujube HCA andPCAwere used to differentiate and classify the sample contentof the 10 triterpenoid The PCA technique was also used toanalyse HPLC data generated for the authentication of theflavonoid content of different basil cultivars [112 117]

The linear discriminant analysis (LDA) k-nearest neigh-bour (k-NN) soft independent modelling of class anal-ogy (SIMCA) artificial neural network (ANN) partial leastsquaresndashdiscriminant analysis (PLSDA) and orthogonal pro-jections to latent structuresndashdiscriminant analysis (OPLS-DA) are the most intensively used supervised pattern recog-nition in the analysis of HMP [213] However before embark-ing on chemometrics technique analysis the data are usuallypretreated and the goal is to remove unwanted componentsor unclear interference which can cause overlap of peaks andshifted baseline on the HPLC chromatogram [180] Examplesof some chemometric methods used for the analysis of HMPare shown in Table 4

26 Limitation of HPLC in the Authentication of HerbalMedicinal Products The HPLC chemical fingerprint is thetechnique that is mostly used as a chemical method forauthentication of HMP [214] However despite its wideacceptance it has a number of limitations One of the cruciallimitations of this method is the unavailability of manybiologically active compounds (reference standard) and evenif present they are often expensive to purchase [215]

The use of HPLC analysis for authentication of HMPsmay also be compromised due to the fact that their testresults can be significantly influenced by many factors suchas variation in climate phenotype manufacturing processand storage condition and even variation in the type of planttissue used [171 180 216ndash218] The authentication of theHMP using HPLC is also affected by age and cultivationtime A study reported by Zhang et al [219] showed thatthe best harvesting time for shihu ginseng is the fifth year ofcultivation because of the high content of ginsenosides in thefifth year Therefore optimization of the numerous variablesto ensure reproducibility in the chromatographyrsquos fingerprintis always difficult

The inability ofHPLCalone to remarkably identify closelyrelated species that shared similar morphological and chem-ical properties is one of the most concerning shortcomingsof this technique [27] HPLC can only produce indirectevidence of fraud as it lacks the capability of determiningthe identity of the given species Certainly it is very clearthat standardization of HMP to comply with internationalstandard is of enormous importance [16] However this pro-cess is inherently difficult to achieve with chromatographyrsquosfingerprint because there is no universally accepted industrialstandard [180]

3 DNA Barcoding

DNA barcoding is a novel technique which was first coinedby Canadian zoologist Paul Herbert of the University ofGuelph in 2003 and it identifies closely related allies of

lepidopterans using short DNA sequences from the standardpart of the genome [44] He recommended the use of a partialregion ofmitochondrial cytochrome c oxidase which ismadeup of approximately 650 bp for animal identification as theregion is sufficient enough to generate the DNA barcodeThe mitochondrial region is suitable for DNA barcode as itfulfilled the basic criteria necessary for species identificationThe three basic criteria include (i) universality so that itcan be easier to be amplified (ii) high discriminatory poweror specification so as to distinguish it from closely relatedspecies and finally (iii) high sequence quality for easyidentification [220] However this makes the mitochondrialregion of the plants a poor choice for species identificationdue to the fact that their genes evolved slowly and have a lowrate of species discrimination which makes it not suitable forDNA barcoding [45 221]

For that reason attention moved to chloroplast andnuclear region in order to find which one among them canserve as a standard barcode for species identification in theplant After several assessments and exploitation of these lociregions it was later concluded that multilocus barcode is arequisite for plant barcoding as most of them have one ormore limitation as such their combination was suggested[45] For example the two chloroplast regions of maturase K(matK) and large subunit of ribulose-biphosphate carboxy-lase (rbcL) have been found to be suitable barcode regionwhen combined together a situation that makes the Consor-tium for the Barcode of Life (CBOL) plant working group rec-ommend them as the universal barcode [222]

The rbcL barcode region which consists of approximately1430 bp is easy to be amplified and has the ability to generategood quality sequence [223] The limitation of using thisregion is its length which in some cases needs to be entirelysequenced for species discrimination [224] However otherrbcL primers such as 1F724R which has been proposed asa universal primer in gymnosperm due to its high rate ofuniversality [225] can be used to amplify part or half of therbcL gene The length region of the rbcL has made it havepoor discriminatory power more especially at the specieslevel [226 227] The long size and limiting factor of rbcLregion makes it not suitable for species identification becausean ideal DNA barcode region should be short to be amplifiedfrom degraded DNA [228] in terms of the latter universalbarcode region which has the closest analogue to the COIanimal barcode [229] that consists of 841 bp at the centre ofthe gene (Staats et al 2016b) The chloroplastic matK regionis one of the most rapidly evolving genes with suitable lengthand high level of discrimination among angiosperm [230]The difficulty in the amplification of matK more especiallyin nonangiosperm is one of the limitations of using thisbarcode region and this can be linked as a result of itsinsufficient universal primers [231 232] The combination ofthese two loci is found suitable for DNA barcode due to thehigh amplification rate of the rbcL region and discriminatorypower ofmatK region [233]

However despite the fact that the combination of rbcL +matKoffers slightly higher identification efficiency than othercombinations their ability to discriminate between closelyrelated species is lower than that of COI in animals [224 234]


Table 4 Example of some chemometric methods used for the analysis of HMPs

Types of HMPs Medicinal value Methods of analysis Chemometricsmethod Reference

Basil (Ocimum sp) Antimicrobial andantioxidant properties HPLC-DAD PCA Grayer et al [112]



encephalitis BRP-HPLC HCA Zou et al [73]

Artemisia selengensisTurcz

Diminishinginflammation relievinga cough and stimulating

the appetite

HPLCndashPAD PCA HCA Peng et al [113]

Flos Lonicerae JaponicaeTreatment of bacterialand virus diseases

inflammation and feverHPLC-DAD HCA Li et al [114]

Polygala japonicaHouttAnti-inflammatoryantibacterial and

antidepressant agentHPLC-DAD-ELSD PCA Hong-Lan et al

[115]

Cassia obtusifolia L orCassia tora

Improving eyesight andmedicinal values as acathartic and diuretic

HPLC-DADPCA PLS

BP-ANN andRBF-ANN

Lai et al [116]

Ocimum americanum Ocitriodorum

Strong fungicidalactivity antioxidant

activityHPLC-DAD PCA Vieira et al [117]

Ziziphus jujubaMill Immunity stimulant andantitumour activities HPLC-DAD PCA HCA Guo et al [118]


Eliminating sputum anddispersing painfulabdominal mass

HPLC-DAD PCA HCA SA Xu et al [87]

PCA principal component analysis HCA hierarchical clustering analysis PLS partial least squares BP-ANN back propagation artificial neural networkRBF-ANN radial basis function artificial neural network HPLC-DAD liquid chromatography-diode-array detector HPLC-PDA liquid chromatogra-phy photodiode-array HPLC-DAD-ELSD liquid chromatography-diode-array detector-evaporative light scattering detection HPLC-APCI-MS liquidchromatographyndashatmospheric-pressure chemical ionization-mass spectrometry RP-HPLC reverse-phase liquid chromatography LDA linear discriminantanalysis SA similarity analysis PLS partial least square

In addition to that the combined barcode also has a lowPCR efficiency of matK and this explains the fact that therbcL + matK barcode still failed to meet the original goal ofuniversal barcode [224] Increase in analytical difficulties isalways an issue in combining barcodes compared to singlelocus-marker more especially when one of the target regionsfailed to be amplified

These developments opted China plant Barcode of Life(BOL) working group to propose the use of another nuclearregion (ITS) in addition to the standard barcode [45] Theyargued that the ITS regions have more discriminatory powerthan the plastid barcodes [233] despite the limitations asso-ciated with the regions such as difficulties of amplificationsequencing and incomplete concerted evolution [235] Inorder to resolve the difficulties in amplification and sequenc-ing of the ITS region they suggested the use of the short ITS2region as a backup because of the presence of it conservesequences [224] The ITS region in addition to anothernoncoding chloroplast region (psbA-trnH) is themost widelyused supplementary loci for species identification [236]

Subsequently the options of using either three or twobarcodes were later discussed in the fourth international

Barcode of Life conference held inAustralia It was concludedthat the two-barcode region was preferred in order to avoidthe low cost of sequencing [237] Therefore the use ofrbcL and matK DNA barcodes regions as standard barcodesregion together with ITS and psbA-trnH regions servingas complementary barcodes has helped tremendously inidentifying herbal medicines and their adulterants [220]Further standard DNA barcodes regions which have beenused in authentication of HMP include trnL-trnF [125] andrpoC1 [146]

The chloroplasts are very important region used for theidentification of HMPs This is due to the fact that thisregion contains highly conserved and variable genes whichare fundamental and informative to plants over broad timescale Nock et al [238] use complete chloroplast genometo identify species and this has become a universal methodused for single locus barcode identification taxonomy andPhylogenetic analysis of plant species The authors felt thatthe massive parallel sequencing (MPS) which they used cansignificantly improve the ability of distinguishing betweenand identifying different species and it is simple and cost-effective


31 DNA Barcoding for Identification of HMPs The processof identification of species using DNA barcoding can beachieved by building the DNA barcode library of knownspecies and matching or assigning the unknown barcodesequence against the barcode library [239] In terms ofHMPsthese processes can be achieved in five basic steps whichare of enormous importance as error or misinterpretationcan arise within each step The steps followed include (1)homogenisation of the medicinal herbal product to groundpowder (2) extraction of the genomicDNA (3) amplificationof the specific DNA barcodes region using PCR (4) sequenc-ing of the amplified region and finally (5) identification ofthe unknown sequence against known standard referencematerial [49]

311 DNA Extraction Extraction of DNA of high quality isthe most crucial step and prerequisite for proper identifica-tion of HMPs Extraction of any cellular components usuallyinvolves three central steps (1) disruption of the cell walls(2) removal of insoluble particulates from DNA and finally(3) DNA precipitation with integrity maintenance [240] Dueto the physical nature of the plant cell wall mechanicaldisruption such as the use of pestle and mortar glass beadssteel or glass rod and enzymatic digestion must occur beforeextraction [241] A physical disruption method using liquidnitrogen is more preferred as it will help to prevent crosscontamination more especially when multiple samples areused [242] The use of chemical disruptors is also convenientas it can bypass the possibility of DNA shearing due to themechanical force applied [243]The prime limitation of usingchemical disruptors is that they are expensive [240]

Like any other type of molecular technique DNA bar-coding relies on the availability of high quality DNA forspecies identification [50] Therefore any extraction methodthat will produce higher DNA yield and quality from HMPwill be of enormous importance This is due to the factthat HMP undergoes a series of heavy processing activitiessuch as drying and stewing and this may result in thefragmentation or degradation of theDNA to be used forDNAbarcoding In addition most HMPs contain high amountof secondary metabolites derived from raw materials (egpolysaccharides polyphenolic compounds and pigments)within various tissues and organs of the plants [244 245]The presence of these metabolites may prove to be the maincause for extraction of low quality DNA by binding with itand precipitating along with it [246]

The different types of extraction methods which can beused for extraction of DNA of high quality from HMP arenow available this can be based on conventionally developedprotocols or commercially available kits (Table 5) The useof commercial kits for extraction of DNA from HMP issometimes necessary as it used to extract DNA of high purityAnother advantage of using commercially available kits is theminimal requirement for laboratory equipment as such itcan easily be implemented in any laboratory The majorityof the commercially available kits used for DNA extractionin HMP use silica binding extraction methods As such theaffinity of the column through which the membrane bindswith the DNA is very important The prime limitation with

commercially available kit is that it may not be available forlow funded laboratory or routinely used for extraction ofa large number of samples [247] Furthermore it does notprovide room for researchers tomodify the standard protocolas the kits provided are limited

However other in-house DNA extraction protocols suchas Cetyltrimethylammonium bromide (CTAB) extractionbuffer utilize salting out precipitationmethodThis extractionmethod has been shown to be cost-effective compared tocommercially available kits although it is time consuming[248 249] The combined use of two extraction protocolssuch as CTAB with silica binding or resin has showed apromising result in a wide range of plant and plant derivedproducts [250ndash252]

However like the CTAB method sodium dodecyl sulfate(SDS) which is an anionic detergent is also used in theextraction of DNA from plant derived products The SDS isused in the disruption of the cell membrane to release theDNA into the extraction buffer after breaking or digestingaway the cell wall and nuclear membrane [253] The use ofSDS in the extraction of DNA developed by Edwards et al[134] is a simpler faster and inexpensive technique Besidesthe digesting of the cell wall and nuclear membrane SDStogether with EDTA present in the extraction buffer protectsthe DNA from endogenous nucleases by inhibiting theiractivities [254] The activities of endonucleases are inhibitedin the presence of EDTA which immediately chelates withdivalent ions (mg+2 Ca+2 andMn+2) the necessary cofactorsrequired for the enzyme structure and function [255]

312 Amplification of DNA Barcoding Region Amplificationof the DNA barcode region is one of the most importantsteps for species identification and authenticity and this issolely dependent on the quality of the DNA template Therelatively long gene sequence which is required for properDNA barcoding (ranging from 500 to 1000 bp) [49] is mostlynot found in HMPs as they get fragmented into piecesFragmentation of DNA usually occurs as a result of a processin which the herbal starting material may have been dried inthe sun or heavily processed to the extent that the DNA to beisolated is degraded [256] Fragmentation or degradation ofDNAmolecules extracted from the herbalmedicinalmaterialis the primary cause of PCR failure [48]

Therefore to overcome the problem of PCR amplifi-cation appropriate PCR primers (eg primers with highaffinity) such as the novel minibarcode will be of enormousimportance as they have high amplification success comparedto the full length barcode in degraded form of DNA [257]Sarkinen et al [252] who were working on degraded formDNA (herbariumDNA) reported that there is a negative cor-relation between amplicon size and PCR success indicatingthat smaller fragments are easy to be amplified The barcoderegion to be used in authenticating HMPs should not be largeasmost of the failure of the PCR amplification from degradedDNA samples is frequently reported when the amplicons aregreater than 200 bp [48]

313 Sequencing of the Amplified Region Generation ofsequence of high quality is one of the most important criteria


Table 5 Different DNA extraction methods that have been used in HMPs

Method used Extraction method Referencesupplier Medicinal partstype ofHMPs References

Roots leaves powderstem seeds whole plant Vassou et al [47]

Leaves and fruit Srirama et al [119]

Juice Mahadani and Ghosh[120]

Leaves da Costa et al [121]Fruits stem seeds herbcortex flowers rhizome Han et al [27]

Modified CTABmethods

Salting outprecipitation Doyle [122]

Herb Kumar et al [123]Leaves Alexander [124]Herb Li et al [125]Leaves Costa et al [126]

Dry leaves bark fruit Seethapathy et al [127]Leaves Selvaraj et al [128]Powder Sheth andThaker [129]Herbs Cheng et al [130]Roots Zhou et al [131]

Rhizome Wong et al [132]

Modified SDS methods Salting outprecipitation

Leaves flower bud Tamari et al [133]

Edwards et al [134]Leaves Alexander [124]Leaves da Costa et al [121]Herbs Cheng et al (2014)

Wizard Genomic DNApurification kit Silica binding Promega Tea Jian et al [135]

Capsule tablets powderleaf Newmaster et al [20]

Dried and fresh planttissue capsule tea dried

bark

Singtonat andOsathanunkul [136]

NucleoSpin plant II mini Silica binding Macherey-Nagel Tea Uncu et al [137]Plant tissue Osathanunkul et al [138]

Seeds Costa et al [126]Dried plant tissue Osathanunkul et al [139]

Leaves pills cone stemroots bark Llongueras et al [140]

NuceloSpin Tissue Kits Silica binding Macherey-Nagel Tea roots capsule liquidroot pieces tablets Wallace et al [46]

Bark Palhares et al [141]Capsule tablets Baker [142]

Leaves da Costa et al [121]Leaves flower roots Palhares et al [143]

DNeasy Plant mini kit Silica binding Qiagen Leaves pills cone stemroots bark Llongueras et al [140]

Powder Cimino [144]Leaves Alexander [124]Leaves Enan and Ahmed [145]

Fruit powder Parvathy et al [146]


Table 5 Continued


DNeasy96 Plant kit Silica binding Qiagen Tea Stoeckle et al [147]

Plant Genomic DNA Kit Silica binding Tiangen Biotech

Decoction Xin et al [148]Dried bulbus Xiang et al [149]Leaves cortex Zhang et al [150]

Crude drug dry leaf root Zhu et al [151]Root pills capsule tablets Liu et al [152]

Dry fruit leaves Dian-Yun et al [153]Dry leaves roots Hu et al [154]Dry flower bud Hou et al [155]

IBI genomic DNA minikit (plant GP1) Silica binding IBI Leaves pills cone stem

roots bark Llongueras et al [140]

for species identification and authentication The level ofDNA degradation at which useful sequence can be generatedfor species identification varies with the type of methodsused for instance high-throughput sequencing approach canbe effective with a small fragment size (around 50ndash400 bp)[50] However in other sequencing techniques such asSanger the PCR needed to amplify the DNA requires thefragment of at least the size of amplified fragment [50]So also higher chances of successful amplification of thetarget barcode marker are possible with less fragmented totalDNA yield Next-Generation Sequencing (NGS) technologyhas several key advantages over the conventional Sangersequencing [258] as such it will offer a tremendous impactin the authentication of the herbal products Some of theadvantages of NGS technique include (1) high-throughput(2) massive parallelization of sequencing reaction (3) lowcost and operation difficulties (4) superior sensitivity and(5) also the ability to be used to sequence large and morecomplex sequences [258 259] This in turns makes NGS themost preferred method for analysing samples with variousdegrees of DNA degradation

314 Reference Sequence Data The identification and auth-entication of herbalmedicinal plant byDNAbarcoding solelydepend on the availability of reference sequence data This isvery important because in the absence of reliable referencesequence identification of unknown samples may not beaccurate [49]The sequence data that are used for this identi-fication are currently deposited in the public library and arefree to access The most common ones among them includethe following

(1) BOLD (The Barcode of Life Data System) This is aninformation workbench which was created and maintainedby the Guelph in Canada Presently it contains over 370000plant barcodes representing over 58510 species of plant [50]and this includes vouchers images and maps BOLD alsopartners with other public reference libraries which includeCBOL [Consortium for the Barcode of Life] IBOL [Interna-tional Barcode of Life (httpwwwibolorg)] GBIF [GlobalBiodiversity Information Facility (httpwwwgbiforg)] and

NCBI GenBank [National Centre for Biotechnology Infor-mation (httpwwwncbinlmnihgov)] One limitation ofthe BOLDdatabase is thatmanymedicinal plants aremissingSo also various important barcodes regions such as ITS2 donot have complete coverage

(2) NCBI GenBank GenBank is probably one of the biggestand most important publicly available online databasesbuilt by NCBI GenBank is one of the most frequentlyused databases of genetic information [260] as such theassembled sequence in its database has exceeded 1 Terabasein October 2014 [50] GenBank contains BLAST (basiclocal alignment search tool) algorithm [261] through whichan unknown (query) DNA sequence can be rapidly andaccurately compared with known (reference) sequence

(3) MMDBD (Medicinal Materials DNA Barcode Database)This is a database whichmostly containedDNA sequence andinformation on medicinal plants listed in both Chinese andAmerican pharmacopeia Presently this database containsover 15375 sequences representing 1660 species of medicinalmaterial and also contains multiple regions including 4nuclear and mitochondria regions each and 7 chloroplastregions [50]

32 Limitation of DNA Barcoding in the Authentication ofHMPs Basically there are various limitations of DNA bar-coding in the authentication of HMP and the most crucialamong them is the low quality extraction of DNA templateDifferent drying methods and series of heavily processingactivities in addition to the presence of secondary metabolitein the plants might result in the extraction of degraded orfragmented form of DNA thus resulting in broken strandsDNA [262] If one of these breakages occurs at the primerannealing site amplification will not occur and the reactionwill fail Fragmented or degraded form of DNA is notsuitable for DNA barcoding [263] This is due to the factthat the extraction of DNA of high quality is the prerequisitefor the identification and authentication of HMPs Usefulsequence which can be generated for species identificationand authentication in DNA barcoding varies with the level of


DNAdegradation and type of sequencingmethods used [50]High-throughput sequencing methods can work effectivelywith fragment size of 50ndash400 bp while other approaches suchas Sangerwill require fragment of at least the size of the ampli-fied fragment (gt500 bp) but high chances of amplificationsuccess of the target DNA barcodes are found in less frag-mented extracted DNA

The Sanger sequencing always required pure PCR prod-uct for effective and reliable sequencing that is PCR prod-uct from multiple or complex mixture is not suitable forsequencing using Sanger sequencing [50] This is due to itsinability to resolve mixed signals from such kind of samples[258]However such limitation can be overcome by involvingthe use of next-generation sequencing (NGS) which hasseveral advantages over the Sanger sequencing Yieldingmillions of DNA reads in a small period of time massiveparallelization of sequencing reaction better sensitivity andclonal separation of templates are among the factors thatmake NGS technology perform effectively compared to theSanger sequencing (Staats et al 2016a)The coupling of DNAbarcoding together with NGS is referred to as metabarcoding[264] and has been applied in the diverse identification ofspecies However only few publications demonstrated thepower of metabarcoding in the authentication of complexHMPs Coghlan et al [265] and Cheng et al [266] per-formed a DNA metabarcoding analysis of different typesof complex traditional Chinese medicine (TCM) and bothconcluded that metabarcoding can serve as a prospectiveway to authenticate complex HMPs Ivanova et al [258] alsorecently reported that DNA metabarcoding can be reliablyused in the authentication of complex HMPs They furtheremphasized that that Sanger sequencing should not be usedas they lack the ability to resolve mixed signals samplecontaining multiple species

The processes by which standardized extracts are pro-duced may lead to the degradation or removal of plant DNAfrom the raw material therefore making DNA barcodingunfeasible for the authentication of such HMPs [50 258]This is due to the fact that the process involves multiplesteps [171] In addition some artificial adulterants used donot have DNA thus making the DNA barcoding unfeasiblefor verifying their identity in any given HMP sample [27]Solid state fermentation which is one of the strategies usedto improve the yield of active compounds [267 268] can alsoresult in the extraction of low quality DNA as they can bepartially or fully degraded [49] The presence of foreign orcontaminating DNA frommicrobial organisms such as fungiwhich may accumulate on improperly stored plant materialcan be amplified particularly when using nuclear fragment(ITS or ITS2) primers [49 269 270]This situation can resultin the generation of PCR product which might be difficult toanalyse [269]

Another disadvantage of using DNA barcoding inauthenticating the HMP is that it cannot differentiate tissuewithin the same plant that is it cannot identify the plantpart used tomake theHMP For exampleEurycoma longifoliais a famous medicinal plant in Malaysia solely known forits energetic and aphrodisiac property Most of the chemicalcompounds responsible for this therapeutic effect are present

on the root part as such most of the research on this plantis conducted on the root part [271] Substitution within thesame species part of this plant (eg substituting the root partwith leaf part) cannot be detected using DNA barcoding Soalso despite the effectiveness of DNA barcoding in identi-fying various adulterants used in the production of HMPit lacks the ability of providing information regarding thepresence and concentration of active ingredients responsiblefor their therapeutic properties [27] This explains the factthat DNA barcoding cannot provide information on whethera particular HMP has met the pharmacopeia standard as thisis necessary for overall quality controlThus DNA barcodingcan only determine the authenticity of HMPs but cannot beused to evaluate their quality

The affinity of the primer is also very important as thiscan yield a false negative result [50] If the products thatneed to be authenticated contain a DNA from another plantwhich has great affinity with the barcode primer it cantherefore be preferentially amplified thus leading to a falseamplification and sequencing This situation mostly occursin target species whose DNA is degraded into short pieces[258] The relatively long gene region of 500ndash1000 bases thatis required for DNA barcoding is not mostly intact in HMPsdue to the fact their DNA is mostly degraded or fragmentedinto short pieces as a result of a series of processing activitieswhich they underwent [49] Therefore it is recommendedthat shorter DNA barcodes region so-called minibarcodeof less than 150 bp should be created as this will help toovercome the problem of PCR amplification [50] Failureof PCR amplification has been frequently reported whenthe amplicon size is greater than 200 bp [48 142 272 273]Minibarcode in general has a high amplification success indegraded sample compared to standard or full length DNAbarcode [252 257]

Sequencing is also one of the main limitations of DNAbarcoding as different sequencing methods have differ-ent limitations Low throughput and the requirement ofhigh concentration of DNA amplicon template of about100ndash500 ng are the inherent limitation of using this approachso as to avoid bias and error [274] The Sanger sequencingis the most commonly used method in the authentication ofHMP to date [148 275] This approach is mostly affected byprimer bias even if serial dilution and multiple extractionmethods are used [258] The effects of PCR bias and ampli-fication can be partly solved by ligation of the PCR fragmentinto vector and cloned in bacteria or by performing parallelPCR [50]

DNA barcoding cannot identify adulterants present inHMP that do not contain DNA [27] For example the useof hazardous sulphur fumigated processing which has beenreported to be used by herbal farmers or wholesalers in orderto extend the storage time and prevent insect infestation [14]cannot sufficiently confirmusingDNAbarcodingTheHMPstreated with sulphur which appeared very clean and brightin colour do not only destroy the chemical or biologicalproperties of the HMP but also have toxic effects as thesulphur is realised to the environment [276]

The use of public database such as the GenBank forspecies identification is another limitation of using DNA


Table 6 Overview of chemical and DNA barcoding methods for herbal medicinal plant identification

Analysed material Chemical methods applied Genomic region analysed ReferencesH virginiana M recutita M ilicifolia Mglomerata P ginseng C P incarnate Pboldus and V officinalis

HPLC rbcLmatK and ITS2 Palhares et al [143]

Solanum lyratum and Aristolochiamollissima HPLC and TLC rbcLmatK ITS2 trnH-psbA

and trnL-trnF Li et al [125]

Hedyotis diffusa HPLC and TLC ITS2 Li et al [156]Cinchona pubescens HPLC and TLC rbcL andmatK Palhares et al [141]Salvia L HPLC ITS2 Jian-ping et al [157]

barcoding for the authentication of HMPs This is due to thefact that the reference sequences of some medicinal plantsare insufficient or not available and this makes the authenti-cation process difficult [277] The GenBank mostly providesuseful information when doing a preliminary research orwhen dealing with limited resources The scarcity of thecomplete sequence in the GenBank is another limitationwhen using GenBank for species identification The partialsequences from a less variable portion of a gene may resultin an inaccurate identification of the query sequence [277]Another potential problem of using GenBank database is thewrong submission of sequences which can lead to inaccurateidentification of the query sequence [278 279]The provisionof barcodes for many medicinal plant species by GenBankand MMDBD but without information about the voucherspecimen will likely result in some misidentification [277280]

Ensuring of unequivocal species identification of rawmaterials that will be used by herbal industries in themanufacturing of HMPs is always a challenge (Palhares etal 2015a) This is due to the fact that the various conven-tional methods of identifying herbal medicinal plant such asmacroscopic and microscopic methods and the organolepticmethod cannot identify the species in a processed productas both methods require human expertise [45] So also theuse of chromatography (eg HPLC) or molecular (eg DNAbarcoding) fingerprints alone may not provide the detailedinformation about the composition of the herbal medicinalplants despite their wide acceptance This is because eachof the methods has its own limitation The combination ofboth methods can help to provide detailed knowledge aboutthe main composition of the HMPs Some studies suggestedthat the DNA barcoding should be used in a complementarymanner with chemical analyses for species analysis as thesewill tremendously help to ascertain their efficacy and safetyas these are very important for quality control (Table 6)

Li et al [125] used DNA barcoding HPLC TLC andcytotoxicity assay to authenticate 10 samples retailed asBaiying and Xungufeng HMPs Baiying is a natural productused for the treatment of cancer derived from Solanumlyratum Baiying has a common substitute present in themarket called Xungufeng a carcinogenic aristolochic acid-containing herb derived from Aristolochia mollissimaThunb

A total of 30 sequences were generated using 5 DNAbarcode regions (ITSmatK rbcL trnH-psbA and trnL-trnF)

to differentiate these two plants The five barcode regionswere also applied to authenticate the 10 samples of Baiyingand Xungufeng HMPs The sequence result of all the fivebarcodes regions revealed that two samples of Baiying werederived from Solanum lyratum while the remaining threewere substituted with Aristochia mollissima Thunb whichcontained the carcinogenic aristolochic acid So also for theHMP labelled asXungufeng three sampleswere derived fromAristochia mollissimaThunb while two were substituted withSolanum lyratum Further authentication of the HMPs usingHPLC and TLC showed that the results are in agreement withthat obtained from DNA barcoding analysis

The most recent of such studies is that reported byPalhares et al [143]They analysed 257 samples which consistof dried leaves flowers and roots from 8 distinct speciesof medicinal plants sold in Brazil using three barcoding(rbcL matK and ITS2) regions The result revealed that42 of the samples belong to the correct genus while thelevel of substitution was high being around 71 Qualitativeand quantitative analysis revealed that the correct ones aresometimes sold but the chemical compounds are not presentThe result from this study shows that both of the techniqueshave their own limitation but by combining them togethermore information can be obtained

4 Conclusion

DNA barcoding is a reliable and suitable technique usedfor the identification of HMPs and for the determinationof various adulterants under specific conditions even ifthey existed in a processed products form such as capsulespowder tablets or dried form However in the absence ofany conditions such as good quality genomic DNA goodprimer affinity for successful amplification good sequencingmethods and authentic reference library identification usingDNA barcoding can be challenging Another limitation ofusing DNA barcoding for identification of HMPs is theinability to provide information about the presence and con-centration of a specific active ingredient so as to determinewhether it has met the pharmacopeia standard This showsthat DNA barcoding can only authenticate HMPs but cannotevaluate their quality On the other hand chromatographyfingerprint can be used to evaluate the quality of HMPs dueto the fact that it can provide information about the presenceand concentration of a specific bioactive compound Like


the DNA barcoding chromatography fingerprint may alsobe affected by some factors such as physiological and storageconditions Another limitation of using chromatographyfingerprint in the authentication of HMP is that it can onlyproduce indirect evidence of fraud as it cannot determine theidentity of the given species

Taking account of all the advantages and inherent lim-itation of DNA barcoding and chromatography fingerprintanalysis the combination of the two techniques could bean added advantage for comprehensive quality assessment ofHMPs as these are necessary for quality control Thereforewith the global increase in the demand and consumptionof HMP a combination of these great techniques to workin synergy with each other will help to check their qualityas this is critical for efficacy and safety The informationwill in turn help to change the consumerrsquos perception thateverything natural is safe as they tend to have relatively poorunderstanding of their safety They will tend to understandthat some of the HMPs are not effective and some cancause serious health problems The information may alsohelp to increase the consumersrsquo overall confidence in theconsumption of HMP more especially if they are of qualitystandard

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was partially supported by Ministry of Educa-tion Grant Tier 1 QJ130000254505H97 and FundamentalResearch Grant Scheme (RJ13000078454F893) Thanks aredue to all staff members of Plant Biotechnology LaboratoryFaculty of Biosciences and Medical Engineering UTM

References

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[2] V Razmovski-Naumovski W Tongkao-on B Kimble et alldquoMultiple chromatographic and chemometric methods forquality standardisation of Chinese herbal medicinesrdquo WorldScience and Technology vol 12 no 1 pp 99ndash106 2010

[3] P Posadzki L Watson and E Ernst ldquoContamination and adul-teration of herbal medicinal products (HMPs) an overview ofsystematic reviewsrdquo European Journal of Clinical Pharmacologyvol 69 no 3 pp 295ndash307 2013

[4] T Agbabiaka B Wider L K Watson and C Goodman ldquoCon-current use of prescription drugs and herbal medicinal prod-ucts in older adults a systematic review protocolrdquo SystematicReviews vol 5 no 1 article 65 2016

[5] L Tripathi and J N Tripathi ldquoRole of biotechnology in med-icinal plantsrdquo Tropical Journal of Pharmaceutical Research vol2 pp 243ndash253 2003

[6] S Goud ldquoScreening for antibacterial and antifungal activity ofsome medicinal plants of Nallamalais Andhra pradesh IndiardquoJournal of Economic andTaxonomic Botany vol 29 p 704 2005

[7] K Ramawat and S Goyal The Indian Herbal Drugs Scenarioin Global Perspectives Bioactive Molecules ad Medicinal PlantsSpringer Berlin Germany 2008

[8] M Y Khan S Aliabbas V Kumar and S Rajkumar ldquoRecentadvances in medicinal plant biotechnologyrdquo Indian Journal ofBiotechnology vol 8 no 1 pp 9ndash22 2009

[9] S N HM Azmin Z AManan S RW Alwi L S Chua A AMustaffa and N A Yunus ldquoHerbal processing and extractiontechnologiesrdquo Separation and Purification Reviews vol 45 no4 pp 305ndash320 2016

[10] M I Avigan R P Mozersky and L B Seeff ldquoScientific andregulatory perspectives in herbal and dietary supplement asso-ciated hepatotoxicity in the United Statesrdquo International Journalof Molecular Sciences vol 17 no 3 article 331 2016

[11] E Nyirimigabo Y Xu Y Li Y Wang K Agyemang andY Zhang ldquoA review on phytochemistry pharmacology andtoxicology studies of Aconitumrdquo Journal of Pharmacy andPharmacology vol 67 no 1 pp 1ndash19 2015

[12] H Thatoi and J K Patra ldquoBiotechnology and pharmacologicalevaluation of medicinal plants an overviewrdquo Journal of HerbsSpices and Medicinal Plants vol 17 no 3 pp 214ndash248 2011

[13] V Brower ldquoBack to nature extinction of medicinal plants thr-eatens drug discoveryrdquo Journal of the National Cancer Institutevol 100 no 12 pp 838ndash839 2008

[14] S-H Liu W-C Chuang W Lam Z Jiang and Y-C ChengldquoSafety surveillance of Traditional Chinese Medicine currentand futurerdquo Drug Safety vol 38 no 2 pp 117ndash128 2015

[15] R L Nahin P M Barnes B J Stussman and B Bloom ldquoCostsof complementary and alternative medicine (CAM) and freq-uency of visits to CAM practitioners United States 2007rdquoNational Health Statistics Reports no 18 pp 1ndash14 2009

[16] H M Heyman and J J M Meyer ldquoNMR-based metabolomicsas a quality control tool for herbal productsrdquo South AfricanJournal of Botany vol 82 pp 21ndash32 2012

[17] FDavidhttpknowledgeckgsbeducn20130108chinatradi-tional-chinese-medicine-business-blockbuster-or-false-fad

[18] R R Mendonca-Filho ldquoBioactive phytocompounds new app-roaches in the phytosciencesrdquo inModern Phytomedicine Turn-ing Medicinal Plants into Drugs pp 1ndash25 John Wiley amp Sons2006

[19] J Zhang B Wider H Shang X Li and E Ernst ldquoQualityof herbal medicines challenges and solutionsrdquo ComplementaryTherapies in Medicine vol 20 no 1-2 pp 100ndash106 2012

[20] S GNewmasterMGrguric D Shanmughanandhan S Rama-lingam and S Ragupathy ldquoDNA barcoding detects contamina-tion and substitution inNorthAmerican herbal productsrdquoBMCMedicine vol 11 no 1 article 222 2013

[21] W Sperl H Stuppner I Gassner W Judmaier O Dietzeand W Vogel ldquoReversible hepatic veno-occlusive disease inan infant after consumption of pyrrolizidine-containing herbalteardquo European Journal of Pediatrics vol 154 no 2 pp 112ndash1161995

[22] E S D Johanns L E VanDer Kolk HM A VanGemert A EJ Sijben PW J Peters and IDeVries ldquoAn epidemic of epilepticseizures after consumption of herbal teardquoNederlands Tijdschriftvoor Geneeskunde vol 146 no 17 pp 813ndash816 2002

[23] T Y Chan B Tomlinson and J A Critchley ldquoAconitine poison-ing following the ingestion of Chinese herbal medicines a rep-ort of eight casesrdquo Internal Medicine Journal vol 23 no 3 pp268ndash271 1993


[24] M Ekor ldquoThe growing use of herbal medicines issues relatingto adverse reactions and challenges in monitoring safetyrdquoFrontiers in Neurology vol 4 article 177 2014

[25] P Dhesi R Ng M M Shehata and P K Shah ldquoVentric-ular tachycardia after ingestion of Ayurveda herbal antidiar-rheal medication containing Aconitumrdquo Archives of InternalMedicine vol 170 no 3 pp 303ndash305 2010

[26] T YKChan ldquoAconite poisoning presenting as hypotension andbradycardiardquo Human and Experimental Toxicology vol 28 no12 pp 795ndash797 2009

[27] J Han X Pang B Liao H Yao J Song and S Chen ldquoAnauthenticity survey of herbal medicines from markets in Chinausing DNA barcodingrdquo Scientific Reports vol 6 Article ID18723 2016

[28] R D Marini E Rozet M L A Montes et al ldquoReliable low-costcapillary electrophoresis device for drug quality control andcounterfeit medicinesrdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 53 no 5 pp 1278ndash1287 2010

[29] P K Mukherjee Evidence-based Validation of Herbal MedicineElsevier Amsterdam The Netherlands 2015

[30] B Wang L Shen W Cong et al ldquoA simple HPLC methodfor simultaneous analysis of 7 bioactive constituents for LiuweiDihuang Pill and its application in quality consistency evalua-tionrdquo Analytical Methods vol 5 no 9 pp 2384ndash2390 2013

[31] S G Kim A Poudel Y-K Kim H-K Jo and H-J JungldquoDevelopment of simultaneous analysis for marker constituentsin Hwangryunhaedok-tang and its application in commercialherbal formulasrdquo Journal of Natural Medicines vol 67 no 2 pp390ndash398 2013

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[40] M D G L Brandao G P Cosenza F L Pereira A S Vasco-ncelos and C W Fagg ldquoChanges in the trade in native med-icinal plants in Brazilian public marketsrdquo Environmental Moni-toring and Assessment vol 185 no 8 pp 7013ndash7023 2013

[41] J B Calixto ldquoEfficacy safety quality control marketing andregulatory guidelines for herbal medicines (phytotherapeuticagents)rdquo Brazilian Journal of Medical and Biological Researchvol 33 no 2 pp 179ndash189 2000

[42] G Heubl ldquoNew aspects of DNA-based authentication of Chi-nese medicinal plants by molecular biological techniquesrdquoPlanta Medica vol 76 no 17 pp 1963ndash1974 2010

[43] M L S Silva ldquoComprehensive analysis of phytopharmaceu-tical formulations an emphasis on two-dimensional liquid

chromatographyrdquo Journal of Chromatography and SeparationTechniques vol 6 article 262 2015

[44] P D N Hebert A Cywinska S L Ball and J R DeWaardldquoBiological identifications through DNA barcodesrdquo Proceedingsof the Royal Society B Biological Sciences vol 270 no 1512 pp313ndash321 2003

[45] N Techen I Parveen Z Pan and I A Khan ldquoDNA barcodingof medicinal plant material for identificationrdquo Current Opinionin Biotechnology vol 25 pp 103ndash110 2014

[46] L J Wallace S M A L Boilard S H C Eagle J L SpallS Shokralla and M Hajibabaei ldquoDNA barcodes for everydaylife routine authentication of Natural Health Productsrdquo FoodResearch International vol 49 no 1 pp 446ndash452 2012

[47] S L Vassou G Kusuma and M Parani ldquoDNA barcoding forspecies identification from dried and powdered plant parts acase study with authentication of the raw drug market samplesof Sida cordifoliardquo Gene vol 559 no 1 pp 86ndash93 2015

[48] D P Little and M L Jeanson ldquoDNA barcode authenticationof saw palmetto herbal dietary supplementsrdquo Scientific Reportsvol 3 article 3518 2013

[49] D T H Reynaud A L Ceo B D Mishler J Neal-Kababickand P N Brown ldquoThe capabilities and limitations of DNAbarcoding of botanical dietary supplementsrdquo 2015

[50] H J De Boer M C Ichim and S G Newmaster ldquoDNA bar-coding and pharmacovigilance of herbal medicinesrdquo DrugSafety vol 38 no 7 pp 611ndash620 2015

[51] I Ali H Y Aboul-Enein and J Cazes ldquoA journey frommikhailtswett to nano-liquid chromatographyrdquo Journal of Liquid Chro-matography and Related Technologies vol 33 no 5 pp 645ndash6532010

[52] R EMajors andPWCarr ldquoGlossary of liquid-phase separationtermsrdquo LCGC vol 19 pp 124ndash163 2001

[53] Y V Kazakevich and R Lobrutto HPLC for PharmaceuticalScientists John Wiley amp Sons Inc Somerset NJ USA 2007

[54] J Zhou L Qi and P Li ldquoQuality control of Chinese herbalmedicines with chromatographic fingerprintsrdquo Chinese Journalof Chromatography vol 26 no 2 pp 153ndash159 2008

[55] F Gong Y-Z Liang P-S Xie and F-T Chau ldquoInformation the-ory applied to chromatographic fingerprint of herbal medicinefor quality controlrdquo Journal of Chromatography A vol 1002 no1-2 pp 25ndash40 2003

[56] M Goodarzi P J Russell and Y Vander Heyden ldquoSimilarityanalyses of chromatographic herbal fingerprints a reviewrdquoAnalytica Chimica Acta vol 804 pp 16ndash28 2013

[57] C-Z Liu H-Y Zhou and Q Yan ldquoFingerprint analysis ofDioscorea nipponica by high-performance liquid chromatog-raphy with evaporative light scattering detectionrdquo AnalyticaChimica Acta vol 582 no 1 pp 61ndash68 2007

[58] G-H Lu K Chan Y-Z Liang et al ldquoDevelopment of high-performance liquid chromatographic fingerprints for distin-guishing Chinese Angelica from related umbelliferae herbsrdquoJournal of Chromatography A vol 1073 no 1-2 pp 383ndash3922005

[59] Y Li T Wu J Zhu et al ldquoCombinative method using HPLCfingerprint and quantitative analyses for quality consistencyevaluation of an herbal medicinal preparation produced by dif-ferent manufacturersrdquo Journal of Pharmaceutical and Biomedi-cal Analysis vol 52 no 4 pp 597ndash602 2010

[60] J B Weon H J Yang B Lee J Y Ma and C J Ma ldquoSim-ultaneous quantification of eleven chemical components intraditional herbal medicinal formula socheongryongtang by


HPLC-DAD and LC-MSrdquo Journal of Liquid Chromatographyand Related Technologies vol 35 no 16 pp 2243ndash2254 2012

[61] Y-Z Liang P Xie and K Chan ldquoQuality control of herbalmedicinesrdquo Journal of Chromatography B Analytical Technolo-gies in the Biomedical and Life Sciences vol 812 no 1-2 pp 53ndash70 2004

[62] P P Fu H-M Chiang Q Xia et al ldquoQuality assurance andsafety of herbal dietary supplementsrdquo Journal of EnvironmentalScience and Health Part C Environmental Carcinogenesis andEcotoxicology Reviews vol 27 no 2 pp 91ndash119 2009

[63] D D Joshi Herbal Drugs and Fingerprints Evidence BasedHerbal Drugs Springer New Delhi India 2012

[64] L R Snyder J J Kirkland and J W Dolan Introduction ToModern Liquid Chromatography JohnWiley amp Sons New YorkNY USA 2011

[65] H-J Zhang and Y-Y Cheng ldquoAn HPLCMS method for iden-tifying major constituents in the hypocholesterolemic extractsof Chinese medicine formula lsquoXue-Fu-Zhu-Yu decoctionrsquordquoBiomedical Chromatography vol 20 no 8 pp 821ndash826 2006

[66] P Akarasereenont P Thitilertdecha S Chotewuttakorn et alldquoChromatographic fingerprint development for quality assess-ment of lsquoayurved Siriraj prasachandaengrsquo antipyretic drugrdquoSiriraj Medical Journal vol 62 pp 4ndash8 2010

[67] Y Yan C-Z Chai D-WWang X-Y Yue D-N Zhu and B-YYu ldquoHPLC-DAD-Q-TOF-MSMS analysis andHPLCquantita-tion of chemical constituents in traditional Chinese medicinalformula Ge-Gen Decoctionrdquo Journal of Pharmaceutical andBiomedical Analysis vol 80 pp 192ndash202 2013

[68] L Chen J Qi Y-X Chang D Zhu and B Yu ldquoIdentificationand determination of the major constituents in TraditionalChinese Medicinal formula Danggui-Shaoyao-San by HPLC-DAD-ESI-MSMSrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 50 no 2 pp 127ndash137 2009

[69] D K Kajaria M Gangwar D Kumar et al ldquoEvaluation of anti-microbial activity and bronchodialator effect of a polyherbaldrug-Shrishadirdquo Asian Pacific Journal of Tropical Biomedicinevol 2 no 11 pp 905ndash909 2012

[70] L Zhu X Yang J Tan BWang andX Zhang ldquoA validated highperformance liquid chromatograph-photodiode array methodfor simultaneous determination of 10 bioactive components incompound hongdoushan capsulerdquo Pharmacognosy Magazinevol 10 no 37 pp 83ndash86 2014

[71] S Ghafari S Esmaeili H Aref F Naghibi and M MosaddeghldquoQualitative andquantitative analysis of somebrands of valerianpharmaceutical productsrdquo Studies on Ethno-Medicine vol 3 no1 pp 61ndash64 2009

[72] R Sudani B Akbari G Vidyasagar and P Sharma ldquoQuantita-tive and chromatographic fingerprint analysis of Embelia ribeschurna formulations by HPLC Methodrdquo International Journalof Pharmaceutical and Biological Archives vol 2 pp 657ndash6632011

[73] P Zou Y Hong and H L Koh ldquoChemical fingerprinting ofIsatis indigotica root by RP-HPLC and hierarchical clusteringanalysisrdquo Journal of Pharmaceutical and Biomedical Analysisvol 38 no 3 pp 514ndash520 2005

[74] E-H Liu L-W Qi Y-B Peng et al ldquoRapid separation andidentification of 54 major constituents in Buyang Huanwudecoction by ultra-fast HPLC system coupled with DAD-TOFMSrdquo Biomedical Chromatography vol 23 no 8 pp 828ndash842 2009

[75] B-S Sun L-J Gu Z-M Fang et al ldquoSimultaneous quan-tification of 19 ginsenosides in black ginseng developed from

Panax ginseng by HPLC-ELSDrdquo Journal of Pharmaceutical andBiomedical Analysis vol 50 no 1 pp 15ndash22 2009

[76] S Xu L Yang R Tian et al ldquoSpecies differentiation and qualityassessment of Radix Paeoniae Rubra (Chi-shao) by means ofhigh-performance liquid chromatographic fingerprintrdquo Journalof Chromatography A vol 1216 no 11 pp 2163ndash2168 2009

[77] W Li S Chen D Fabricant et al ldquoHigh-performance liquidchromatographic analysis of Black Cohosh (Cimicifuga race-mosa) constituents with in-line evaporative light scattering andphotodiode array detectionrdquo Analytica Chimica Acta vol 471no 1 pp 61ndash75 2002

[78] S Qiu W-Z Yang C-L Yao et al ldquoNontargeted metabolomicanalysis and lsquocommercial-homophyleticrsquo comparison-inducedbiomarkers verification for the systematic chemical differen-tiation of five different parts of Panax ginsengrdquo Journal ofChromatography A vol 1453 pp 78ndash87 2016

[79] N Basar A D Talukdar L Nahar et al ldquoA simple semi-pre-parative reversed-phaseHPLCPDAmethod for separation andquantification of glycyrrhizin in nine samples of Glycyrrhizaglabra root collected from different geographical originsrdquo Phy-tochemical Analysis vol 25 no 5 pp 399ndash404 2014

[80] A-J Lau B-H Seo S-O Woo and H-L Koh ldquoHigh-per-formance liquid chromatographic method with quantitativecomparisons of whole chromatograms of raw and steamedPanax notoginsengrdquo Journal of Chromatography A vol 1057 no1-2 pp 141ndash149 2004

[81] X-Q Ma X-M Liang Q Xu X-Z Zhang and H-B XiaoldquoIdentification of ginsenosides in roots of Panax ginseng byHPLC-APCIMSrdquoPhytochemical Analysis vol 16 no 3 pp 181ndash187 2005

[82] Y Zhao Z Li X Zhou Z Cai X Gong and C ZhouldquoQuality evaluation of Evodia rutaecarpa (Juss) Benth by highperformance liquid chromatography with photodiode-arraydetectionrdquo Journal of Pharmaceutical and Biomedical Analysisvol 48 no 4 pp 1230ndash1236 2008

[83] P Li L-J Zeng S-L Li and G Lin ldquoThe extraction of imperi-aline and imperialine-3120573-glucoside from Fritillaria pallidifloraSchrenk and quantitative determination by HPLC-evaporativelight scattering detectionrdquo Phytochemical Analysis vol 13 no 3pp 158ndash161 2002

[84] X Ma H Xiao and X Liang ldquoIdentification of ginsenosides inPanax quinquefolium by LC-MSrdquo Chromatographia vol 64 no1-2 pp 31ndash36 2006

[85] W Kong Z Li X Xiao and Y Zhao ldquoQuality control forCoptidis rhizoma through the determination of five alkaloidsby HPLC-ELSD coupled with chemometricsrdquo Natural ProductResearch vol 24 no 17 pp 1616ndash1629 2010

[86] GWangMWei JWang Y Lu GMahady andD Liu ldquoHigh-performance liquid chromatography with photodiode array(HPLC-PDA) quality control of menoprogen a traditionalChinese medicine (TCM) formula used for the management ofmenopauserdquo International Journal of Medicinal Plants Researchvol 2 pp 146ndash151 2013

[87] X Xu J Jiang Y Liang L Yi and J Cheng ldquoChemical finger-print analysis for quality control of Fructus Aurantii Immaturusbased on HPLC-DAD combined with chemometric methodsrdquoAnalytical Methods vol 2 no 12 pp 2002ndash2010 2010

[88] Z Yan X Yang J Wu H Su C Chen and Y Chen ldquoQual-itative and quantitative analysis of chemical constituents intraditional Chinese medicinal formula Tong-Xie-Yao-Fang by


high-performance liquid chromatographydiode array detec-tionelectrospray ionization tandem mass spectrometryrdquo Ana-lytica Chimica Acta vol 691 no 1-2 pp 110ndash118 2011

[89] DChanda A Pal andK Shanker ldquoApplication ofHPLCfinger-prints for defining in vivo safety profile of Tulsi (Ocimum San-ctum)rdquoMedicinal Chemistry Research vol 22 no 1 pp 219ndash2242013

[90] S Zhang R Chen H Wu and C Wang ldquoGinsenoside extrac-tion from Panax quinquefolium L (American ginseng) rootby using ultrahigh pressurerdquo Journal of Pharmaceutical andBiomedical Analysis vol 41 no 1 pp 57ndash63 2006

[91] Y M Han M Jang I S Kim S H Kim and H H Yoo ldquoSim-ultaneous quantitation of six major quassinoids in Tongkat Alidietary supplements by liquid chromatography with tandemmass spectrometryrdquo Journal of Separation Science vol 38 no13 pp 2260ndash2266 2015

[92] L Duan H Li J Yuan et al ldquoFingerprint analysis and quan-titative determination of 16 constituents of Antike capsule byhigh-performance liquid chromatography-photodiode arraydetectionrdquo Analytical Methods vol 7 no 16 pp 6695ndash67042015

[93] T Tan C-J Lai S-L Zeng P Li and E-H Liu ldquoComprehen-sive profiling and characterization of quassinoids from the seedsof Brucea javanica via segment and exposure strategy coupledwith modified mass defect filterrdquo Analytical and BioanalyticalChemistry vol 408 no 2 pp 527ndash533 2016

[94] Y-Y Chen G Jiao F Hui and L-H Huang ldquoSimultane-ous determination of seven alkaloids in Fufang ZhenzhuTiaozhi capsule by HPLC coupled with DADrdquo Chinese HerbalMedicines vol 4 pp 230ndash236 2012

[95] M Ganzera E Bedir and I Khan ldquoSeparation of Cimicifugaracemosa triterpene glycosides by reversed phase high perfor-mance liquid chromatography and evaporative light scatteringdetectionrdquoChromatographia vol 52 no 5-6 pp 301ndash304 2000

[96] G Tan T Yang H Miao H Chen Y Chai and HWu ldquoChara-cterization of compounds in psoralea corylifolia using high-performance liquid chromatography diode array detectiontime-of-flightmass spectrometry and quadrupole ion trapmassspectrometryrdquo Journal of Chromatographic Science vol 53 no9 pp 1455ndash1462 2015

[97] L Kong X Li H Zou et al ldquoAnalysis of terpene compoundsin Cimicifuga foetida L by reversed-phase high-performanceliquid chromatography with evaporative light scattering detec-tionrdquo Journal of Chromatography A vol 936 no 1-2 pp 111ndash1182001

[98] Q Bian H Yang C-O Chan D Jin D K-W Mok and SChen ldquoFingerprint analysis and simultaneous determinationof phenolic compounds in extracts of Curculiginis rhizomaby HPLC-diode array detectorrdquo Chemical and PharmaceuticalBulletin vol 61 no 8 pp 802ndash808 2013

[99] J-H Kim C-S Seo S-S Kim and H-K Shin ldquoQuality assess-ment of Ojeok-san a traditional herbal formula using high-performance liquid chromatography combined with chemo-metric analysisrdquo Journal of AnalyticalMethods in Chemistry vol2015 Article ID 607252 11 pages 2015

[100] SWangHWang Y Liu YWang X Fan andYCheng ldquoRapiddiscovery and identification of anti-inflammatory constituentsfrom traditional Chinese medicine formula by activity indexLC-MS and NMRrdquo Scientific Reports vol 6 Article ID 310002016

[101] Y-C Zhang Z-F Pi C-M Liu F-R Song Z-Q Liu and S-Y Liu ldquoAnalysis of low-polar ginsenosides in steamed Panax

ginseng at high-temperature by HPLC-ESI-MSMSrdquo ChemicalResearch in Chinese Universities vol 28 no 1 pp 31ndash36 2012

[102] C-J Lai T Tan S-L Zeng et al ldquoAn integrated high resolutionmass spectrometric data acquisitionmethod for rapid screeningof saponins in Panax notoginseng (Sanqi)rdquo Journal of Pharma-ceutical and Biomedical Analysis vol 109 pp 184ndash191 2015

[103] C R Horvath P A Martos and P K Saxena ldquoIdentificationand quantification of eight flavones in root and shoot tissues ofthe medicinal plant Huang-qin (Scutellaria baicalensis Georgi)using high-performance liquid chromatography with diodearray andmass spectrometric detectionrdquo Journal of Chromatog-raphy A vol 1062 no 2 pp 199ndash207 2005

[104] R Liu M Ye H Guo K Bi and D-A Guo ldquoLiquid chromat-ographyelectrospray ionization mass spectrometry for thecharacterization of twenty-three flavonoids in the extract ofDalbergia odoriferardquo Rapid Communications inMass Spectrom-etry vol 19 no 11 pp 1557ndash1565 2005

[105] X Zheng X Zhang X Sheng et al ldquoSimultaneous characteriz-ation and quantitation of 11 coumarins in Radix AngelicaeDahuricae by high performance liquid chromatography withelectrospray tandemmass spectrometryrdquo Journal of Pharmaceu-tical and Biomedical Analysis vol 51 no 3 pp 599ndash605 2010

[106] A Skhirtladze A Perrone PMontoro et al ldquoSteroidal saponinsfromYucca gloriosa L rhizomes LC-MSprofiling isolation andquantitative determinationrdquo Phytochemistry vol 72 no 1 pp126ndash135 2011

[107] L Wang and H Qu ldquoDevelopment and optimization of SPE-HPLC-UVELSD for simultaneous determination of nine bio-active components in Shenqi Fuzheng Injection based on Qua-lity by Design principlesrdquo Analytical and Bioanalytical Chem-istry vol 408 no 8 pp 2133ndash2145 2016

[108] X-Y Gao Y Jiang J-Q Lu and P-F Tu ldquoOne single stan-dard substance for the determination of multiple anthraquin-one derivatives in rhubarb using high-performance liquid chro-matography-diode array detectionrdquo Journal of ChromatographyA vol 1216 no 11 pp 2118ndash2123 2009

[109] J-L Zhou P Li H-J Li Y Jiang M-T Ren and Y Liu ldquoDevel-opment and validation of a liquid chromatographyelectrosprayionization time-of-flightmass spectrometrymethod for relativeand absolute quantification of steroidal alkaloids in Fritillariaspeciesrdquo Journal of Chromatography A vol 1177 no 1 pp 126ndash137 2008

[110] T Tan C-J-S Lai S-L Zeng E-H Liu and P Li ldquoEnzymatichydrolysis-based absolute quantification of triacylglycerols inplant oil by use of a single markerrdquo Analytical and BioanalyticalChemistry vol 406 no 20 pp 4921ndash4929 2014

[111] C Cavaliere P Foglia E Pastorini R Samperi and ALagana ldquoIdentification and mass spectrometric characteriza-tion of glycosylated flavonoids in Triticum durum plants byhigh-performance liquid chromatography with tandem massspectrometryrdquo Rapid Communications in Mass Spectrometryvol 19 no 21 pp 3143ndash3158 2005

[112] R J Grayer R F Vieira A M Price G C Kite J E Simon andA J Paton ldquoCharacterization of cultivars within species ofOcimumby exudate flavonoid profilesrdquoBiochemical Systematicsand Ecology vol 32 no 10 pp 901ndash913 2004

[113] L Peng Y Wang H Zhu and Q Chen ldquoFingerprint profile ofactive components for Artemisia selengensis Turcz by HPLC-PAD combined with chemometricsrdquo Food Chemistry vol 125no 3 pp 1064ndash1071 2011


[114] F Li B Yuan Z Xiong et al ldquoFingerprint analysis of FlosLonicerae japonicae using binary HPLC profilingrdquo BiomedicalChromatography vol 20 no 6-7 pp 634ndash641 2006

[115] H-L Wang W-F Yao D-N Zhu and Y-Z Hu ldquoChemicalfingerprinting by HPLC-DAD-ELSD and principal componentanalysis of Polygala japonica from different locations in ChinardquoChinese Journal of Natural Medicines vol 8 no 5 pp 343ndash3482010

[116] Y H Lai Y N Ni and S Kokot ldquoAuthentication of Cassia seedson the basis of two-wavelength HPLC fingerprinting with theuse of chemometricsrdquo Chinese Chemical Letters vol 21 no 2pp 213ndash216 2010

[117] R F Vieira R J Grayer and A J Paton ldquoChemical profilingofOcimum americanum using external flavonoidsrdquo Phytochem-istry vol 63 no 5 pp 555ndash567 2003

[118] S Guo J-A Duan Y Tang et al ldquoHigh-performance liquidchromatographymdashtwo wavelength detection of triterpenoidacids from the fruits of Ziziphus jujuba containing various cul-tivars in different regions and classification using chemometricanalysisrdquo Journal of Pharmaceutical and Biomedical Analysisvol 49 no 5 pp 1296ndash1302 2009

[119] R Srirama U Senthilkumar N Sreejayan et al ldquoAssessing spe-cies admixtures in raw drug trade of Phyllanthus a hepato-protective plant using molecular toolsrdquo Journal of Ethnophar-macology vol 130 no 2 pp 208ndash215 2010

[120] P Mahadani and S K Ghosh ldquoDNA barcoding a tool forspecies identification from herbal juicesrdquo DNA Barcodes vol 1pp 35ndash38 2013

[121] L S Da Costa L R S Reiniger V M Stefenon B M Heinz-mann andADos SantosOliveira ldquoComparative analysis of fiveDNA isolation protocols and three drying methods for leavessamples of Nectandra megapotamica (Spreng) Mezrdquo SeminaCiencias Agrarias vol 37 no 3 pp 1177ndash1188 2016

[122] J J Doyle ldquoA rapid DNA isolation procedure for small quanti-ties of fresh leaf tissuerdquo Phytochemical Bulletin vol 19 pp 11ndash151987

[123] J S Kumar V KrishnaG S Seethapathy et al ldquoDNAbarcodingto assess species adulteration in raw drug trade of lsquoBalarsquo(genus Sida L) herbal products in South Indiardquo BiochemicalSystematics and Ecology vol 61 pp 501ndash509 2015

[124] L Alexander ldquoRapid effective DNA isolation from osmanthusvia modified alkaline lysisrdquo Journal of Biomolecular Techniquesvol 27 no 2 pp 53ndash60 2016

[125] M Li K-Y Au H Lam et al ldquoIdentification of Baiying (HerbaSolani Lyrati) commodity and its toxic substitute Xungufeng(Herba Aristolochiae Mollissimae) using DNA barcoding andchemical profiling techniquesrdquo Food Chemistry vol 135 no 3pp 1653ndash1658 2012

[126] J Costa B Campos J S Amaral M E Nunes M B P P Oli-veira and I Mafra ldquoHRM analysis targeting ITS1 and matKloci as potential DNA mini-barcodes for the authentication ofHypericum perforatum and Hypericum androsaemum in herbalinfusionsrdquo Food Control vol 61 pp 105ndash114 2016

[127] G S Seethapathy D Ganesh J U Santhosh Kumar et alldquoAssessing product adulteration in natural health products forlaxative yielding plants Cassia Senna and Chamaecrista inSouthern India using DNA barcodingrdquo International Journal ofLegal Medicine vol 129 no 4 pp 693ndash700 2015

[128] D Selvaraj D Shanmughanandhan R K Sarma J C Joseph RV Srinivasan and S Ramalingam ldquoDNAbarcode ITs effectivelydistinguishes the medicinal plant Boerhavia diffusa from its

adulterantsrdquo Genomics Proteomics and Bioinformatics vol 10no 6 pp 364ndash367 2012

[129] B P Sheth and V S Thaker ldquoIdentification of a herbal powderby deoxyribonucleic acid barcoding and structural analysesrdquoPharmacognosyMagazine vol 11 supplement 4 pp S570ndashS5742015

[130] X Cheng X Chen X Su et al ldquoDNA extraction protocol forbiological ingredient analysis of LiuWei DiHuang Wanrdquo Geno-mics Proteomics and Bioinformatics vol 12 no 3 pp 137ndash1432014

[131] J ZhouWWangM Liu and Z Liu ldquoMolecular authenticationof the traditional medicinal plant Peucedanum praeruptorumand its substitutes and adulterants by dnamdashbarcoding tech-niquerdquo Pharmacognosy Magazine vol 10 no 40 pp 385ndash3902014

[132] K-L Wong P P-H But and P-C Shaw ldquoEvaluation of sevenDNA barcodes for differentiating closely related medicinalGentiana species and their adulterantsrdquo Chinese Medicine vol8 no 1 article 16 2013

[133] F Tamari C S Hinkley and N Ramprashad ldquoA comparison ofDNA extraction methods using Petunia hybrida tissuesrdquo Jour-nal of Biomolecular Techniques vol 24 no 3 pp 113ndash118 2013

[134] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 article 13491991

[135] C Jian Q Deyi Y Qiaoyun et al ldquoA successful case of DNAbarcoding used in an international trade disputerdquo DNA Bar-codes vol 2 no 1 pp 21ndash28 2014

[136] S Singtonat and M Osathanunkul ldquoFast and reliable detectionof toxic Crotalaria spectabilis Roth in Thunbergia laurifoliaLindl herbal products using DNA barcoding coupled withHRM analysisrdquo BMCComplementary and AlternativeMedicinevol 15 no 1 article 162 2015

[137] A T Uncu A O Uncu A Frary and S Doganlar ldquoAuthenti-cation of botanical origin in herbal teas by plastid noncodingDNA length polymorphismsrdquo Journal of Agricultural and FoodChemistry vol 63 no 25 pp 5920ndash5929 2015

[138] MOsathanunkul C Suwannapoom K Osathanunkul PMad-esis and H De Boer ldquoEvaluation of DNA barcoding coupledhigh resolution melting for discrimination of closely relatedspecies in phytopharmaceuticalsrdquo Phytomedicine vol 23 no 2pp 156ndash165 2016

[139] M Osathanunkul C Suwannapoom S Ounjai J A Rora PMadesis and H De Boer ldquoRefining DNA barcoding coupledhigh resolution melting for discrimination of 12 closely relatedcroton speciesrdquo PLoS ONE vol 10 no 9 Article ID e01388882015

[140] J P Llongueras S Nair D Salas-Leiva and A E SchwarzbachldquoComparing DNA extraction methods for analysis of botan-ical materials found in anti-diabetic supplementsrdquo MolecularBiotechnology vol 53 no 3 pp 249ndash256 2013

[141] R M Palhares M G Drummond B S A F Brasil A U Kre-ttli G C Oliveira and M G L Brandao ldquoThe use of an inte-gratedmolecular- chemical- and biological-based approach forpromoting the better use and conservation ofmedicinal speciesa case study of Brazilian quinasrdquo Journal of Ethnopharmacologyvol 155 no 1 pp 815ndash822 2014

[142] D A Baker ldquoDNA barcode identification of black cohoshherbal dietary supplementsrdquo Journal of AOAC International vol95 no 4 pp 1023ndash1034 2012


[143] R M Palhares M G Drummond B Dos Santos Alves Figuei-redo Brasil G P Cosenza M Das Gracas Lins Brandao andG Oliveira ldquoMedicinal plants recommended by the world hea-lth organization DNA barcode identification associated withchemical analyses guarantees their qualityrdquo PLoS ONE vol 10no 5 Article ID e0127866 2015

[144] M T Cimino ldquoSuccessful isolation and PCR amplification ofDNA fromnational institute of standards and technology herbaldietary supplement standard reference material powders andextractsrdquo Planta Medica vol 76 no 5 pp 495ndash497 2010

[145] M R Enan and A Ahmed ldquoDNA barcoding based on plastidmatK and RNA polymerase for assessing the genetic identity ofdate (Phoenix dactylifera L) cultivarsrdquo Genetics and MolecularResearch vol 13 no 2 pp 3527ndash3536 2014

[146] V A Parvathy V P Swetha T E Sheeja N K Leela B Chem-pakam and B Sasikumar ldquoDNAbarcoding to detect chilli adul-teration in traded black pepper powderrdquo Food Biotechnologyvol 28 no 1 pp 25ndash40 2014

[147] M Y Stoeckle C C Gamble R Kirpekar G Young S Ahmedand D P Little ldquoCommercial teas highlight plant DNA barcodeidentification successes and obstaclesrdquo Scientific Reports vol 1article 42 2011

[148] T Xin X Li H Yao et al ldquoSurvey of commercial Rhodiolaproducts revealed species diversity and potential safety issuesrdquoScientific Reports vol 5 article 8337 2015

[149] L Xiang Y Su X Li et al ldquoIdentification of Fritillariae bulbusfrom adulterants using ITS2 regionsrdquo Plant Gene vol 7 pp 42ndash49 2016

[150] Z Zhang Y Zhang Z Zhang et al ldquoComparative analysis ofDNA barcoding and HPLC fingerprint to trace species of Phel-lodendri Cortex an important traditional Chinese medicinefrom multiple sourcesrdquo Biological and Pharmaceutical Bulletinvol 39 no 8 pp 1325ndash1330 2016

[151] X Zhu Y Zhang X Liu D Hou and T Gao ldquoAuthenticationof commercial processed Glehniae Radix (Beishashen) by DNAbarcodesrdquo Chinese Medicine vol 10 article 35 2015

[152] Y Liu X Wang L Wang X Chen X Pang and J Han ldquoAnucleotide signature for the identification of American ginsengand its productsrdquo Frontiers in Plant Science vol 7 article 3192016

[153] D-Y Hou J-Y Song H Yao et al ldquoMolecular identificationof Corni Fructus and its adulterants by ITSITS2 sequencesrdquoChinese Journal of Natural Medicines vol 11 no 2 pp 121ndash1272013

[154] Z Hu Y Tu Y Xia et al ldquoRapid identification and verifica-tion of indirubin-containing medicinal plantsrdquo Evidence-basedComplementary and Alternative Medicine vol 2015 Article ID484670 9 pages 2015

[155] D Hou J Song L Shi et al ldquoStability and accuracy assessmentof identification of traditional chinese materia medica usingDNA barcoding a case study on Flos Lonicerae JaponicaerdquoBioMed Research International vol 2013 Article ID 549037 8pages 2013

[156] M Li R-W Jiang P-M Hon et al ldquoAuthentication of theanti-tumor herb Baihuasheshecao with bioactive marker com-pounds and molecular sequencesrdquo Food Chemistry vol 119 no3 pp 1239ndash1245 2010

[157] H Jian-Ping L Chang LMin-Hui et al ldquoRelationship betweenDNA barcoding and chemical classification of Salvia medicinalherbsrdquo Chinese Herbal Medicines vol 2 pp 16ndash29 2010

[158] B Nickerson ldquoProperties that impact sample preparation andextraction of pharmaceutical dosage formsrdquo in Sample Prepa-ration of Pharmaceutical Dosage Forms pp 3ndash19 Springer US2011

[159] Y He X Xiao Y Cheng and G Li ldquoProgress in field-assistedextraction and its application to solid sample analysisrdquo Journalof Separation Science vol 39 no 1 pp 177ndash187 2016

[160] Y Wen L Chen J Li D Liu and L Chen ldquoRecent advancesin solid-phase sorbents for sample preparation prior to chro-matographic analysisrdquo TrACmdashTrends in Analytical Chemistryvol 59 pp 26ndash41 2014

[161] J M Rosenfeld ldquoIntroduction current techniques and issuesin sample preparationrdquo Sample Preparation for HyphenatedAnalytical Techniques pp 1ndash6 2009

[162] P ChenMOzcan and JHarnly ldquoChromatographic fingerprintanalysis for evaluation of Ginkgo biloba productsrdquo Analyticaland Bioanalytical Chemistry vol 389 no 1 pp 251ndash261 2007

[163] CKChoi andMWDong ldquoSample preparation forHPLCana-lysis of drug productsrdquo Separation Science and Technology vol6 pp 123ndash144 2005

[164] Z Latif and S D Sarker ldquoIsolation of natural products bypreparative high performance liquid chromatography (prep-HPLC)rdquo Methods in Molecular Biology vol 864 pp 255ndash2742012

[165] N Khari A F A Aisha and Z Ismail ldquoReverse phase highperformance liquid chromatography for the quantification ofeurycomanone in Eurycoma longifolia Jack (Simaroubaceae)extracts and their commercial productsrdquo Tropical Journal ofPharmaceutical Research vol 13 no 5 pp 801ndash807 2014

[166] P Wang L Li H Yang et al ldquoChromatographic fingerprintingand quantitative analysis for the quality evaluation of Xinkeshutabletrdquo Journal of Pharmaceutical Analysis vol 2 no 6 pp 422ndash430 2012

[167] Y Xie Z-H Jiang H Zhou et al ldquoCombinative methodusing HPLC quantitative and qualitative analyses for qualityconsistency assessment of a herbal medicinal preparationrdquoJournal of Pharmaceutical and Biomedical Analysis vol 43 no1 pp 204ndash212 2007

[168] T J Mason F Chemat and M Vinatoru ldquoThe extraction ofnatural products using ultrasound or microwavesrdquo CurrentOrganic Chemistry vol 15 no 2 pp 237ndash247 2011

[169] T JMason ldquoUltrasonically assisted extraction of bioactive prin-ciples from plants and their constituentsrdquo in Advances inSonochemistry vol 5 pp 209ndash248 JAI PressElsevier 1999

[170] M Vinatoru ldquoAn overview of the ultrasonically assisted extrac-tion of bioactive principles from herbsrdquo Ultrasonics Sonochem-istry vol 8 no 3 pp 303ndash313 2001

[171] J Azmir I S M Zaidul M M Rahman et al ldquoTechniques forextraction of bioactive compounds from plant materials a rev-iewrdquo Journal of Food Engineering vol 117 no 4 pp 426ndash4362013

[172] B Zygmunt and J Namiesnik ldquoPreparation of samples of plantmaterial for chromatographic analysisrdquo Journal of Chromato-graphic Science vol 41 no 3 pp 109ndash116 2003

[173] J Ronowicz B Kupcewicz and E Budzisz ldquoChemometric ana-lysis of antioxidant properties of herbal products containingGinkgo biloba extractrdquoCentral European Journal of Biology vol8 no 4 pp 374ndash385 2013

[174] Z Liu Z Xu H Zhou et al ldquoSimultaneous determination offour bioactive compounds in Verbena officinalis L by usinghigh-performance liquid chromatographyrdquo PharmacognosyMagazine vol 8 no 30 pp 162ndash165 2012


[175] K-J Mao P Huang F-Q Li X Li and J-W Chen ldquoSimul-taneous determination of 10 active components in Hulisantablets by a high-performance liquid chromatographic-diodearray detectorrdquo Journal of Chromatographic Science vol 53 no9 pp 1449ndash1454 2015

[176] Y Liu H Bi F You J Hu X Chai and Y Du ldquoDeterminationof the fingerprint chromatogram of Sanhuang tablet by HPLCrdquoAsian Journal of Chemistry vol 24 no 10 pp 4522ndash4524 2012

[177] J J Kirkland ldquoDevelopment of some stationary phases forreversed-phase HPLCrdquo Journal of Chromatography A vol 1060no 1-2 pp 9ndash21 2004

[178] O Nunez T Ikegami K Miyamoto and N Tanaka ldquoStudy of amonolithic silica capillary column coated with poly(octadecylmethacrylate) for the reversed-phase liquid chromatographicseparation of some polar and non-polar compoundsrdquo Journalof Chromatography A vol 1175 no 1 pp 7ndash15 2007

[179] M Waksmundzka-Hajnos J Sherma and I Chinou ldquoHerbaldrugs and the role of chromatographic methods in theiranalysisrdquo inHigh Performance Liquid Chromatography in Phyto-chemical Analysis pp 13ndash22 CRC Press Boca Raton Fla USA2010

[180] A Bansal V Chhabra R K Rawal and S Sharma ldquoChemomet-rics a new scenario in herbal drug standardizationrdquo Journal ofPharmaceutical Analysis vol 4 no 4 pp 223ndash233 2014

[181] C Tistaert B Dejaegher and Y V Heyden ldquoChromatographicseparation techniques and data handling methods for herbalfingerprints a reviewrdquo Analytica Chimica Acta vol 690 no 2pp 148ndash161 2011

[182] S Rj B Akbari G Vidyasagar and P Sharma ldquoQuantitativeand chromatographic fingerprint analysis of Embelia ribeschurna formulations byHPLCmethodrdquo International Journal ofPharmaceutical and Biological Archive vol 2 pp 657ndash663 2010

[183] U Sanyal S Bhattacharyya A Patra and B Hazra ldquoLiquidchromatographic separation of derivatives of diospyrin a bio-active bisnaphthoquinonoid plant-product and analogous nap-hthyl compoundsrdquo Journal of Chromatography A vol 1017 no1-2 pp 225ndash232 2003

[184] S-J Sheu and K-L Li ldquoLiquid chromatographic determinationof the constituents in Shao-yao-tang and related chinese herbalpreparationsrdquo Journal of High Resolution Chromatography vol21 no 10 pp 569ndash573 1998

[185] J BWeonH J Yang B Lee J YMa andC JMa ldquoQuantitativeanalysis of the eight major compounds in the Samsoeum usinga high-performance liquid chromatography coupled with diodearray detection and electrospray ionization mass spectrometerrdquoPharmacognosy Magazine vol 11 no 42 pp 320ndash326 2015

[186] X Yang S-P Yang X Zhang X-D Yu Q-Y He and B-CWang ldquoStudy on the multi-marker components quantitativeHPLC fingerprint of the compound Chinese medicine WuweiChangyanning granulerdquo Iranian Journal of PharmaceuticalResearch vol 13 no 4 pp 1191ndash1201 2014

[187] W F Chan and C W Lin ldquoHigh-pressure liquid chromatogra-phy quantitative analysis of Chinese herbal medicinerdquo Journalof Chemical Education vol 84 no 12 pp 1982ndash1984 2007

[188] R K Patel V R Patel and M G Patel ldquoDevelopment andvalidation of a RP-HPLC method for the simultaneous deter-mination of Embelin Rottlerin and Ellagic acid in Vidangadichurnardquo Journal of Pharmaceutical Analysis vol 2 no 5 pp366ndash371 2012

[189] B Duan J Hu L Huang X Yang and F Chen ldquoChemicalfingerprint analysis of Gentianae Radix et Rhizoma by high-performance liquid chromatographyrdquo Acta PharmaceuticaSinica B vol 2 pp 46ndash52 2012

[190] S C Moldoveanu and V David Essentials in Modern HPLCSeparations Elsevier Amsterdam The Netherlands 2012

[191] W Markowski and M Waksmundzka-Hajnos ldquoPhotodiodearray (PDA) and other detection methods in HPLC of plantmetabolitesrdquo in High Performance Liquid Chromatography inPhytochemical Analysis CRC Press 2010

[192] R-T Tian P-S Xie and H-P Liu ldquoEvaluation of traditionalChinese herbal medicine Chaihu (Bupleuri Radix) byboth high-performance liquid chromatographic and high-performance thin-layer chromatographic fingerprint andchemometric analysisrdquo Journal of Chromatography A vol 1216no 11 pp 2150ndash2155 2009

[193] S N Kim Y W Ha H Shin S H Son S J Wu and Y SKim ldquoSimultaneous quantification of 14 ginsenosides in Panaxginseng CA Meyer (Korean red ginseng) by HPLC-ELSD andits application to quality controlrdquo Journal of Pharmaceutical andBiomedical Analysis vol 45 no 1 pp 164ndash170 2007

[194] H Wang J Gao D Zhu and B Yu ldquoQuality evaluation ofPolygala japonica through simultaneous determination of sixbioactive triterpenoid saponins by HPLC-ELSDrdquo Journal ofPharmaceutical and Biomedical Analysis vol 43 no 4 pp 1552ndash1556 2007

[195] N Y Morgan and P D Smith ldquoHPLC detectorsrdquo in Handbookof HPLC pp 207ndash232 CRC Press 2nd edition 2010

[196] S D Sarker and L Nahar ldquoHyphenated techniques and theirapplications in natural products analysisrdquo Natural ProductsIsolation vol 864 pp 301ndash340 2012

[197] J Kingston D Orsquoconnor T Sparey and S Thomas ldquoChapter 6Hyphenated techniques in drug discovery purity assessmentpurification quantitative analysis and metabolite identifica-tionrdquo in Sample Preparation for Hyphenated Analytical Tech-niques pp 114ndash149 John Wiley amp Sons Hoboken NJ USA2004

[198] E P Springfield P K F Eagles and G Scott ldquoQuality assess-ment of South African herbal medicines by means of HPLCfingerprintingrdquo Journal of Ethnopharmacology vol 101 no 1ndash3pp 75ndash83 2005

[199] M Pereira da Costa and C A Conte-Junior ldquoChromatographicmethods for the determination of carbohydrates and organicacids in foods of animal originrdquoComprehensive Reviews in FoodScience and Food Safety vol 14 no 5 pp 586ndash600 2015

[200] M-J Dubber and I Kanfer ldquoDetermination of terpene trilac-tones inGinkgo biloba solid oral dosage forms usingHPLCwithevaporative light scattering detectionrdquo Journal of Pharmaceuti-cal and Biomedical Analysis vol 41 no 1 pp 135ndash140 2006

[201] B T Schaneberg R J Molyneux and I A Khan ldquoEvaporativelight scattering detection of pyrrolizidine alkaloidsrdquo Phyto-chemical Analysis vol 15 no 1 pp 36ndash39 2004

[202] V Verhelst and P Vandereecken ldquoAnalysis of organosiliconecopolymers by gradient polymer elution chromatography withevaporative light scattering detectionrdquo Journal of Chromatogra-phy A vol 871 no 1-2 pp 269ndash277 2000

[203] L Carbognani ldquoFast monitoring of C20-C160 crude oil alkanesby size-exclusion chromatography-evaporative light scatteringdetection performed with silica columnsrdquo Journal of Chro-matography A vol 788 pp 63ndash73 1997

[204] Y-J Hsieh L-C Lin and T-H Tsai ldquoDetermination and iden-tification of plumbagin from the roots of Plumbago zeylanica L


by liquid chromatography with tandem mass spectrometryrdquoJournal of Chromatography A vol 1083 no 1-2 pp 141ndash1452005

[205] M Yang J Sun Z Lu et al ldquoPhytochemical analysis of tradi-tional Chinese medicine using liquid chromatography coupledwith mass spectrometryrdquo Journal of Chromatography A vol1216 no 11 pp 2045ndash2062 2009

[206] LWu H Hao andGWang ldquoLCMS based tools and strategieson qualitative and quantitative analysis of herbal components incomplex matrixesrdquo Current Drug Metabolism vol 13 no 9 pp1251ndash1265 2012

[207] X-M Liang Y Jin Y-P Wang G-W Jin Q Fu and Y-SXiao ldquoQualitative and quantitative analysis in quality control oftraditional Chinese medicinesrdquo Journal of Chromatography Avol 1216 no 11 pp 2033ndash2044 2009

[208] H Qu Y Ma K Yu and Y Cheng ldquoSimultaneous determina-tion of eight active components in Chinese medicine rsquoYIQINGrsquocapsule using high-performance liquid chromatographyrdquo Jour-nal of Pharmaceutical and Biomedical Analysis vol 43 no 1 pp66ndash72 2007

[209] X-Y Chai S-L Li and P Li ldquoQuality evaluation of FlosLonicerae through a simultaneous determination of sevensaponins by HPLC with ELSDrdquo Journal of Chromatography Avol 1070 no 1-2 pp 43ndash48 2005

[210] W Li and J F Fitzloff ldquoA validated method for quantitativedetermination of saponins in notoginseng (Panax notoginseng)using high-performance liquid chromatography with evap-orative light-scattering detectionrdquo Journal of Pharmacy andPharmacology vol 53 no 12 pp 1637ndash1643 2001

[211] D Jing W Deguang H Linfang C Shilin and Q MinjianldquoApplication of chemometrics in quality evaluation ofmedicinalplantsrdquo Journal of Medicinal Plants Research vol 5 no 17 pp4001ndash4008 2011

[212] H A Gad S H El-Ahmady M I Abou-Shoer and M M Al-Azizi ldquoApplication of chemometrics in authentication of herbalmedicines a reviewrdquo Phytochemical Analysis vol 24 no 1 pp1ndash24 2013

[213] C Tistaert L Thierry A Szandrach et al ldquoQuality control ofCitri reticulatae pericarpium exploratory analysis and discrim-inationrdquo Analytica Chimica Acta vol 705 no 1-2 pp 111ndash1222011

[214] J-H Kim C-S Seo and H-K Shin ldquoDevelopment of val-idated determination of the eleven marker compounds inGyejibokryeong-hwan for the quality assessment using HPLCanalysisrdquo Archives of Pharmacal Research vol 38 no 1 pp 52ndash62 2015

[215] N J Lazarowych and P Pekos ldquoUse of fingerprinting andmarker compounds for identification and standardization ofbotanical drugs strategies for applying pharmaceutical HPLCanalysis to herbal productsrdquo Drug Information Journal vol 32no 2 pp 497ndash512 1998

[216] G Cimpan and S Gocan ldquoAnalysis of medicinal plants byHPLC recent approachesrdquo Journal of Liquid ChromatographyandRelated Technologies vol 25 no 13ndash15 pp 2225ndash2292 2002

[217] H Xiong L X Yu and H Qu ldquoBatch-to-batch quality consis-tency evaluation of botanical drug products using multivariatestatistical analysis of the chromatographic fingerprintrdquo AAPSPharmSciTech vol 14 no 2 pp 802ndash810 2013

[218] F Jiang Y Tao and Y Shao ldquoFingerprinting quality control ofQianghuo by high-performance liquid chromatography-pho-todiode array detectionrdquo Journal of Ethnopharmacology vol 111no 2 pp 265ndash270 2007

[219] Y-C ZhangG Li C Jiang et al ldquoTissue-specific distribution ofginsenosides in different aged ginseng and antioxidant activityof ginseng leafrdquoMolecules vol 19 no 11 pp 17381ndash17399 2014

[220] M Li H Cao P P-H But and P-C Shaw ldquoIdentification ofherbal medicinal materials using DNA barcodesrdquo Journal ofSystematics and Evolution vol 49 no 3 pp 271ndash283 2011

[221] J D Palmer and L A Herbon ldquoPlant mitochondrial DNAevolved rapidly in structure but slowly in sequencerdquo Journal ofMolecular Evolution vol 28 no 1-2 pp 87ndash97 1988

[222] M Ajmal Ali G Gyulai N Hidvegi et al ldquoThe changing epit-ome of species identificationmdashDNA barcodingrdquo Saudi Journalof Biological Sciences vol 21 no 3 pp 204ndash231 2014

[223] KVijayan andCHTsou ldquoDNAbarcoding in plants taxonomyin a new perspectiverdquo Current Science vol 99 no 11 pp 1530ndash1541 2010

[224] X Li Y Yang R J Henry M Rossetto Y Wang and S ChenldquoPlant DNA barcoding from gene to genomerdquo BiologicalReviews vol 90 no 1 pp 157ndash166 2015

[225] Y Li L-M Gao R C Poudel D-Z Li and A Forrest ldquoHighuniversality of matK primers for barcoding gymnospermsrdquoJournal of Systematics and Evolution vol 49 no 3 pp 169ndash1752011

[226] D E Soltis and P S SoltisChoosing an Approach and an Appro-priate Gene for Phylogenetic Analysis Molecular Systematics ofPlants II Springer New York NY USA 1998

[227] A J Fazekas K S Burgess P R Kesanakurti et al ldquoMultiplemultilocus DNA barcodes from the plastid genome discrimi-nate plant species equally wellrdquo PLoS ONE vol 3 no 7 ArticleID e2802 2008

[228] M W Chase R S Cowan P M Hollingsworth et al ldquoA pro-posal for a standardised protocol to barcode all land plantsrdquoTaxon vol 56 no 2 pp 295ndash299 2007

[229] K W Hilu and H Liang ldquoThe matK gene sequence variationand application in plant systematicsrdquo American Journal of Bot-any vol 84 no 6 pp 830ndash839 1997

[230] R LahayeM VanDer Bank D Bogarin et al ldquoDNA barcodingthe floras of biodiversity hotspotsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 105 no8 pp 2923ndash2928 2008

[231] W J Kress and D L Erickson ldquoA two-locus global DNA bar-code for land plants the coding rbcL gene complements thenon-coding trnH-psbA spacer regionrdquo PLoS ONE vol 2 no6 article e508 2007

[232] P Cuenoud V Savolainen L W Chatrou M Powell R JGrayer and M W Chase ldquoMolecular phylogenetics of Caryo-phyllales based on nuclear 18S rDNAand plastid rbcL atpB andmatK DNA sequencesrdquo American Journal of Botany vol 89 no1 pp 132ndash144 2002

[233] P M Hollingsworth L L Forrest J L Spouge et al ldquoA DNAbarcode for land plantsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 31 pp12794ndash12797 2009

[234] H Singh I Parveen S Raghuvanshi and S B Babbar ldquoTheloci recommended as universal barcodes for plants on the basisof floristic studies may not work with congeneric species asexemplified by DNA barcoding of Dendrobium speciesrdquo BMCResearch Notes vol 5 article 42 2012

[235] P M Hollingsworth S W Graham and D P Little ldquoChoosingand using a plantDNAbarcoderdquo PLoSONE vol 6 no 5 ArticleID e19254 2011


[236] S Chen H Yao J Han et al ldquoValidation of the ITS2 region asa novel DNA barcode for identifying medicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010

[237] K R Sundari ldquoDNA barcoding a genomic-based tool for auth-entication of phytomedicinals and its productsrdquo Botanics Tar-gets and Therapy vol 2015 no 5 pp 77ndash84 2015

[238] C J Nock D L E Waters M A Edwards et al ldquoChloroplastgenome sequences from total DNA for plant identificationrdquoPlant Biotechnology Journal vol 9 no 3 pp 328ndash333 2011

[239] W J Kress and D L Erickson ldquoDNA barcodes methods andprotocolsrdquoMethods inMolecular Biology vol 858 pp 3ndash8 2012

[240] A Varma H Padh and N Shrivastava ldquoPlant genomic DNAisolation an art or a sciencerdquo Biotechnology Journal vol 2 no3 pp 386ndash392 2007

[241] G C AllenM A Flores-Vergara S Krasynanski S Kumar andW F Thompson ldquoA modified protocol for rapid DNA isolationfrom plant tissues using cetyltrimethylammonium bromiderdquoNature Protocols vol 1 no 5 pp 2320ndash2325 2006

[242] D D Kuta E Kwon-Ndung S Dachi O Bakare and L AOgunkanmi ldquoOptimization of protocols for DNA extractionand RAPD analysis in West African fonio (Digitaria exilis andDigitaria iburua ) germplasm characterizationrdquo African Journalof Biotechnology vol 4 no 12 pp 1368ndash1371 2005

[243] J-F Manen O Sinitsyna L Aeschbach A V Markov andA Sinitsyn ldquoA fully automatable enzymatic method for DNAextraction from plant tissuesrdquo BMC Plant Biology vol 5 article23 2005

[244] M Moyo S O Amoo M W Bairu J F Finnie and J VanStaden ldquoOptimising DNA isolation formedicinal plantsrdquo SouthAfrican Journal of Botany vol 74 no 4 pp 771ndash775 2008

[245] M R Lum and A M Hirsch ldquoDetecting the componentsof botanical mixtures by single-strand conformation polymor-phism analysisrdquo ACS Symposium Series vol 1081 pp 351ndash3622011

[246] A M Pirttila M Hirsikorpi T Kamarainen L Jaakola and AHohtola ldquoDNA isolation methods for medicinal and aromaticplantsrdquo Plant Molecular Biology Reporter vol 19 no 3 p 2732001

[247] F Tamari and C S Hinkley ldquoExtraction of DNA from planttissue review and protocolsrdquo in Sample Preparation Techniquesfor Soil Plant and Animal Samples pp 245ndash263 Springer 2016

[248] N Gryson K Messens and K Dewettinck ldquoEvaluation andoptimisation of five different extraction methods for soy DNAin chocolate and biscuits Extraction of DNA as a first step inGMO analysisrdquo Journal of the Science of Food and Agriculturevol 84 no 11 pp 1357ndash1363 2004

[249] A Zimmermann J Luthy and U Pauli ldquoQuantitative andqualitative evaluation of nine different extraction methodsfor nucleic acids on soya bean food samplesrdquo Zeitschrift furLebensmittel-Untersuchung und -Forschung vol 207 no 2 pp81ndash90 1998

[250] N Gryson ldquoEffect of food processing on plant DNA degrada-tion and PCR-based GMO analysis a reviewrdquo Analytical andBioanalytical Chemistry vol 396 no 6 pp 2003ndash2022 2010

[251] L Olexova Lrsquo Dovicovicova and T Kuchta ldquoComparisonof three types of methods for the isolation of DNA fromflours biscuits and instant papsrdquo European Food Research andTechnology vol 218 no 4 pp 390ndash393 2004

[252] T Sarkinen M Staats J E Richardson R S Cowan and FT Bakker ldquoHow to open the treasure chest Optimising DNAextraction from herbarium specimensrdquo PLoS ONE vol 7 no 8Article ID e43808 2012

[253] D Puchooa ldquoA simple rapid and efficient method for theextraction of genomic DNA from lychee (Litchi chinensisSonn)rdquo African Journal of Biotechnology vol 3 no 4 pp 253ndash255 2004

[254] SMAhmadMMGanaie PHQazi VVerma S F Basir andG N Qazi ldquoRapid DNA isolation protocol for angiospermicplantsrdquo Bulgarian Journal of Plant Physiology vol 30 pp 25ndash332004

[255] G B Barra T H Santa Rita J D A Vasques C F ChiancaL F A Nery and S S S Costa ldquoEDTA-mediated inhibitionof DNases protects circulating cell-free DNA from ex vivo deg-radation in blood samplesrdquoClinical Biochemistry vol 48 no 15pp 976ndash981 2015

[256] D F CoutinhoMoraes DW StillM R Lum andAMHirschldquoDNA-based authentication of botanicals and plant-deri-ved dietary supplements where have we been and where are wegoingrdquo Planta Medica vol 81 no 9 pp 687ndash695 2015

[257] D P Little ldquoA DNA mini-barcode for land plantsrdquo MolecularEcology Resources vol 14 no 3 pp 437ndash446 2014

[258] N V Ivanova M L Kuzmina T W A Braukmann A VBorisenko and E V Zakharov ldquoAuthentication of herbal supp-lements using next-generation sequencingrdquo PLoS ONE vol 11no 5 Article ID e0156426 2016

[259] Y Yang B Xie and J Yan ldquoApplication of next-generation seq-uencing technology in forensic sciencerdquo Genomics Proteomicsand Bioinformatics vol 12 no 5 pp 190ndash197 2014

[260] J R Hennell P M DrsquoAgostino S Lee C S Khoo and N JSucher ldquoUsing genbank for genomic authentication a tuto-rialrdquoMethods in Molecular Biology vol 862 pp 181ndash200 2012

[261] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[262] T Kazi N Hussain P Bremner A Slater and C Howard ldquoTheapplication of a DNA-based identification technique to over-the-counter herbal medicinesrdquo Fitoterapia vol 87 no 1 pp 27ndash30 2013

[263] H J De Boer A Ouarghidi G Martin A Abbad and A KoolldquoDNA barcoding reveals limited accuracy of identificationsbased on folk taxonomyrdquo PLoS ONE vol 9 no 1 Article IDe84291 2014

[264] Y Ji L Ashton S M Pedley et al ldquoReliable verifiable andefficientmonitoring of biodiversity viametabarcodingrdquo EcologyLetters vol 16 no 10 pp 1245ndash1257 2013

[265] M L Coghlan J Haile J Houston et al ldquoDeep sequencing ofplant and animal DNA contained within traditional Chinesemedicines reveals legality issues and health safety concernsrdquoPLoS Genetics vol 8 no 4 Article ID e1002657 2012

[266] X Cheng X Su X Chen et al ldquoBiological ingredient analysisof traditional Chinese medicine preparation based on high-throughput sequencing the story for Liuwei Dihuang WanrdquoScientific Reports vol 4 article 5147 2014

[267] SMartins J A Teixeira and S IMussatto ldquoSolid-state fermen-tation as a strategy to improve the bioactive compounds reco-very from larrea tridentata leavesrdquo Applied Biochemistry andBiotechnology vol 171 no 5 pp 1227ndash1239 2013

[268] T N Huynh G Smagghe G B Gonzales J Van Camp and KRaes ldquoExtraction and bioconversion of kaempferol metabolitesfrom cauliflower outer leaves through fungal fermentationrdquoBiochemical Engineering Journal vol 116 pp 27ndash33 2016


[269] M R Lum and A M Hirsch ldquoMolecular methods for the auth-entication of botanicals and detection of potential contaminantsand adulterantsrdquo in Proceedings of the 4th International Confer-ence on Quality and Safety Issues Related to Botanicals pp 59ndash72 2005

[270] D F CMoraes DW StillM R Lum andAMHirsch ldquoDNA-based authentication of botanicals and plant-derived dietarysupplements where have we been and where are we goingrdquoPlanta Medica vol 81 no 9 pp 687ndash695 2015

[271] A N Mohamed J Vejayan and M M Yusoff ldquoReview onEurycoma longifolia pharmacological and phytochemical pro-pertiesrdquo Journal of Applied Sciences vol 15 no 6 pp 831ndash8442015

[272] S R Murray R C Butler A K Hardacre and G M Timmer-man-Vaughan ldquoUse of quantitative real-time PCR to estimatemaize endogenous DNA degradation after cooking and extru-sion or in food productsrdquo Journal of Agricultural and FoodChemistry vol 55 no 6 pp 2231ndash2239 2007

[273] T J R Fernandes M B P P Oliveira and I Mafra ldquoTracingtransgenic maize as affected by breadmaking process and rawmaterial for the production of a traditional maize bread broardquoFood Chemistry vol 138 no 1 pp 687ndash692 2013

[274] M F Polz and C M Cavanaugh ldquoBias in template-to-productratios in multitemplate PCRrdquo Applied and EnvironmentalMicrobiology vol 64 no 10 pp 3724ndash3730 1998

[275] J Novak S Grausgruber-Groger and B Lukas ldquoDNA-basedauthentication of plant extractsrdquo Food Research Internationalvol 40 no 3 pp 388ndash392 2007

[276] W L T Kan BMa andG Lin ldquoSulfur fumigation processing oftraditional Chinesemedicinal herbs beneficial or detrimentalrdquoFrontiers in Pharmacology vol 2 article 84 2011

[277] N B Zahra Z K Shinwari and M Qaiser ldquoDna barcoding atool for standardization of Herbal Medicinal Products (HMPS)of Lamiaceae From Pakistanrdquo Pakistan Journal of Botany vol48 pp 2167ndash2174 2016

[278] R Vilgalys ldquoTaxonomic misidentification in public DNA data-basesrdquo New Phytologist vol 160 no 1 pp 4ndash5 2003

[279] B Gemeinholzer C Oberprieler and K Bachmann ldquoUsingGenBank data for plant identification possibilities and limita-tions using the ITS 1 of Asteraceae species belonging to the tri-bes Lactuceae and Anthemideaerdquo Taxon vol 55 no 1 pp 173ndash187 2006

[280] D Shanmughanandhan S Ragupathy S G Newmaster SMohanasundaram and R Sathishkumar ldquoEstimating herbalproduct authentication and adulteration in india using avouchered DNA-based biological reference material libraryrdquoDrug Safety vol 39 no 12 pp 1211ndash1227 2016

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


reported that the annual sales of herbal medicine in Germanyfor the year 2002 have reached US$2432 billion [14] Nahinet al [15] also reported that the market of HMPs in USAhas expanded tremendously from US$29 billion to US$48billion from 1995 to 2008

So also the average import of these HMPs in USA in2004ndash2008 is more than US$220 million and the highestexporters for that very period were China and India valuedat around US$348 and US$93 million respectively [16] Aftera period of 3 years the sales of herbal medicine in Chinaincreased tremendously as reported by National Bureau ofStatistics [14] They reported that the sale value in herbalindustries has reached US$68 billion (RMB418 billion) withan annual growth rate of 379 So also the export annualrate of the herbal medicine from China to US in the sameyears has reached up to 663 [17]

Despite the extensive usage of HMP for a long period oftime issues related to their assessment and quality controlare always a major problem because active phytocompoundshave inherent risk just like all the active chemical compounds[18] The problems encountered are mostly attributed tothe poor quality of raw material or finished products andmay result in different types of adverse effects Basically thequality of the herbal products can be categorised into externaland internal categories [19] The external issue affectingthe quality includes contamination cultivation processingadulteration and misidentification while internal issues arebasically caused by the presence of bioactive chemicals in theherbal products

Both the external and internal qualities of the raw mate-rial are very important due to the fact that both safety andefficacy of the herbal products depend on them Accordingto the WHO adulteration of the herbal product is notonly a fraud but a threat to the consumerrsquos health as thismight result in adverse effects and eventually death [20]False identification of HMPs has resulted in several toxiceffects to consumers A dramatic example of such incidentoccurs in an 18-month-old baby who was diagnosed withvenoocculusive disease after regularly consuming a herbaltea containing high amount of pyrrolizidine alkaloid [21]Other cases include adulteration of herbal medicine teawith a neurotoxin compound from Illicium anisatum [22]Aconitum carmichaelii and Aconitum kusnezoffii have longbeen used to relief pain conditions such as rheumatismarthritis bruises and fractures [23] Most of the toxicity ofthese HMPs is primarily derived as a result of the presenceof diterpene alkaloids particularly aconitine others includemesaconitine and hypaconitine [24] In traditional Chinesemedicine (TCM) practices aconitine has been effectivelyused to treat various ailments despite their toxic effects Sucheffects were related to that causing ventricular tachycardia[25] bradycardia and hypotension [26]

The proportion of adulterated HMPs sold in developingcountries can account for up to 10 but may increase up to50 when purchased through unregulated channels such asonline transaction [27] The popular demand in such marketespecially in developing countries may pose further a seriousproblemwhere drug regulation is weak Reports have showedthatmore than 80of theHMPs sold inAfrica are counterfeit

or adulterated [28] All these incidences keep compoundingand increasing frequently due to lack of proper identificationmethods for primary plant source [27]

In general HMPs are classified into two products mono-herbal and polyherbal products [29] Monoherbal productscontained only one herb or herbal component for exampleEurycoma longifolia capsule which consists of its extract onlyThe polyherbal normally can be found either in the formof capsule tablets or tea which consists of more than one(two or more) herb component which can work in synergy todeliver their therapeutic effects Liuwei Dihuang pill (LDP)a well known TCM widely used for the treatment of kidneyyin deficiency contains 6 different Chinese medicinal herbs[30] Hwangryunhaedok-tang (HHT) a traditional herbalmedicine that is used for the treatment of inflammation feverhypertension and gastritis in China and Japan is composedof four herbal substances [31] Other examples of polyherbalproducts are shown in Table 1

Due to the widespread use of medicinal plants forthe past decades tandem with the assurance of its qualitysafety and efficacy of medicinal plant the WHO has pub-lished monographs [32ndash35] for selected medicinal plants forreferences Each volume of these monographs consists ofdifferent species of plant with their synonyms and vernac-ular names commonly used parts geographical locationknown active compounds dosage forms potential adverseeffects contraindication recognised medicinal benefits andhow to correctly and effectively use them Some countrieshave also established a series of monographs for their localusage such as Chinese pharmacopeia [36] United Statespharmacopeia [37] Ayurvedic Pharmacopoeia of India [38]Japanese pharmacopeia [39] and other countries The mainpurpose of developing all these monographs is to provideaccurate scientific information on the safety efficacy andquality assurance of the widely used medicinal plants and tohelp other countries to develop their own

Certain steps are of enormous importance in order toguarantee the quality of HMPs and these include (1) correctidentification of the plant species and (2) correct identifi-cation and analyses of the purity of the pharmacologicallyactive constituent (biomarkers with their required minimumconcentration) [40] In this aspect the identification of theexact and correct raw material used for the production ofHMPs is always challenging Correct identification of thesource of raw materials of medicinal herbal plants used isthe prerequisite and is of utmost criticalness to ensure theirquality safety and therapeutic efficacy [41 42] Generallythere are different types of identification methods most arecommonly based on botanical and morphological analysesby taxonomist expert thus identification by common peoplemay lead to incorrect specieThis is also true in the sense thatmost of the HMP consists of dried or unidentifiable part andthis makes the species identification process difficult even tohighly trained and experienced professional taxonomist [27]

The identification of herbal medicinal plant for qualitycontrol and standardization of raw materials should com-ply with the requirement specified in pharmacopeia theseinclude organoleptic evaluation (identification of sensestouch smell sight and taste) morphological characteristics














Ge-Gen Decoction



dysmenorrhea




Yan et al [67]



dysmenorrhea



Chen et al [68]

































































VidangaAnthelmintic






Sudani et al [72]



encephalitis B


5 120583m)HPLC-DAD


c35∘C d10 120583LZou et al [73]





HPLC-DAD-TOFMS



Liu et al [74]

GinsengAntidiabetic






c40∘C d10 120583L

Sun et al [75]



(46 times 250mm id5 120583m)

HPLC-Q-TOF-MSMS


c30∘C d10 120583L

Yan et al [67]


blood flow




Xu et al [76]






c43∘C d20 120583LLi et al [77]



cancer


CH3CN-H

2O


Qiu et al [78]






Basar et al [79]







Lau et al [80]



cancer


5 120583m)

HPLC-APCIMS



Ma et al [81]



5 120583m)HPLC-DAD


c25∘C d10 120583LZhao et al [82]




c88∘C d20 120583L

Li et al [83]


Table 2 Continued





c25∘C d5 120583LZhu et al [70]




HPLC-APCI-MS



Ma et al [84]




(250mm times 46mm5 120583m)

HPLC-ELSD



d20 120583L

Kong et al [85]





Wang et al [86]




46mm)HPLC-DAD



Xu et al [87]



5 120583m)

HPLC-DAD-ESI-M


c35∘C d5120583L

Yan et al [88]




Waters USA)HPLC-PDA









Zhang et al [90]


infertility


27 120583m)LC-MSMS



c40∘C d2120583L

Han et al [91]



2PO4


with H3PO4)


Duan et al [92]


malaria


5 120583m)





HPLC-DAD



d20 120583L

Chen et al [94]


Table 2 Continued




activities



Ganzera et al [95]






Tan et al [96]






c30∘C d10 120583LKong et al [97]



ZorbaxSB-C18 column


a(CH3COOHH

2O)-


Zhang and Cheng[65]



disorders


5 120583m)HPLC-DAD



Bian et al [98]

Danggui-Shaoyao-San



5 120583m)

HPLC-DAD-ESI-MS







Kim et al [99]



gastrospasm


LC-MS and NMR



Wang et al [100]




HPLC-ESI-MS



Zhang et al [101]




5 120583m)

HPLC-QTOFM



Tan et al [102]


































malaria


Triticum durum


anticancer













3 DNA Barcoding















the appetite






[115]



HPLC-DADPCA PLS

BP-ANN andRBF-ANN

Lai et al [116]











































bark












Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Ge-Gen Decoction



dysmenorrhea




Yan et al [67]



dysmenorrhea



Chen et al [68]

































































VidangaAnthelmintic






Sudani et al [72]



encephalitis B


5 120583m)HPLC-DAD


c35∘C d10 120583LZou et al [73]





HPLC-DAD-TOFMS



Liu et al [74]

GinsengAntidiabetic






c40∘C d10 120583L

Sun et al [75]



(46 times 250mm id5 120583m)

HPLC-Q-TOF-MSMS


c30∘C d10 120583L

Yan et al [67]


blood flow




Xu et al [76]






c43∘C d20 120583LLi et al [77]



cancer


CH3CN-H

2O


Qiu et al [78]






Basar et al [79]







Lau et al [80]



cancer


5 120583m)

HPLC-APCIMS



Ma et al [81]



5 120583m)HPLC-DAD


c25∘C d10 120583LZhao et al [82]




c88∘C d20 120583L

Li et al [83]


Table 2 Continued





c25∘C d5 120583LZhu et al [70]




HPLC-APCI-MS



Ma et al [84]




(250mm times 46mm5 120583m)

HPLC-ELSD



d20 120583L

Kong et al [85]





Wang et al [86]




46mm)HPLC-DAD



Xu et al [87]



5 120583m)

HPLC-DAD-ESI-M


c35∘C d5120583L

Yan et al [88]




Waters USA)HPLC-PDA









Zhang et al [90]


infertility


27 120583m)LC-MSMS



c40∘C d2120583L

Han et al [91]



2PO4


with H3PO4)


Duan et al [92]


malaria


5 120583m)





HPLC-DAD



d20 120583L

Chen et al [94]


Table 2 Continued




activities



Ganzera et al [95]






Tan et al [96]






c30∘C d10 120583LKong et al [97]



ZorbaxSB-C18 column


a(CH3COOHH

2O)-


Zhang and Cheng[65]



disorders


5 120583m)HPLC-DAD



Bian et al [98]

Danggui-Shaoyao-San



5 120583m)

HPLC-DAD-ESI-MS







Kim et al [99]



gastrospasm


LC-MS and NMR



Wang et al [100]




HPLC-ESI-MS



Zhang et al [101]




5 120583m)

HPLC-QTOFM



Tan et al [102]


































malaria


Triticum durum


anticancer













3 DNA Barcoding















the appetite






[115]



HPLC-DADPCA PLS

BP-ANN andRBF-ANN

Lai et al [116]











































bark












Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































VidangaAnthelmintic






Sudani et al [72]



encephalitis B


5 120583m)HPLC-DAD


c35∘C d10 120583LZou et al [73]





HPLC-DAD-TOFMS



Liu et al [74]

GinsengAntidiabetic






c40∘C d10 120583L

Sun et al [75]



(46 times 250mm id5 120583m)

HPLC-Q-TOF-MSMS


c30∘C d10 120583L

Yan et al [67]


blood flow




Xu et al [76]






c43∘C d20 120583LLi et al [77]



cancer


CH3CN-H

2O


Qiu et al [78]






Basar et al [79]







Lau et al [80]



cancer


5 120583m)

HPLC-APCIMS



Ma et al [81]



5 120583m)HPLC-DAD


c25∘C d10 120583LZhao et al [82]




c88∘C d20 120583L

Li et al [83]


Table 2 Continued





c25∘C d5 120583LZhu et al [70]




HPLC-APCI-MS



Ma et al [84]




(250mm times 46mm5 120583m)

HPLC-ELSD



d20 120583L

Kong et al [85]





Wang et al [86]




46mm)HPLC-DAD



Xu et al [87]



5 120583m)

HPLC-DAD-ESI-M


c35∘C d5120583L

Yan et al [88]




Waters USA)HPLC-PDA









Zhang et al [90]


infertility


27 120583m)LC-MSMS



c40∘C d2120583L

Han et al [91]



2PO4


with H3PO4)


Duan et al [92]


malaria


5 120583m)





HPLC-DAD



d20 120583L

Chen et al [94]


Table 2 Continued




activities



Ganzera et al [95]






Tan et al [96]






c30∘C d10 120583LKong et al [97]



ZorbaxSB-C18 column


a(CH3COOHH

2O)-


Zhang and Cheng[65]



disorders


5 120583m)HPLC-DAD



Bian et al [98]

Danggui-Shaoyao-San



5 120583m)

HPLC-DAD-ESI-MS







Kim et al [99]



gastrospasm


LC-MS and NMR



Wang et al [100]




HPLC-ESI-MS



Zhang et al [101]




5 120583m)

HPLC-QTOFM



Tan et al [102]


































malaria


Triticum durum


anticancer













3 DNA Barcoding















the appetite






[115]



HPLC-DADPCA PLS

BP-ANN andRBF-ANN

Lai et al [116]











































bark












Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 2 Continued





c25∘C d5 120583LZhu et al [70]




HPLC-APCI-MS



Ma et al [84]




(250mm times 46mm5 120583m)

HPLC-ELSD



d20 120583L

Kong et al [85]





Wang et al [86]




46mm)HPLC-DAD



Xu et al [87]



5 120583m)

HPLC-DAD-ESI-M


c35∘C d5120583L

Yan et al [88]




Waters USA)HPLC-PDA









Zhang et al [90]


infertility


27 120583m)LC-MSMS



c40∘C d2120583L

Han et al [91]



2PO4


with H3PO4)


Duan et al [92]


malaria


5 120583m)





HPLC-DAD



d20 120583L

Chen et al [94]


Table 2 Continued




activities



Ganzera et al [95]






Tan et al [96]






c30∘C d10 120583LKong et al [97]



ZorbaxSB-C18 column


a(CH3COOHH

2O)-


Zhang and Cheng[65]



disorders


5 120583m)HPLC-DAD



Bian et al [98]

Danggui-Shaoyao-San



5 120583m)

HPLC-DAD-ESI-MS







Kim et al [99]



gastrospasm


LC-MS and NMR



Wang et al [100]




HPLC-ESI-MS



Zhang et al [101]




5 120583m)

HPLC-QTOFM



Tan et al [102]


































malaria


Triticum durum


anticancer













3 DNA Barcoding















the appetite






[115]



HPLC-DADPCA PLS

BP-ANN andRBF-ANN

Lai et al [116]











































bark












Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 2 Continued




activities



Ganzera et al [95]






Tan et al [96]






c30∘C d10 120583LKong et al [97]



ZorbaxSB-C18 column


a(CH3COOHH

2O)-


Zhang and Cheng[65]



disorders


5 120583m)HPLC-DAD



Bian et al [98]

Danggui-Shaoyao-San



5 120583m)

HPLC-DAD-ESI-MS







Kim et al [99]



gastrospasm


LC-MS and NMR



Wang et al [100]




HPLC-ESI-MS



Zhang et al [101]




5 120583m)

HPLC-QTOFM



Tan et al [102]


































malaria


Triticum durum


anticancer













3 DNA Barcoding















the appetite






[115]



HPLC-DADPCA PLS

BP-ANN andRBF-ANN

Lai et al [116]











































bark












Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































malaria


Triticum durum


anticancer













3 DNA Barcoding















the appetite






[115]



HPLC-DADPCA PLS

BP-ANN andRBF-ANN

Lai et al [116]











































bark












Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 DNA Barcoding















the appetite






[115]



HPLC-DADPCA PLS

BP-ANN andRBF-ANN

Lai et al [116]











































bark












Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























the appetite






[115]



HPLC-DADPCA PLS

BP-ANN andRBF-ANN

Lai et al [116]











































bark












Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































bark












Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 5 Continued







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































4 Conclusion







Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References














































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































































































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Review: DNA Barcoding and Chromatography Fingerprints for the Authentication...

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Transcript of Review: DNA Barcoding and Chromatography Fingerprints for the Authentication...