Research ArticleIntegrated Analysis for Identifying Radix Astragali and ItsAdulterants Based on DNA Barcoding
Sihao Zheng1 Dewang Liu2 Weiguang Ren1 Juan Fu1 Linfang Huang1 and Shilin Chen3
1 Institute ofMedicinal Plant Development Chinese Academy ofMedical Sciences Peking UnionMedical College Beijing 100193 China2 School of Pharmacy Inner Mongolia Medical University Inner Mongolia 010080 China3 Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences Beijing 100700 China
Correspondence should be addressed to Linfang Huang lfhuangimpladaccn
Received 15 June 2014 Accepted 22 July 2014 Published 27 August 2014
Academic Editor Robert Henry
Copyright copy 2014 Sihao Zheng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Radix Astragali is a popular herb used in traditional Chinese medicine for its proimmune and antidiabetic properties Howevermethods are needed to help distinguish Radix Astragali from its varied adulterants DNAbarcoding is a widely applicablemolecularmethod used to identify medicinal plants Yet its use has been hampered by genetic distance base variation and limitations of thebio-NJ tree Herein we report the validation of an integrated analysis method for plant species identification using DNA barcodingthat focuses on genetic distance identification efficiency inter- and intraspecific variation and barcoding gap We collected 478sequences from six candidate DNA barcodes (ITS2 ITS psbA-trnH rbcL matK and COI) from 29 species of Radix Astragaliand adulterants The internal transcribed spacer (ITS) sequence was demonstrated as the optimal barcode for identifying RadixAstragali and its adulterants This new analysis method is helpful in identifying Radix Astragali and expedites the utilization anddata mining of DNA barcoding
1 Introduction
Radix Astragali (Huang Qi) a commonly used Chinesemedicinal material is mainly sourced from the plantsof Astragalus membranaceus and Astragalus mongholicusaccording to Chinese Pharmacopoeia (2010 edition) RadixAstragali is widely used for its antiperspirant antidiureticand antidiabetic properties and as a tonic drug [1ndash3] Itpossesses various beneficial compounds including astraga-losides isoflavonoids isoflavones isoflavan and pterocarpanglycosides [4ndash6]
Due to the high market demand for Radix Astragalia diverse group of adulterants with similar-morphologicalcharacteristics from genuses such as AstragalusHedysarumand Malva are often used in its stead [7] The traditionalmethods used to identify Radix Astragali for use as amedicinal material such as morphological and microscopicidentification [8] thin-layer chromatography and Ultravioletspectroscopy [9] Fourier Transform infrared spectroscopy(FTIR) [10] and high performance liquid chromatography(HPLC) [11] all require specialized equipment and training
Several PCR-based molecular methods have been developedproviding an alternative means of identification MultiplexPCR methods of DNA fragment analysis such as randomlyamplified polymorphic DNA (RAPD) [12] or amplified frag-ment length polymorphism (AFLP) [13] are unstable for theresults to identify DNA barcoding is a widely usedmolecularmarker technology first proposed by Hebert et al [14 15]It uses a standardized and conserved but diverse DNAsequence to identify species and uncover biological diversity[16 17] In previous studies various coding sequences foridentifying Radix Astragali and its adulterants have beenused such as the 5S-rRNA spacer domain [18] 31015840 untrans-lated region (31015840 UTR) [19] ITS (internal transcribed spacerregion) and 18S rRNA [3 20 21] ITS2 [22] ITS1 [6] matK(maturase K) and rbcL (ribulose 1 5-bisphosphate carboxy-lase) of chloroplast genome and coxI (cytochrome c oxidase1) of the mitochondrial genome [23] However sequenceanalysis was mainly focused on genetic distance variablesites amplified polymorphisms and the use of a modifiedneighbor-joining (NJ) algorithm Bio-NJ tree which werebasic analyses limited to particular species A more effective
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 843923 11 pageshttpdxdoiorg1011552014843923
2 Evidence-Based Complementary and Alternative Medicine
Table 1 Taxon sampling information of astragalus and its adulterants
method of molecular identification is necessary The currentstudy evaluates the identification reliability and efficiency ofDNAbarcoding for the identification of RadixAstragali usingsix indicators of genetic distance identification efficiencyintra- and interspecific variation gap rate and barcoding gapSix barcodes were selected for identification because theyare commonly used in plant especially in medicinal plantWe collected Radix Astragaliand several of its adulterantsreported in previous research and downloaded the geneticsequences from the GenBank database A total of 29 species(including 19 species of Astragalus) and 478 sequences fromsix barcodes were used to validate the new method foridentifying Radix Astragali and adulterants and to acceleratethe data utilization of DNA barcoding
2 Materials and Methods
21 Materials Information A total of 77 specimens werecollected from two origins of Radix Astragali along withseven adulterants Radix Astragali specimens were collectedfrom Inner Mongolia Shaan xi and Gan su provinces in thePeoplersquos Republic of China which are the main producingareas The collection information is shown in Table 1 Allcorresponding voucher specimens were deposited in theHerbariumof the Institute ofMedicinal PlantDevelopment atthe Chinese Academy of Medical Sciences in Beijing ChinaThe GenBank accession number of the ITS2 in this experi-ment was orderly KJ999296ndashKJ999344 the accession num-ber of ITS sequences was orderly KJ999345ndashKJ999416 andthe accession number of psbA-trnH was orderly KJ999256ndashKJ999295 The sequences added in the subsequent analysisincluding ITS ITS2 psbA-trnHmatK and rbcL were down-loaded from the GenBank database
22 DNA Extraction PCR Amplification and SequencingThe material specimens were naturally dried and 30mgof dried plant material was used for the DNA extractionSamples were rubbed for two minutes at a frequency of30 rs in a FastPrep bead mill (Retsch MM400 Germany)and total genomic DNA was isolated from the crushedmaterial according to the manufacturerrsquos instructions (Plant
Genomic DNA Kit Tiangen Biotech Co China) We madethe following modifications to the protocol chloroform wasdiluted with isoamyl alcohol (24 1 in the same volume) andbuffer solution GP2 with isopropanol (same volume) Thepowder 700120583L of 65∘C GP1 and 1 120583L 120573-mercaptoethanolweremixed for 10ndash20 s before being incubated for 60minutesat 65∘C Then 700120583L of the chloroformisoamyl alcoholmixture was added and the solution was centrifuged for 5minutes at 12000 rpm (sim13400timesg) Supernatant was removedand placed into a new tube before adding 700 120583L isopropanoland blending for 15ndash20minutesThemixture was centrifugedin CB3 spin columns for 40 s at 12000 rpm The filtratewas discarded and 500120583L GD (adding quantitative anhy-drous ethanol before use) was added before centrifuging at12000 rpm for 40 s The filtrate was discarded and 700120583LPW (adding quantitative anhydrous ethanol before use) wasused to wash the membrane before centrifuging for 40 s at12000 rpm This step was repeated with 500120583L PW followedby a final centrifuge for 2 minutes at 12000 rpm to removeresidual wash buffer The spin column was dried at roomtemperature for 3ndash5 minutes and then centrifuged for 2minutes at 12000 rpm to obtain the total DNA
General PCR reaction conditions and universal DNAbarcode primers were used for the ITS ITS2 and psbA-trnHbarcodes as presented in Table 2 [24ndash26] PCR amplificationwas performed on 25-120583L reaction mixtures containing 2120583LDNA template (20ndash100 ng) 85 120583L ddH2O 125 120583L 2times TaqPCRMaster Mix (Beijing TransGen Biotech Co China) and11-120583L forwardreverse (FR) primers (25 120583M) The reactionmixtures were amplified in a 9700 GeneAmp PCR system(Applied Biosystems USA) Amplicons were visualized byelectrophoresis on 1 agarose gels Purified PCR productswere sequenced in both directions using the ABI 3730XLsequencer (Applied Biosystems USA)
23 Sequence Assembly Alignment and Analysis Sequencingpeak diagramswere obtained and proofread and then contigswere assembled using a CodonCode Aligner 501 (Codon-Code Co USA) Complete ITS2 sequences were obtainedusing the HMMer annotation method based on the HiddenMarkov model (HMM) [27] All of the sequences were
Evidence-Based Complementary and Alternative Medicine 3
Table 2 Primers and PCR reaction conditions
Primer name Primer sequences (51015840-31015840) PCR reaction conditionITS2
2F ATGCGATACTTGGTGTGAAT 94∘C 5min
3R GACGCTTCTCCAGACTACAAT94∘C 30 s 56∘C 30 s72∘C 45 s 40 cycles
72∘C 10minITS
4R TCCTCCGCTTATTGATATGC 94∘C 5min
5F GGAAGTAAAAGTCGTAACAAGG94∘C 1min 50∘C 1min
72∘C 15min + 3 scycle 30 cycles72∘C 7min
psbAfwdPA GTTATGCATGAACGTAATGCTC 94∘C 4min
trnH
rev TH CGCGCATGGTGGATTCACAATCC94∘C 30 s 55∘C 1min72∘C 1min 35 cycles
72∘C 10min
aligned using ClustalW in combination with 317 sequencesfrom six commonly used barcodes (ITS2 ITS psbA-trnHmatK rbcL and COI) which were downloaded from theGenBank database (Table 3) Sequence genetic distance andGC content were calculated using the maximum compositelikelihoodmodel Maximum likelihood (ML) trees were con-structed based on the Tamura-Nei model and bootstrap testswere conducted using 1000 repeats to assess the confidenceof the phylogenetic relationships byMEGA 60 software [28]The barcoding gap defined as the spacer region betweenintra- and interspecific genetic variations and identificationefficiency based on BLAST1 and K2P nearest distance wereperformed by the Perl language algorithm (Putty) [25 29 30]
3 Results
31 Sequence Information and Identification Efficiency Atotal of 478 sequences for six barcodes were analyzed fromwhich 161 sequences were obtained from Astragalus Radixand its adulterants Sequence information and identificationsuccess rates are listed in Table 4 The average GC contentof six barcodes was discrepant and ITS and ITS2 regionsfrom nuclear ribosomal DNA performed higher than otherbarcodes (5297 versus 5080) Among the six barcodesITS2 provided the largest average genetic distance (10792)and rbcL was the smallest (00349) All of the six barcodesobtained a zero value for the minimum genetic distance Interms of identification efficiency the nearest distancemethodwas superior to the BLAST1method for all of the six barcodesMoreover ITS and the psbA-trnHandmatK regions provideda higher rate of success than the other three barcodes usingthe BLAST1 method However matK ITS and psbA-trnHperformed better than the other three barcodes based on thenearest distancemethod ITS and psbA-trnHobtained highergenetic distances so thematK ITS and psbA-trnH barcodeswere the preferable methods for identifying Radix Astragali
and its adulterants based on superior sequencing efficiencyand identification efficiency
32 Intra- and Interspecific Variation Analysis Using SixParameters Six parameters to analyze intraspecific variationand interspecific divergence were employed to assess theutility of six DNA barcodes (Table 5) We expected theldquominimum interspecific distancerdquo would be higher than theldquocoalescent depthrdquo (maximum intraspecific distance) There-fore we first utilized the ldquogap raterdquo to indicate the distinctnesscalculated by the formula (minimum interspecific distance minusmaximum intraspecific distance)minimum interspecific dis-tance Results show that the ITS2 COI matK and rbcLregions outperformed the ITS and psbA-trnH regions for gaprates However when we compared all of the average inter-and intraspecific distances the ITS2 rbcL matK and psbA-trnH regions performed better than the ITS and COI regionsTherefore in terms of intra- and interspecific variation ITS2matK and rbcL are the preferable options for identifyingRadix Astragali and its adulterants
33 Barcoding Gap Analysis Analysis of the DNA barcod-ing gap presents the divergence of inter- and intraspeciesand indicates separate nonoverlapping distribution betweenspecimens in an ideal situation [25] In our study (Figure 1)the rbcL COI ITS and matK regions possessed less relativedistribution of inter- and intraspecific variation than psbA-trnH and ITS2 although there were no nonoverlappingregions for the six barcodes Hence the rbcL COI ITS andmatK regions are more successful at identifying Radix Astra-gali and its adulterants from the standpoint of barcoding gapanalysis
34MLTreeAnalysis Maximum likelihood (ML) is a generalstatistical criterion in widespread use for the inference ofmolecular phylogenies [31] An ML tree visually revealed therelationship between species As the results show (Figure 2)
4 Evidence-Based Complementary and Alternative Medicine
Table 3 Sequences from GenBank for identifying Astragalus and its adulterants
psbA-trnH successfully differentiated Radix Astragali andits adulterants Furthermore it produced areas of obviousseparation for Radix Astragali The remaining five barcodesalso differentiated Radix Astragali and its adulterants Eachspecies clustered together separate from other species Con-sidering the difficult amplification and sequencing and fastand accurate identification purpose of DNA barcoding wedid not add all the sequence data of ITS2 and psbA-trnH tobuild ML tree and subsequent analysis
4 Discussion and Conclusions
Radix Astragali is reported to possess 47 bioactive com-pounds and has many bioactive properties [32ndash37] VariousRadix Astragali preparations are commercially availablenot only in China as a TCM component but also in theUnited States as dietary supplements [38] However due toincreasing demand substitutes and adulterants have floodedthe market Traditional identification methods such as mor-phological and microscopic methods are limited by the lackof explicit criteria for character selection or coding and thusmainly depend on subjective assessments Although chemicalmethods are able to distinguish between different species itis difficult to differentiate sibling species that possess similarchemical compositions In addition chemical methods areunable to provide accurate species authentication Severaltypes of molecular markers for characterizing genotypes areuseful in identifying plant species For example RAPD hasbeen used to estimate genetic diversity in plant populationsbased on amplification of random DNA fragments andcomparisons of common polymorphisms DNA barcoding
is advocated for species identification due to its universalapplicability simplicity and scientific accuracy Howeverthe analysis methods for DNA barcodes were limited Withthe development of molecular biology and bioinformaticsa more improved analytic method for DNA barcoding canbe established to identify Radix Astragali and closely relatedspecies
In this study we validated a new analytical method foridentifying Radix Astragali using DNA barcoding Seventy-seven specimens of Radix Astragali and its adulterants werecollected and the sequences of 29 species reported in theliterature were downloaded from the GenBank databaseBased on the 478 sequences for six barcodes (ITS2 ITS fromnuclear genome psbA-trnH rbcL andmatK fromchloroplastgenome COI from mitochondrial genome) genetic distanceand ML Tree were calculated by MEGA 60 software andidentification efficiency intra- and interspecific variationand barcoding gap were calculated using the Perl languagealgorithm Results of the six indicators assessed are shownin Table 6 ITS and psbA-trnH outperformed other barcodesin terms of identification efficiency ITS2 performed better interms of genetic distance gap rate and inter- and intraspecificvariation RbcL performed better in terms of barcoding gapand inter- and intraspecific variation Although ITS2was partof the ITS sequence it performed poorly in identificationefficiency Therefore we suggest that the ITS sequence isthe optimal barcode and that the psbA-trnH region is acomplementary barcode for identifying Radix Astragali andits adulterants
In conclusion we describe a new analytical method forthe use of DNA barcoding in the identification of Radix
Evidence-Based Complementary and Alternative Medicine 7
0102030405060708090100 COI
0
IntraspeciesInterspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
(a)
0
10
20
30
40
50
60ITS2
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(b)
0102030405060
ITS
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(c)
010203040506070
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
matK
gt02
Interspecies
(d)
0102030405060708090100
rbcL
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(e)
05101520253035 psbA-trnH
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(f)
Figure 1 Barcoding gap for six barcodes
Astragali Six indicators including average genetic distanceBLAST1 and the nearest distance method for identificationefficiency inter- and intraspecific variation and gap rate weretested to evaluate six DNA barcodes using bioinformaticssoftware and the Perl language algorithm The ITS sequence
was the optimal barcode for identifying Radix Astragali andits adulterants This method provides a novel means foraccurate identification of Radix Astragali and its adulterantsand improves the utilization of DNA barcoding in identifyingmedicinal plant species
8 Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 9
JX017329
JXJX017330
JX0101
7328
JX0177326
JX017325
JX0173324
JX0173
23JX01732
JX01732122
JF736668
0JF736665
HM1
M144
228
H M14
2289
H MM14
2287
H M142228
6H M1142
272
H42
275
H U289
274
G U289
6643
G U289666
G 28U2896623
G U 28
9661
G U 5296
60
G U8059
E
42
EF685
969
SX9SX7SX6SX5SX4SX3NM
8NM
7NM3NM2GS5GS4S2GG S1
AB787166
JF736666
65 JF736669JF736667
61
GS3GS6NM1NM10NM4NNM5NM6S3M9
SSDD1
SD 2SD 3SD 5SX 7SX810
F68AF35975
AF359749EJ572 596 0FM14 044 8
HM14 227
H227 2
HM1422776
H142278M
H1M42279
H14M2280
HM142281
HM142283
M1422
HM1422 84
H
85
HM142288
HM142293
HM142294
HM142295
HM142296
H42M1291
H42M1292
H89127Q8
17331JX0JX017327733JX01
167 2
AF12219 5
KC2675 9
F35952 1
59
AF35975
AF35973
A
B2310919951
55
73
AM142297
HF121681
A AFA
359756F359757
AB741299
AF359755
AF5
AF35975421952
9972
76
HHQ1
HHQ2
HHQ3
HQ4
HHQ5
HHQ6
HQ7
9973
EM14223
0 2H HFJ980292
F68597030
HM144230 1
HM1
32 0
HH
6XJ
9675
AF
8Q1
99
HM142298
HM142299
35975
78
CY1CY2CY
6
CY5
CY4
F68597
1
HM142
303
AB051
988
94
74 719468 80
E
9Q33
82
HQ199
316
GU217
599FJ5372
84
94
Q83 9999HQ
514
GD
78598
F032294
K
HQ1HQ1
1HQ2
HQ10HQ4HQ9Q1 64HH 2
HHQ6Q3
HQ7
142304Q8HM
HM142305JX017333
54
9991 67
JX0173348
GQ24EU591996130
60
S1S26
9999
SSDSD9D8
99
JX017337938697
3KFX0173
7336
J 5
MX
GQ1
JX01AY256392
AF053142
8854
99
4HH2HH5
HH4HH3HH6
HH1
JF461309
F461308
A
J B
6HH7
01JF4613070101
Q31198554679
65
99
95
D AH X73
72J F4195
1967
679744
E EF14
99JK2JK1
F41
EF4194797053
SX24JK14 9
E78
6099
99
ITS
HM142290
(f)
Figure 2 ML tree for six barcodes lowastThe different color and shape for different species in clusters presented the identification of differentbarcodes
Table 6 Six indicators assessed for DNA barcoding
DNA barcodes
Parameters
Average geneticdistance
Identification efficiency Gap rateInter- to
intraspecificvariation
Barcoding gap Total scoreBLAST1 Nearest
distancesITS2 8 12 8 8 8 4 48ITS 6 28 22 0 0 6 62psbA-trnH 6 26 18 0 2 2 54rbcL 4 12 14 4 6 8 48matK 4 14 24 4 4 2 52COI 2 6 10 6 0 6 30lowastThe total score of six parameters was set by 10 30 30 10 10 and 10 in order Identification efficiency based on two methods was set by 30 score because of itsimportance for identification
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
method of molecular identification is necessary The currentstudy evaluates the identification reliability and efficiency ofDNAbarcoding for the identification of RadixAstragali usingsix indicators of genetic distance identification efficiencyintra- and interspecific variation gap rate and barcoding gapSix barcodes were selected for identification because theyare commonly used in plant especially in medicinal plantWe collected Radix Astragaliand several of its adulterantsreported in previous research and downloaded the geneticsequences from the GenBank database A total of 29 species(including 19 species of Astragalus) and 478 sequences fromsix barcodes were used to validate the new method foridentifying Radix Astragali and adulterants and to acceleratethe data utilization of DNA barcoding
2 Materials and Methods
21 Materials Information A total of 77 specimens werecollected from two origins of Radix Astragali along withseven adulterants Radix Astragali specimens were collectedfrom Inner Mongolia Shaan xi and Gan su provinces in thePeoplersquos Republic of China which are the main producingareas The collection information is shown in Table 1 Allcorresponding voucher specimens were deposited in theHerbariumof the Institute ofMedicinal PlantDevelopment atthe Chinese Academy of Medical Sciences in Beijing ChinaThe GenBank accession number of the ITS2 in this experi-ment was orderly KJ999296ndashKJ999344 the accession num-ber of ITS sequences was orderly KJ999345ndashKJ999416 andthe accession number of psbA-trnH was orderly KJ999256ndashKJ999295 The sequences added in the subsequent analysisincluding ITS ITS2 psbA-trnHmatK and rbcL were down-loaded from the GenBank database
22 DNA Extraction PCR Amplification and SequencingThe material specimens were naturally dried and 30mgof dried plant material was used for the DNA extractionSamples were rubbed for two minutes at a frequency of30 rs in a FastPrep bead mill (Retsch MM400 Germany)and total genomic DNA was isolated from the crushedmaterial according to the manufacturerrsquos instructions (Plant
Genomic DNA Kit Tiangen Biotech Co China) We madethe following modifications to the protocol chloroform wasdiluted with isoamyl alcohol (24 1 in the same volume) andbuffer solution GP2 with isopropanol (same volume) Thepowder 700120583L of 65∘C GP1 and 1 120583L 120573-mercaptoethanolweremixed for 10ndash20 s before being incubated for 60minutesat 65∘C Then 700120583L of the chloroformisoamyl alcoholmixture was added and the solution was centrifuged for 5minutes at 12000 rpm (sim13400timesg) Supernatant was removedand placed into a new tube before adding 700 120583L isopropanoland blending for 15ndash20minutesThemixture was centrifugedin CB3 spin columns for 40 s at 12000 rpm The filtratewas discarded and 500120583L GD (adding quantitative anhy-drous ethanol before use) was added before centrifuging at12000 rpm for 40 s The filtrate was discarded and 700120583LPW (adding quantitative anhydrous ethanol before use) wasused to wash the membrane before centrifuging for 40 s at12000 rpm This step was repeated with 500120583L PW followedby a final centrifuge for 2 minutes at 12000 rpm to removeresidual wash buffer The spin column was dried at roomtemperature for 3ndash5 minutes and then centrifuged for 2minutes at 12000 rpm to obtain the total DNA
General PCR reaction conditions and universal DNAbarcode primers were used for the ITS ITS2 and psbA-trnHbarcodes as presented in Table 2 [24ndash26] PCR amplificationwas performed on 25-120583L reaction mixtures containing 2120583LDNA template (20ndash100 ng) 85 120583L ddH2O 125 120583L 2times TaqPCRMaster Mix (Beijing TransGen Biotech Co China) and11-120583L forwardreverse (FR) primers (25 120583M) The reactionmixtures were amplified in a 9700 GeneAmp PCR system(Applied Biosystems USA) Amplicons were visualized byelectrophoresis on 1 agarose gels Purified PCR productswere sequenced in both directions using the ABI 3730XLsequencer (Applied Biosystems USA)
23 Sequence Assembly Alignment and Analysis Sequencingpeak diagramswere obtained and proofread and then contigswere assembled using a CodonCode Aligner 501 (Codon-Code Co USA) Complete ITS2 sequences were obtainedusing the HMMer annotation method based on the HiddenMarkov model (HMM) [27] All of the sequences were
Evidence-Based Complementary and Alternative Medicine 3
Table 2 Primers and PCR reaction conditions
Primer name Primer sequences (51015840-31015840) PCR reaction conditionITS2
2F ATGCGATACTTGGTGTGAAT 94∘C 5min
3R GACGCTTCTCCAGACTACAAT94∘C 30 s 56∘C 30 s72∘C 45 s 40 cycles
72∘C 10minITS
4R TCCTCCGCTTATTGATATGC 94∘C 5min
5F GGAAGTAAAAGTCGTAACAAGG94∘C 1min 50∘C 1min
72∘C 15min + 3 scycle 30 cycles72∘C 7min
psbAfwdPA GTTATGCATGAACGTAATGCTC 94∘C 4min
trnH
rev TH CGCGCATGGTGGATTCACAATCC94∘C 30 s 55∘C 1min72∘C 1min 35 cycles
72∘C 10min
aligned using ClustalW in combination with 317 sequencesfrom six commonly used barcodes (ITS2 ITS psbA-trnHmatK rbcL and COI) which were downloaded from theGenBank database (Table 3) Sequence genetic distance andGC content were calculated using the maximum compositelikelihoodmodel Maximum likelihood (ML) trees were con-structed based on the Tamura-Nei model and bootstrap testswere conducted using 1000 repeats to assess the confidenceof the phylogenetic relationships byMEGA 60 software [28]The barcoding gap defined as the spacer region betweenintra- and interspecific genetic variations and identificationefficiency based on BLAST1 and K2P nearest distance wereperformed by the Perl language algorithm (Putty) [25 29 30]
3 Results
31 Sequence Information and Identification Efficiency Atotal of 478 sequences for six barcodes were analyzed fromwhich 161 sequences were obtained from Astragalus Radixand its adulterants Sequence information and identificationsuccess rates are listed in Table 4 The average GC contentof six barcodes was discrepant and ITS and ITS2 regionsfrom nuclear ribosomal DNA performed higher than otherbarcodes (5297 versus 5080) Among the six barcodesITS2 provided the largest average genetic distance (10792)and rbcL was the smallest (00349) All of the six barcodesobtained a zero value for the minimum genetic distance Interms of identification efficiency the nearest distancemethodwas superior to the BLAST1method for all of the six barcodesMoreover ITS and the psbA-trnHandmatK regions provideda higher rate of success than the other three barcodes usingthe BLAST1 method However matK ITS and psbA-trnHperformed better than the other three barcodes based on thenearest distancemethod ITS and psbA-trnHobtained highergenetic distances so thematK ITS and psbA-trnH barcodeswere the preferable methods for identifying Radix Astragali
and its adulterants based on superior sequencing efficiencyand identification efficiency
32 Intra- and Interspecific Variation Analysis Using SixParameters Six parameters to analyze intraspecific variationand interspecific divergence were employed to assess theutility of six DNA barcodes (Table 5) We expected theldquominimum interspecific distancerdquo would be higher than theldquocoalescent depthrdquo (maximum intraspecific distance) There-fore we first utilized the ldquogap raterdquo to indicate the distinctnesscalculated by the formula (minimum interspecific distance minusmaximum intraspecific distance)minimum interspecific dis-tance Results show that the ITS2 COI matK and rbcLregions outperformed the ITS and psbA-trnH regions for gaprates However when we compared all of the average inter-and intraspecific distances the ITS2 rbcL matK and psbA-trnH regions performed better than the ITS and COI regionsTherefore in terms of intra- and interspecific variation ITS2matK and rbcL are the preferable options for identifyingRadix Astragali and its adulterants
33 Barcoding Gap Analysis Analysis of the DNA barcod-ing gap presents the divergence of inter- and intraspeciesand indicates separate nonoverlapping distribution betweenspecimens in an ideal situation [25] In our study (Figure 1)the rbcL COI ITS and matK regions possessed less relativedistribution of inter- and intraspecific variation than psbA-trnH and ITS2 although there were no nonoverlappingregions for the six barcodes Hence the rbcL COI ITS andmatK regions are more successful at identifying Radix Astra-gali and its adulterants from the standpoint of barcoding gapanalysis
34MLTreeAnalysis Maximum likelihood (ML) is a generalstatistical criterion in widespread use for the inference ofmolecular phylogenies [31] An ML tree visually revealed therelationship between species As the results show (Figure 2)
4 Evidence-Based Complementary and Alternative Medicine
Table 3 Sequences from GenBank for identifying Astragalus and its adulterants
psbA-trnH successfully differentiated Radix Astragali andits adulterants Furthermore it produced areas of obviousseparation for Radix Astragali The remaining five barcodesalso differentiated Radix Astragali and its adulterants Eachspecies clustered together separate from other species Con-sidering the difficult amplification and sequencing and fastand accurate identification purpose of DNA barcoding wedid not add all the sequence data of ITS2 and psbA-trnH tobuild ML tree and subsequent analysis
4 Discussion and Conclusions
Radix Astragali is reported to possess 47 bioactive com-pounds and has many bioactive properties [32ndash37] VariousRadix Astragali preparations are commercially availablenot only in China as a TCM component but also in theUnited States as dietary supplements [38] However due toincreasing demand substitutes and adulterants have floodedthe market Traditional identification methods such as mor-phological and microscopic methods are limited by the lackof explicit criteria for character selection or coding and thusmainly depend on subjective assessments Although chemicalmethods are able to distinguish between different species itis difficult to differentiate sibling species that possess similarchemical compositions In addition chemical methods areunable to provide accurate species authentication Severaltypes of molecular markers for characterizing genotypes areuseful in identifying plant species For example RAPD hasbeen used to estimate genetic diversity in plant populationsbased on amplification of random DNA fragments andcomparisons of common polymorphisms DNA barcoding
is advocated for species identification due to its universalapplicability simplicity and scientific accuracy Howeverthe analysis methods for DNA barcodes were limited Withthe development of molecular biology and bioinformaticsa more improved analytic method for DNA barcoding canbe established to identify Radix Astragali and closely relatedspecies
In this study we validated a new analytical method foridentifying Radix Astragali using DNA barcoding Seventy-seven specimens of Radix Astragali and its adulterants werecollected and the sequences of 29 species reported in theliterature were downloaded from the GenBank databaseBased on the 478 sequences for six barcodes (ITS2 ITS fromnuclear genome psbA-trnH rbcL andmatK fromchloroplastgenome COI from mitochondrial genome) genetic distanceand ML Tree were calculated by MEGA 60 software andidentification efficiency intra- and interspecific variationand barcoding gap were calculated using the Perl languagealgorithm Results of the six indicators assessed are shownin Table 6 ITS and psbA-trnH outperformed other barcodesin terms of identification efficiency ITS2 performed better interms of genetic distance gap rate and inter- and intraspecificvariation RbcL performed better in terms of barcoding gapand inter- and intraspecific variation Although ITS2was partof the ITS sequence it performed poorly in identificationefficiency Therefore we suggest that the ITS sequence isthe optimal barcode and that the psbA-trnH region is acomplementary barcode for identifying Radix Astragali andits adulterants
In conclusion we describe a new analytical method forthe use of DNA barcoding in the identification of Radix
Evidence-Based Complementary and Alternative Medicine 7
0102030405060708090100 COI
0
IntraspeciesInterspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
(a)
0
10
20
30
40
50
60ITS2
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(b)
0102030405060
ITS
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(c)
010203040506070
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
matK
gt02
Interspecies
(d)
0102030405060708090100
rbcL
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(e)
05101520253035 psbA-trnH
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(f)
Figure 1 Barcoding gap for six barcodes
Astragali Six indicators including average genetic distanceBLAST1 and the nearest distance method for identificationefficiency inter- and intraspecific variation and gap rate weretested to evaluate six DNA barcodes using bioinformaticssoftware and the Perl language algorithm The ITS sequence
was the optimal barcode for identifying Radix Astragali andits adulterants This method provides a novel means foraccurate identification of Radix Astragali and its adulterantsand improves the utilization of DNA barcoding in identifyingmedicinal plant species
8 Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 9
JX017329
JXJX017330
JX0101
7328
JX0177326
JX017325
JX0173324
JX0173
23JX01732
JX01732122
JF736668
0JF736665
HM1
M144
228
H M14
2289
H MM14
2287
H M142228
6H M1142
272
H42
275
H U289
274
G U289
6643
G U289666
G 28U2896623
G U 28
9661
G U 5296
60
G U8059
E
42
EF685
969
SX9SX7SX6SX5SX4SX3NM
8NM
7NM3NM2GS5GS4S2GG S1
AB787166
JF736666
65 JF736669JF736667
61
GS3GS6NM1NM10NM4NNM5NM6S3M9
SSDD1
SD 2SD 3SD 5SX 7SX810
F68AF35975
AF359749EJ572 596 0FM14 044 8
HM14 227
H227 2
HM1422776
H142278M
H1M42279
H14M2280
HM142281
HM142283
M1422
HM1422 84
H
85
HM142288
HM142293
HM142294
HM142295
HM142296
H42M1291
H42M1292
H89127Q8
17331JX0JX017327733JX01
167 2
AF12219 5
KC2675 9
F35952 1
59
AF35975
AF35973
A
B2310919951
55
73
AM142297
HF121681
A AFA
359756F359757
AB741299
AF359755
AF5
AF35975421952
9972
76
HHQ1
HHQ2
HHQ3
HQ4
HHQ5
HHQ6
HQ7
9973
EM14223
0 2H HFJ980292
F68597030
HM144230 1
HM1
32 0
HH
6XJ
9675
AF
8Q1
99
HM142298
HM142299
35975
78
CY1CY2CY
6
CY5
CY4
F68597
1
HM142
303
AB051
988
94
74 719468 80
E
9Q33
82
HQ199
316
GU217
599FJ5372
84
94
Q83 9999HQ
514
GD
78598
F032294
K
HQ1HQ1
1HQ2
HQ10HQ4HQ9Q1 64HH 2
HHQ6Q3
HQ7
142304Q8HM
HM142305JX017333
54
9991 67
JX0173348
GQ24EU591996130
60
S1S26
9999
SSDSD9D8
99
JX017337938697
3KFX0173
7336
J 5
MX
GQ1
JX01AY256392
AF053142
8854
99
4HH2HH5
HH4HH3HH6
HH1
JF461309
F461308
A
J B
6HH7
01JF4613070101
Q31198554679
65
99
95
D AH X73
72J F4195
1967
679744
E EF14
99JK2JK1
F41
EF4194797053
SX24JK14 9
E78
6099
99
ITS
HM142290
(f)
Figure 2 ML tree for six barcodes lowastThe different color and shape for different species in clusters presented the identification of differentbarcodes
Table 6 Six indicators assessed for DNA barcoding
DNA barcodes
Parameters
Average geneticdistance
Identification efficiency Gap rateInter- to
intraspecificvariation
Barcoding gap Total scoreBLAST1 Nearest
distancesITS2 8 12 8 8 8 4 48ITS 6 28 22 0 0 6 62psbA-trnH 6 26 18 0 2 2 54rbcL 4 12 14 4 6 8 48matK 4 14 24 4 4 2 52COI 2 6 10 6 0 6 30lowastThe total score of six parameters was set by 10 30 30 10 10 and 10 in order Identification efficiency based on two methods was set by 30 score because of itsimportance for identification
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
Evidence-Based Complementary and Alternative Medicine 3
Table 2 Primers and PCR reaction conditions
Primer name Primer sequences (51015840-31015840) PCR reaction conditionITS2
2F ATGCGATACTTGGTGTGAAT 94∘C 5min
3R GACGCTTCTCCAGACTACAAT94∘C 30 s 56∘C 30 s72∘C 45 s 40 cycles
72∘C 10minITS
4R TCCTCCGCTTATTGATATGC 94∘C 5min
5F GGAAGTAAAAGTCGTAACAAGG94∘C 1min 50∘C 1min
72∘C 15min + 3 scycle 30 cycles72∘C 7min
psbAfwdPA GTTATGCATGAACGTAATGCTC 94∘C 4min
trnH
rev TH CGCGCATGGTGGATTCACAATCC94∘C 30 s 55∘C 1min72∘C 1min 35 cycles
72∘C 10min
aligned using ClustalW in combination with 317 sequencesfrom six commonly used barcodes (ITS2 ITS psbA-trnHmatK rbcL and COI) which were downloaded from theGenBank database (Table 3) Sequence genetic distance andGC content were calculated using the maximum compositelikelihoodmodel Maximum likelihood (ML) trees were con-structed based on the Tamura-Nei model and bootstrap testswere conducted using 1000 repeats to assess the confidenceof the phylogenetic relationships byMEGA 60 software [28]The barcoding gap defined as the spacer region betweenintra- and interspecific genetic variations and identificationefficiency based on BLAST1 and K2P nearest distance wereperformed by the Perl language algorithm (Putty) [25 29 30]
3 Results
31 Sequence Information and Identification Efficiency Atotal of 478 sequences for six barcodes were analyzed fromwhich 161 sequences were obtained from Astragalus Radixand its adulterants Sequence information and identificationsuccess rates are listed in Table 4 The average GC contentof six barcodes was discrepant and ITS and ITS2 regionsfrom nuclear ribosomal DNA performed higher than otherbarcodes (5297 versus 5080) Among the six barcodesITS2 provided the largest average genetic distance (10792)and rbcL was the smallest (00349) All of the six barcodesobtained a zero value for the minimum genetic distance Interms of identification efficiency the nearest distancemethodwas superior to the BLAST1method for all of the six barcodesMoreover ITS and the psbA-trnHandmatK regions provideda higher rate of success than the other three barcodes usingthe BLAST1 method However matK ITS and psbA-trnHperformed better than the other three barcodes based on thenearest distancemethod ITS and psbA-trnHobtained highergenetic distances so thematK ITS and psbA-trnH barcodeswere the preferable methods for identifying Radix Astragali
and its adulterants based on superior sequencing efficiencyand identification efficiency
32 Intra- and Interspecific Variation Analysis Using SixParameters Six parameters to analyze intraspecific variationand interspecific divergence were employed to assess theutility of six DNA barcodes (Table 5) We expected theldquominimum interspecific distancerdquo would be higher than theldquocoalescent depthrdquo (maximum intraspecific distance) There-fore we first utilized the ldquogap raterdquo to indicate the distinctnesscalculated by the formula (minimum interspecific distance minusmaximum intraspecific distance)minimum interspecific dis-tance Results show that the ITS2 COI matK and rbcLregions outperformed the ITS and psbA-trnH regions for gaprates However when we compared all of the average inter-and intraspecific distances the ITS2 rbcL matK and psbA-trnH regions performed better than the ITS and COI regionsTherefore in terms of intra- and interspecific variation ITS2matK and rbcL are the preferable options for identifyingRadix Astragali and its adulterants
33 Barcoding Gap Analysis Analysis of the DNA barcod-ing gap presents the divergence of inter- and intraspeciesand indicates separate nonoverlapping distribution betweenspecimens in an ideal situation [25] In our study (Figure 1)the rbcL COI ITS and matK regions possessed less relativedistribution of inter- and intraspecific variation than psbA-trnH and ITS2 although there were no nonoverlappingregions for the six barcodes Hence the rbcL COI ITS andmatK regions are more successful at identifying Radix Astra-gali and its adulterants from the standpoint of barcoding gapanalysis
34MLTreeAnalysis Maximum likelihood (ML) is a generalstatistical criterion in widespread use for the inference ofmolecular phylogenies [31] An ML tree visually revealed therelationship between species As the results show (Figure 2)
4 Evidence-Based Complementary and Alternative Medicine
Table 3 Sequences from GenBank for identifying Astragalus and its adulterants
psbA-trnH successfully differentiated Radix Astragali andits adulterants Furthermore it produced areas of obviousseparation for Radix Astragali The remaining five barcodesalso differentiated Radix Astragali and its adulterants Eachspecies clustered together separate from other species Con-sidering the difficult amplification and sequencing and fastand accurate identification purpose of DNA barcoding wedid not add all the sequence data of ITS2 and psbA-trnH tobuild ML tree and subsequent analysis
4 Discussion and Conclusions
Radix Astragali is reported to possess 47 bioactive com-pounds and has many bioactive properties [32ndash37] VariousRadix Astragali preparations are commercially availablenot only in China as a TCM component but also in theUnited States as dietary supplements [38] However due toincreasing demand substitutes and adulterants have floodedthe market Traditional identification methods such as mor-phological and microscopic methods are limited by the lackof explicit criteria for character selection or coding and thusmainly depend on subjective assessments Although chemicalmethods are able to distinguish between different species itis difficult to differentiate sibling species that possess similarchemical compositions In addition chemical methods areunable to provide accurate species authentication Severaltypes of molecular markers for characterizing genotypes areuseful in identifying plant species For example RAPD hasbeen used to estimate genetic diversity in plant populationsbased on amplification of random DNA fragments andcomparisons of common polymorphisms DNA barcoding
is advocated for species identification due to its universalapplicability simplicity and scientific accuracy Howeverthe analysis methods for DNA barcodes were limited Withthe development of molecular biology and bioinformaticsa more improved analytic method for DNA barcoding canbe established to identify Radix Astragali and closely relatedspecies
In this study we validated a new analytical method foridentifying Radix Astragali using DNA barcoding Seventy-seven specimens of Radix Astragali and its adulterants werecollected and the sequences of 29 species reported in theliterature were downloaded from the GenBank databaseBased on the 478 sequences for six barcodes (ITS2 ITS fromnuclear genome psbA-trnH rbcL andmatK fromchloroplastgenome COI from mitochondrial genome) genetic distanceand ML Tree were calculated by MEGA 60 software andidentification efficiency intra- and interspecific variationand barcoding gap were calculated using the Perl languagealgorithm Results of the six indicators assessed are shownin Table 6 ITS and psbA-trnH outperformed other barcodesin terms of identification efficiency ITS2 performed better interms of genetic distance gap rate and inter- and intraspecificvariation RbcL performed better in terms of barcoding gapand inter- and intraspecific variation Although ITS2was partof the ITS sequence it performed poorly in identificationefficiency Therefore we suggest that the ITS sequence isthe optimal barcode and that the psbA-trnH region is acomplementary barcode for identifying Radix Astragali andits adulterants
In conclusion we describe a new analytical method forthe use of DNA barcoding in the identification of Radix
Evidence-Based Complementary and Alternative Medicine 7
0102030405060708090100 COI
0
IntraspeciesInterspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
(a)
0
10
20
30
40
50
60ITS2
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(b)
0102030405060
ITS
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(c)
010203040506070
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
matK
gt02
Interspecies
(d)
0102030405060708090100
rbcL
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(e)
05101520253035 psbA-trnH
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(f)
Figure 1 Barcoding gap for six barcodes
Astragali Six indicators including average genetic distanceBLAST1 and the nearest distance method for identificationefficiency inter- and intraspecific variation and gap rate weretested to evaluate six DNA barcodes using bioinformaticssoftware and the Perl language algorithm The ITS sequence
was the optimal barcode for identifying Radix Astragali andits adulterants This method provides a novel means foraccurate identification of Radix Astragali and its adulterantsand improves the utilization of DNA barcoding in identifyingmedicinal plant species
8 Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 9
JX017329
JXJX017330
JX0101
7328
JX0177326
JX017325
JX0173324
JX0173
23JX01732
JX01732122
JF736668
0JF736665
HM1
M144
228
H M14
2289
H MM14
2287
H M142228
6H M1142
272
H42
275
H U289
274
G U289
6643
G U289666
G 28U2896623
G U 28
9661
G U 5296
60
G U8059
E
42
EF685
969
SX9SX7SX6SX5SX4SX3NM
8NM
7NM3NM2GS5GS4S2GG S1
AB787166
JF736666
65 JF736669JF736667
61
GS3GS6NM1NM10NM4NNM5NM6S3M9
SSDD1
SD 2SD 3SD 5SX 7SX810
F68AF35975
AF359749EJ572 596 0FM14 044 8
HM14 227
H227 2
HM1422776
H142278M
H1M42279
H14M2280
HM142281
HM142283
M1422
HM1422 84
H
85
HM142288
HM142293
HM142294
HM142295
HM142296
H42M1291
H42M1292
H89127Q8
17331JX0JX017327733JX01
167 2
AF12219 5
KC2675 9
F35952 1
59
AF35975
AF35973
A
B2310919951
55
73
AM142297
HF121681
A AFA
359756F359757
AB741299
AF359755
AF5
AF35975421952
9972
76
HHQ1
HHQ2
HHQ3
HQ4
HHQ5
HHQ6
HQ7
9973
EM14223
0 2H HFJ980292
F68597030
HM144230 1
HM1
32 0
HH
6XJ
9675
AF
8Q1
99
HM142298
HM142299
35975
78
CY1CY2CY
6
CY5
CY4
F68597
1
HM142
303
AB051
988
94
74 719468 80
E
9Q33
82
HQ199
316
GU217
599FJ5372
84
94
Q83 9999HQ
514
GD
78598
F032294
K
HQ1HQ1
1HQ2
HQ10HQ4HQ9Q1 64HH 2
HHQ6Q3
HQ7
142304Q8HM
HM142305JX017333
54
9991 67
JX0173348
GQ24EU591996130
60
S1S26
9999
SSDSD9D8
99
JX017337938697
3KFX0173
7336
J 5
MX
GQ1
JX01AY256392
AF053142
8854
99
4HH2HH5
HH4HH3HH6
HH1
JF461309
F461308
A
J B
6HH7
01JF4613070101
Q31198554679
65
99
95
D AH X73
72J F4195
1967
679744
E EF14
99JK2JK1
F41
EF4194797053
SX24JK14 9
E78
6099
99
ITS
HM142290
(f)
Figure 2 ML tree for six barcodes lowastThe different color and shape for different species in clusters presented the identification of differentbarcodes
Table 6 Six indicators assessed for DNA barcoding
DNA barcodes
Parameters
Average geneticdistance
Identification efficiency Gap rateInter- to
intraspecificvariation
Barcoding gap Total scoreBLAST1 Nearest
distancesITS2 8 12 8 8 8 4 48ITS 6 28 22 0 0 6 62psbA-trnH 6 26 18 0 2 2 54rbcL 4 12 14 4 6 8 48matK 4 14 24 4 4 2 52COI 2 6 10 6 0 6 30lowastThe total score of six parameters was set by 10 30 30 10 10 and 10 in order Identification efficiency based on two methods was set by 30 score because of itsimportance for identification
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
psbA-trnH successfully differentiated Radix Astragali andits adulterants Furthermore it produced areas of obviousseparation for Radix Astragali The remaining five barcodesalso differentiated Radix Astragali and its adulterants Eachspecies clustered together separate from other species Con-sidering the difficult amplification and sequencing and fastand accurate identification purpose of DNA barcoding wedid not add all the sequence data of ITS2 and psbA-trnH tobuild ML tree and subsequent analysis
4 Discussion and Conclusions
Radix Astragali is reported to possess 47 bioactive com-pounds and has many bioactive properties [32ndash37] VariousRadix Astragali preparations are commercially availablenot only in China as a TCM component but also in theUnited States as dietary supplements [38] However due toincreasing demand substitutes and adulterants have floodedthe market Traditional identification methods such as mor-phological and microscopic methods are limited by the lackof explicit criteria for character selection or coding and thusmainly depend on subjective assessments Although chemicalmethods are able to distinguish between different species itis difficult to differentiate sibling species that possess similarchemical compositions In addition chemical methods areunable to provide accurate species authentication Severaltypes of molecular markers for characterizing genotypes areuseful in identifying plant species For example RAPD hasbeen used to estimate genetic diversity in plant populationsbased on amplification of random DNA fragments andcomparisons of common polymorphisms DNA barcoding
is advocated for species identification due to its universalapplicability simplicity and scientific accuracy Howeverthe analysis methods for DNA barcodes were limited Withthe development of molecular biology and bioinformaticsa more improved analytic method for DNA barcoding canbe established to identify Radix Astragali and closely relatedspecies
In this study we validated a new analytical method foridentifying Radix Astragali using DNA barcoding Seventy-seven specimens of Radix Astragali and its adulterants werecollected and the sequences of 29 species reported in theliterature were downloaded from the GenBank databaseBased on the 478 sequences for six barcodes (ITS2 ITS fromnuclear genome psbA-trnH rbcL andmatK fromchloroplastgenome COI from mitochondrial genome) genetic distanceand ML Tree were calculated by MEGA 60 software andidentification efficiency intra- and interspecific variationand barcoding gap were calculated using the Perl languagealgorithm Results of the six indicators assessed are shownin Table 6 ITS and psbA-trnH outperformed other barcodesin terms of identification efficiency ITS2 performed better interms of genetic distance gap rate and inter- and intraspecificvariation RbcL performed better in terms of barcoding gapand inter- and intraspecific variation Although ITS2was partof the ITS sequence it performed poorly in identificationefficiency Therefore we suggest that the ITS sequence isthe optimal barcode and that the psbA-trnH region is acomplementary barcode for identifying Radix Astragali andits adulterants
In conclusion we describe a new analytical method forthe use of DNA barcoding in the identification of Radix
Evidence-Based Complementary and Alternative Medicine 7
0102030405060708090100 COI
0
IntraspeciesInterspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
(a)
0
10
20
30
40
50
60ITS2
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(b)
0102030405060
ITS
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(c)
010203040506070
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
matK
gt02
Interspecies
(d)
0102030405060708090100
rbcL
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(e)
05101520253035 psbA-trnH
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(f)
Figure 1 Barcoding gap for six barcodes
Astragali Six indicators including average genetic distanceBLAST1 and the nearest distance method for identificationefficiency inter- and intraspecific variation and gap rate weretested to evaluate six DNA barcodes using bioinformaticssoftware and the Perl language algorithm The ITS sequence
was the optimal barcode for identifying Radix Astragali andits adulterants This method provides a novel means foraccurate identification of Radix Astragali and its adulterantsand improves the utilization of DNA barcoding in identifyingmedicinal plant species
8 Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 9
JX017329
JXJX017330
JX0101
7328
JX0177326
JX017325
JX0173324
JX0173
23JX01732
JX01732122
JF736668
0JF736665
HM1
M144
228
H M14
2289
H MM14
2287
H M142228
6H M1142
272
H42
275
H U289
274
G U289
6643
G U289666
G 28U2896623
G U 28
9661
G U 5296
60
G U8059
E
42
EF685
969
SX9SX7SX6SX5SX4SX3NM
8NM
7NM3NM2GS5GS4S2GG S1
AB787166
JF736666
65 JF736669JF736667
61
GS3GS6NM1NM10NM4NNM5NM6S3M9
SSDD1
SD 2SD 3SD 5SX 7SX810
F68AF35975
AF359749EJ572 596 0FM14 044 8
HM14 227
H227 2
HM1422776
H142278M
H1M42279
H14M2280
HM142281
HM142283
M1422
HM1422 84
H
85
HM142288
HM142293
HM142294
HM142295
HM142296
H42M1291
H42M1292
H89127Q8
17331JX0JX017327733JX01
167 2
AF12219 5
KC2675 9
F35952 1
59
AF35975
AF35973
A
B2310919951
55
73
AM142297
HF121681
A AFA
359756F359757
AB741299
AF359755
AF5
AF35975421952
9972
76
HHQ1
HHQ2
HHQ3
HQ4
HHQ5
HHQ6
HQ7
9973
EM14223
0 2H HFJ980292
F68597030
HM144230 1
HM1
32 0
HH
6XJ
9675
AF
8Q1
99
HM142298
HM142299
35975
78
CY1CY2CY
6
CY5
CY4
F68597
1
HM142
303
AB051
988
94
74 719468 80
E
9Q33
82
HQ199
316
GU217
599FJ5372
84
94
Q83 9999HQ
514
GD
78598
F032294
K
HQ1HQ1
1HQ2
HQ10HQ4HQ9Q1 64HH 2
HHQ6Q3
HQ7
142304Q8HM
HM142305JX017333
54
9991 67
JX0173348
GQ24EU591996130
60
S1S26
9999
SSDSD9D8
99
JX017337938697
3KFX0173
7336
J 5
MX
GQ1
JX01AY256392
AF053142
8854
99
4HH2HH5
HH4HH3HH6
HH1
JF461309
F461308
A
J B
6HH7
01JF4613070101
Q31198554679
65
99
95
D AH X73
72J F4195
1967
679744
E EF14
99JK2JK1
F41
EF4194797053
SX24JK14 9
E78
6099
99
ITS
HM142290
(f)
Figure 2 ML tree for six barcodes lowastThe different color and shape for different species in clusters presented the identification of differentbarcodes
Table 6 Six indicators assessed for DNA barcoding
DNA barcodes
Parameters
Average geneticdistance
Identification efficiency Gap rateInter- to
intraspecificvariation
Barcoding gap Total scoreBLAST1 Nearest
distancesITS2 8 12 8 8 8 4 48ITS 6 28 22 0 0 6 62psbA-trnH 6 26 18 0 2 2 54rbcL 4 12 14 4 6 8 48matK 4 14 24 4 4 2 52COI 2 6 10 6 0 6 30lowastThe total score of six parameters was set by 10 30 30 10 10 and 10 in order Identification efficiency based on two methods was set by 30 score because of itsimportance for identification
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
psbA-trnH successfully differentiated Radix Astragali andits adulterants Furthermore it produced areas of obviousseparation for Radix Astragali The remaining five barcodesalso differentiated Radix Astragali and its adulterants Eachspecies clustered together separate from other species Con-sidering the difficult amplification and sequencing and fastand accurate identification purpose of DNA barcoding wedid not add all the sequence data of ITS2 and psbA-trnH tobuild ML tree and subsequent analysis
4 Discussion and Conclusions
Radix Astragali is reported to possess 47 bioactive com-pounds and has many bioactive properties [32ndash37] VariousRadix Astragali preparations are commercially availablenot only in China as a TCM component but also in theUnited States as dietary supplements [38] However due toincreasing demand substitutes and adulterants have floodedthe market Traditional identification methods such as mor-phological and microscopic methods are limited by the lackof explicit criteria for character selection or coding and thusmainly depend on subjective assessments Although chemicalmethods are able to distinguish between different species itis difficult to differentiate sibling species that possess similarchemical compositions In addition chemical methods areunable to provide accurate species authentication Severaltypes of molecular markers for characterizing genotypes areuseful in identifying plant species For example RAPD hasbeen used to estimate genetic diversity in plant populationsbased on amplification of random DNA fragments andcomparisons of common polymorphisms DNA barcoding
is advocated for species identification due to its universalapplicability simplicity and scientific accuracy Howeverthe analysis methods for DNA barcodes were limited Withthe development of molecular biology and bioinformaticsa more improved analytic method for DNA barcoding canbe established to identify Radix Astragali and closely relatedspecies
In this study we validated a new analytical method foridentifying Radix Astragali using DNA barcoding Seventy-seven specimens of Radix Astragali and its adulterants werecollected and the sequences of 29 species reported in theliterature were downloaded from the GenBank databaseBased on the 478 sequences for six barcodes (ITS2 ITS fromnuclear genome psbA-trnH rbcL andmatK fromchloroplastgenome COI from mitochondrial genome) genetic distanceand ML Tree were calculated by MEGA 60 software andidentification efficiency intra- and interspecific variationand barcoding gap were calculated using the Perl languagealgorithm Results of the six indicators assessed are shownin Table 6 ITS and psbA-trnH outperformed other barcodesin terms of identification efficiency ITS2 performed better interms of genetic distance gap rate and inter- and intraspecificvariation RbcL performed better in terms of barcoding gapand inter- and intraspecific variation Although ITS2was partof the ITS sequence it performed poorly in identificationefficiency Therefore we suggest that the ITS sequence isthe optimal barcode and that the psbA-trnH region is acomplementary barcode for identifying Radix Astragali andits adulterants
In conclusion we describe a new analytical method forthe use of DNA barcoding in the identification of Radix
Evidence-Based Complementary and Alternative Medicine 7
0102030405060708090100 COI
0
IntraspeciesInterspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
(a)
0
10
20
30
40
50
60ITS2
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(b)
0102030405060
ITS
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(c)
010203040506070
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
matK
gt02
Interspecies
(d)
0102030405060708090100
rbcL
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(e)
05101520253035 psbA-trnH
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(f)
Figure 1 Barcoding gap for six barcodes
Astragali Six indicators including average genetic distanceBLAST1 and the nearest distance method for identificationefficiency inter- and intraspecific variation and gap rate weretested to evaluate six DNA barcodes using bioinformaticssoftware and the Perl language algorithm The ITS sequence
was the optimal barcode for identifying Radix Astragali andits adulterants This method provides a novel means foraccurate identification of Radix Astragali and its adulterantsand improves the utilization of DNA barcoding in identifyingmedicinal plant species
8 Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 9
JX017329
JXJX017330
JX0101
7328
JX0177326
JX017325
JX0173324
JX0173
23JX01732
JX01732122
JF736668
0JF736665
HM1
M144
228
H M14
2289
H MM14
2287
H M142228
6H M1142
272
H42
275
H U289
274
G U289
6643
G U289666
G 28U2896623
G U 28
9661
G U 5296
60
G U8059
E
42
EF685
969
SX9SX7SX6SX5SX4SX3NM
8NM
7NM3NM2GS5GS4S2GG S1
AB787166
JF736666
65 JF736669JF736667
61
GS3GS6NM1NM10NM4NNM5NM6S3M9
SSDD1
SD 2SD 3SD 5SX 7SX810
F68AF35975
AF359749EJ572 596 0FM14 044 8
HM14 227
H227 2
HM1422776
H142278M
H1M42279
H14M2280
HM142281
HM142283
M1422
HM1422 84
H
85
HM142288
HM142293
HM142294
HM142295
HM142296
H42M1291
H42M1292
H89127Q8
17331JX0JX017327733JX01
167 2
AF12219 5
KC2675 9
F35952 1
59
AF35975
AF35973
A
B2310919951
55
73
AM142297
HF121681
A AFA
359756F359757
AB741299
AF359755
AF5
AF35975421952
9972
76
HHQ1
HHQ2
HHQ3
HQ4
HHQ5
HHQ6
HQ7
9973
EM14223
0 2H HFJ980292
F68597030
HM144230 1
HM1
32 0
HH
6XJ
9675
AF
8Q1
99
HM142298
HM142299
35975
78
CY1CY2CY
6
CY5
CY4
F68597
1
HM142
303
AB051
988
94
74 719468 80
E
9Q33
82
HQ199
316
GU217
599FJ5372
84
94
Q83 9999HQ
514
GD
78598
F032294
K
HQ1HQ1
1HQ2
HQ10HQ4HQ9Q1 64HH 2
HHQ6Q3
HQ7
142304Q8HM
HM142305JX017333
54
9991 67
JX0173348
GQ24EU591996130
60
S1S26
9999
SSDSD9D8
99
JX017337938697
3KFX0173
7336
J 5
MX
GQ1
JX01AY256392
AF053142
8854
99
4HH2HH5
HH4HH3HH6
HH1
JF461309
F461308
A
J B
6HH7
01JF4613070101
Q31198554679
65
99
95
D AH X73
72J F4195
1967
679744
E EF14
99JK2JK1
F41
EF4194797053
SX24JK14 9
E78
6099
99
ITS
HM142290
(f)
Figure 2 ML tree for six barcodes lowastThe different color and shape for different species in clusters presented the identification of differentbarcodes
Table 6 Six indicators assessed for DNA barcoding
DNA barcodes
Parameters
Average geneticdistance
Identification efficiency Gap rateInter- to
intraspecificvariation
Barcoding gap Total scoreBLAST1 Nearest
distancesITS2 8 12 8 8 8 4 48ITS 6 28 22 0 0 6 62psbA-trnH 6 26 18 0 2 2 54rbcL 4 12 14 4 6 8 48matK 4 14 24 4 4 2 52COI 2 6 10 6 0 6 30lowastThe total score of six parameters was set by 10 30 30 10 10 and 10 in order Identification efficiency based on two methods was set by 30 score because of itsimportance for identification
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
psbA-trnH successfully differentiated Radix Astragali andits adulterants Furthermore it produced areas of obviousseparation for Radix Astragali The remaining five barcodesalso differentiated Radix Astragali and its adulterants Eachspecies clustered together separate from other species Con-sidering the difficult amplification and sequencing and fastand accurate identification purpose of DNA barcoding wedid not add all the sequence data of ITS2 and psbA-trnH tobuild ML tree and subsequent analysis
4 Discussion and Conclusions
Radix Astragali is reported to possess 47 bioactive com-pounds and has many bioactive properties [32ndash37] VariousRadix Astragali preparations are commercially availablenot only in China as a TCM component but also in theUnited States as dietary supplements [38] However due toincreasing demand substitutes and adulterants have floodedthe market Traditional identification methods such as mor-phological and microscopic methods are limited by the lackof explicit criteria for character selection or coding and thusmainly depend on subjective assessments Although chemicalmethods are able to distinguish between different species itis difficult to differentiate sibling species that possess similarchemical compositions In addition chemical methods areunable to provide accurate species authentication Severaltypes of molecular markers for characterizing genotypes areuseful in identifying plant species For example RAPD hasbeen used to estimate genetic diversity in plant populationsbased on amplification of random DNA fragments andcomparisons of common polymorphisms DNA barcoding
is advocated for species identification due to its universalapplicability simplicity and scientific accuracy Howeverthe analysis methods for DNA barcodes were limited Withthe development of molecular biology and bioinformaticsa more improved analytic method for DNA barcoding canbe established to identify Radix Astragali and closely relatedspecies
In this study we validated a new analytical method foridentifying Radix Astragali using DNA barcoding Seventy-seven specimens of Radix Astragali and its adulterants werecollected and the sequences of 29 species reported in theliterature were downloaded from the GenBank databaseBased on the 478 sequences for six barcodes (ITS2 ITS fromnuclear genome psbA-trnH rbcL andmatK fromchloroplastgenome COI from mitochondrial genome) genetic distanceand ML Tree were calculated by MEGA 60 software andidentification efficiency intra- and interspecific variationand barcoding gap were calculated using the Perl languagealgorithm Results of the six indicators assessed are shownin Table 6 ITS and psbA-trnH outperformed other barcodesin terms of identification efficiency ITS2 performed better interms of genetic distance gap rate and inter- and intraspecificvariation RbcL performed better in terms of barcoding gapand inter- and intraspecific variation Although ITS2was partof the ITS sequence it performed poorly in identificationefficiency Therefore we suggest that the ITS sequence isthe optimal barcode and that the psbA-trnH region is acomplementary barcode for identifying Radix Astragali andits adulterants
In conclusion we describe a new analytical method forthe use of DNA barcoding in the identification of Radix
Evidence-Based Complementary and Alternative Medicine 7
0102030405060708090100 COI
0
IntraspeciesInterspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
(a)
0
10
20
30
40
50
60ITS2
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(b)
0102030405060
ITS
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(c)
010203040506070
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
matK
gt02
Interspecies
(d)
0102030405060708090100
rbcL
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(e)
05101520253035 psbA-trnH
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(f)
Figure 1 Barcoding gap for six barcodes
Astragali Six indicators including average genetic distanceBLAST1 and the nearest distance method for identificationefficiency inter- and intraspecific variation and gap rate weretested to evaluate six DNA barcodes using bioinformaticssoftware and the Perl language algorithm The ITS sequence
was the optimal barcode for identifying Radix Astragali andits adulterants This method provides a novel means foraccurate identification of Radix Astragali and its adulterantsand improves the utilization of DNA barcoding in identifyingmedicinal plant species
8 Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 9
JX017329
JXJX017330
JX0101
7328
JX0177326
JX017325
JX0173324
JX0173
23JX01732
JX01732122
JF736668
0JF736665
HM1
M144
228
H M14
2289
H MM14
2287
H M142228
6H M1142
272
H42
275
H U289
274
G U289
6643
G U289666
G 28U2896623
G U 28
9661
G U 5296
60
G U8059
E
42
EF685
969
SX9SX7SX6SX5SX4SX3NM
8NM
7NM3NM2GS5GS4S2GG S1
AB787166
JF736666
65 JF736669JF736667
61
GS3GS6NM1NM10NM4NNM5NM6S3M9
SSDD1
SD 2SD 3SD 5SX 7SX810
F68AF35975
AF359749EJ572 596 0FM14 044 8
HM14 227
H227 2
HM1422776
H142278M
H1M42279
H14M2280
HM142281
HM142283
M1422
HM1422 84
H
85
HM142288
HM142293
HM142294
HM142295
HM142296
H42M1291
H42M1292
H89127Q8
17331JX0JX017327733JX01
167 2
AF12219 5
KC2675 9
F35952 1
59
AF35975
AF35973
A
B2310919951
55
73
AM142297
HF121681
A AFA
359756F359757
AB741299
AF359755
AF5
AF35975421952
9972
76
HHQ1
HHQ2
HHQ3
HQ4
HHQ5
HHQ6
HQ7
9973
EM14223
0 2H HFJ980292
F68597030
HM144230 1
HM1
32 0
HH
6XJ
9675
AF
8Q1
99
HM142298
HM142299
35975
78
CY1CY2CY
6
CY5
CY4
F68597
1
HM142
303
AB051
988
94
74 719468 80
E
9Q33
82
HQ199
316
GU217
599FJ5372
84
94
Q83 9999HQ
514
GD
78598
F032294
K
HQ1HQ1
1HQ2
HQ10HQ4HQ9Q1 64HH 2
HHQ6Q3
HQ7
142304Q8HM
HM142305JX017333
54
9991 67
JX0173348
GQ24EU591996130
60
S1S26
9999
SSDSD9D8
99
JX017337938697
3KFX0173
7336
J 5
MX
GQ1
JX01AY256392
AF053142
8854
99
4HH2HH5
HH4HH3HH6
HH1
JF461309
F461308
A
J B
6HH7
01JF4613070101
Q31198554679
65
99
95
D AH X73
72J F4195
1967
679744
E EF14
99JK2JK1
F41
EF4194797053
SX24JK14 9
E78
6099
99
ITS
HM142290
(f)
Figure 2 ML tree for six barcodes lowastThe different color and shape for different species in clusters presented the identification of differentbarcodes
Table 6 Six indicators assessed for DNA barcoding
DNA barcodes
Parameters
Average geneticdistance
Identification efficiency Gap rateInter- to
intraspecificvariation
Barcoding gap Total scoreBLAST1 Nearest
distancesITS2 8 12 8 8 8 4 48ITS 6 28 22 0 0 6 62psbA-trnH 6 26 18 0 2 2 54rbcL 4 12 14 4 6 8 48matK 4 14 24 4 4 2 52COI 2 6 10 6 0 6 30lowastThe total score of six parameters was set by 10 30 30 10 10 and 10 in order Identification efficiency based on two methods was set by 30 score because of itsimportance for identification
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
Evidence-Based Complementary and Alternative Medicine 7
0102030405060708090100 COI
0
IntraspeciesInterspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
(a)
0
10
20
30
40
50
60ITS2
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(b)
0102030405060
ITS
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(c)
010203040506070
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
matK
gt02
Interspecies
(d)
0102030405060708090100
rbcL
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(e)
05101520253035 psbA-trnH
0
Intraspecies
0000ndash0
010
0010ndash0
020
0020ndash0
030
0030ndash0
040
0040ndash0
050
0050ndash0
060
0060ndash0
070
0070ndash0
080
0080ndash0
090
0090ndash0
100
0100ndash0
110
0110ndash0
120
0120ndash0
130
0130ndash0
140
0140ndash0
150
0150ndash0
160
0160ndash0
170
0170ndash0
180
0180ndash0
190
gt02
Interspecies
(f)
Figure 1 Barcoding gap for six barcodes
Astragali Six indicators including average genetic distanceBLAST1 and the nearest distance method for identificationefficiency inter- and intraspecific variation and gap rate weretested to evaluate six DNA barcodes using bioinformaticssoftware and the Perl language algorithm The ITS sequence
was the optimal barcode for identifying Radix Astragali andits adulterants This method provides a novel means foraccurate identification of Radix Astragali and its adulterantsand improves the utilization of DNA barcoding in identifyingmedicinal plant species
8 Evidence-Based Complementary and Alternative Medicine
Evidence-Based Complementary and Alternative Medicine 9
JX017329
JXJX017330
JX0101
7328
JX0177326
JX017325
JX0173324
JX0173
23JX01732
JX01732122
JF736668
0JF736665
HM1
M144
228
H M14
2289
H MM14
2287
H M142228
6H M1142
272
H42
275
H U289
274
G U289
6643
G U289666
G 28U2896623
G U 28
9661
G U 5296
60
G U8059
E
42
EF685
969
SX9SX7SX6SX5SX4SX3NM
8NM
7NM3NM2GS5GS4S2GG S1
AB787166
JF736666
65 JF736669JF736667
61
GS3GS6NM1NM10NM4NNM5NM6S3M9
SSDD1
SD 2SD 3SD 5SX 7SX810
F68AF35975
AF359749EJ572 596 0FM14 044 8
HM14 227
H227 2
HM1422776
H142278M
H1M42279
H14M2280
HM142281
HM142283
M1422
HM1422 84
H
85
HM142288
HM142293
HM142294
HM142295
HM142296
H42M1291
H42M1292
H89127Q8
17331JX0JX017327733JX01
167 2
AF12219 5
KC2675 9
F35952 1
59
AF35975
AF35973
A
B2310919951
55
73
AM142297
HF121681
A AFA
359756F359757
AB741299
AF359755
AF5
AF35975421952
9972
76
HHQ1
HHQ2
HHQ3
HQ4
HHQ5
HHQ6
HQ7
9973
EM14223
0 2H HFJ980292
F68597030
HM144230 1
HM1
32 0
HH
6XJ
9675
AF
8Q1
99
HM142298
HM142299
35975
78
CY1CY2CY
6
CY5
CY4
F68597
1
HM142
303
AB051
988
94
74 719468 80
E
9Q33
82
HQ199
316
GU217
599FJ5372
84
94
Q83 9999HQ
514
GD
78598
F032294
K
HQ1HQ1
1HQ2
HQ10HQ4HQ9Q1 64HH 2
HHQ6Q3
HQ7
142304Q8HM
HM142305JX017333
54
9991 67
JX0173348
GQ24EU591996130
60
S1S26
9999
SSDSD9D8
99
JX017337938697
3KFX0173
7336
J 5
MX
GQ1
JX01AY256392
AF053142
8854
99
4HH2HH5
HH4HH3HH6
HH1
JF461309
F461308
A
J B
6HH7
01JF4613070101
Q31198554679
65
99
95
D AH X73
72J F4195
1967
679744
E EF14
99JK2JK1
F41
EF4194797053
SX24JK14 9
E78
6099
99
ITS
HM142290
(f)
Figure 2 ML tree for six barcodes lowastThe different color and shape for different species in clusters presented the identification of differentbarcodes
Table 6 Six indicators assessed for DNA barcoding
DNA barcodes
Parameters
Average geneticdistance
Identification efficiency Gap rateInter- to
intraspecificvariation
Barcoding gap Total scoreBLAST1 Nearest
distancesITS2 8 12 8 8 8 4 48ITS 6 28 22 0 0 6 62psbA-trnH 6 26 18 0 2 2 54rbcL 4 12 14 4 6 8 48matK 4 14 24 4 4 2 52COI 2 6 10 6 0 6 30lowastThe total score of six parameters was set by 10 30 30 10 10 and 10 in order Identification efficiency based on two methods was set by 30 score because of itsimportance for identification
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
Evidence-Based Complementary and Alternative Medicine 9
JX017329
JXJX017330
JX0101
7328
JX0177326
JX017325
JX0173324
JX0173
23JX01732
JX01732122
JF736668
0JF736665
HM1
M144
228
H M14
2289
H MM14
2287
H M142228
6H M1142
272
H42
275
H U289
274
G U289
6643
G U289666
G 28U2896623
G U 28
9661
G U 5296
60
G U8059
E
42
EF685
969
SX9SX7SX6SX5SX4SX3NM
8NM
7NM3NM2GS5GS4S2GG S1
AB787166
JF736666
65 JF736669JF736667
61
GS3GS6NM1NM10NM4NNM5NM6S3M9
SSDD1
SD 2SD 3SD 5SX 7SX810
F68AF35975
AF359749EJ572 596 0FM14 044 8
HM14 227
H227 2
HM1422776
H142278M
H1M42279
H14M2280
HM142281
HM142283
M1422
HM1422 84
H
85
HM142288
HM142293
HM142294
HM142295
HM142296
H42M1291
H42M1292
H89127Q8
17331JX0JX017327733JX01
167 2
AF12219 5
KC2675 9
F35952 1
59
AF35975
AF35973
A
B2310919951
55
73
AM142297
HF121681
A AFA
359756F359757
AB741299
AF359755
AF5
AF35975421952
9972
76
HHQ1
HHQ2
HHQ3
HQ4
HHQ5
HHQ6
HQ7
9973
EM14223
0 2H HFJ980292
F68597030
HM144230 1
HM1
32 0
HH
6XJ
9675
AF
8Q1
99
HM142298
HM142299
35975
78
CY1CY2CY
6
CY5
CY4
F68597
1
HM142
303
AB051
988
94
74 719468 80
E
9Q33
82
HQ199
316
GU217
599FJ5372
84
94
Q83 9999HQ
514
GD
78598
F032294
K
HQ1HQ1
1HQ2
HQ10HQ4HQ9Q1 64HH 2
HHQ6Q3
HQ7
142304Q8HM
HM142305JX017333
54
9991 67
JX0173348
GQ24EU591996130
60
S1S26
9999
SSDSD9D8
99
JX017337938697
3KFX0173
7336
J 5
MX
GQ1
JX01AY256392
AF053142
8854
99
4HH2HH5
HH4HH3HH6
HH1
JF461309
F461308
A
J B
6HH7
01JF4613070101
Q31198554679
65
99
95
D AH X73
72J F4195
1967
679744
E EF14
99JK2JK1
F41
EF4194797053
SX24JK14 9
E78
6099
99
ITS
HM142290
(f)
Figure 2 ML tree for six barcodes lowastThe different color and shape for different species in clusters presented the identification of differentbarcodes
Table 6 Six indicators assessed for DNA barcoding
DNA barcodes
Parameters
Average geneticdistance
Identification efficiency Gap rateInter- to
intraspecificvariation
Barcoding gap Total scoreBLAST1 Nearest
distancesITS2 8 12 8 8 8 4 48ITS 6 28 22 0 0 6 62psbA-trnH 6 26 18 0 2 2 54rbcL 4 12 14 4 6 8 48matK 4 14 24 4 4 2 52COI 2 6 10 6 0 6 30lowastThe total score of six parameters was set by 10 30 30 10 10 and 10 in order Identification efficiency based on two methods was set by 30 score because of itsimportance for identification
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
Evidence-Based Complementary and Alternative Medicine 9
JX017329
JXJX017330
JX0101
7328
JX0177326
JX017325
JX0173324
JX0173
23JX01732
JX01732122
JF736668
0JF736665
HM1
M144
228
H M14
2289
H MM14
2287
H M142228
6H M1142
272
H42
275
H U289
274
G U289
6643
G U289666
G 28U2896623
G U 28
9661
G U 5296
60
G U8059
E
42
EF685
969
SX9SX7SX6SX5SX4SX3NM
8NM
7NM3NM2GS5GS4S2GG S1
AB787166
JF736666
65 JF736669JF736667
61
GS3GS6NM1NM10NM4NNM5NM6S3M9
SSDD1
SD 2SD 3SD 5SX 7SX810
F68AF35975
AF359749EJ572 596 0FM14 044 8
HM14 227
H227 2
HM1422776
H142278M
H1M42279
H14M2280
HM142281
HM142283
M1422
HM1422 84
H
85
HM142288
HM142293
HM142294
HM142295
HM142296
H42M1291
H42M1292
H89127Q8
17331JX0JX017327733JX01
167 2
AF12219 5
KC2675 9
F35952 1
59
AF35975
AF35973
A
B2310919951
55
73
AM142297
HF121681
A AFA
359756F359757
AB741299
AF359755
AF5
AF35975421952
9972
76
HHQ1
HHQ2
HHQ3
HQ4
HHQ5
HHQ6
HQ7
9973
EM14223
0 2H HFJ980292
F68597030
HM144230 1
HM1
32 0
HH
6XJ
9675
AF
8Q1
99
HM142298
HM142299
35975
78
CY1CY2CY
6
CY5
CY4
F68597
1
HM142
303
AB051
988
94
74 719468 80
E
9Q33
82
HQ199
316
GU217
599FJ5372
84
94
Q83 9999HQ
514
GD
78598
F032294
K
HQ1HQ1
1HQ2
HQ10HQ4HQ9Q1 64HH 2
HHQ6Q3
HQ7
142304Q8HM
HM142305JX017333
54
9991 67
JX0173348
GQ24EU591996130
60
S1S26
9999
SSDSD9D8
99
JX017337938697
3KFX0173
7336
J 5
MX
GQ1
JX01AY256392
AF053142
8854
99
4HH2HH5
HH4HH3HH6
HH1
JF461309
F461308
A
J B
6HH7
01JF4613070101
Q31198554679
65
99
95
D AH X73
72J F4195
1967
679744
E EF14
99JK2JK1
F41
EF4194797053
SX24JK14 9
E78
6099
99
ITS
HM142290
(f)
Figure 2 ML tree for six barcodes lowastThe different color and shape for different species in clusters presented the identification of differentbarcodes
Table 6 Six indicators assessed for DNA barcoding
DNA barcodes
Parameters
Average geneticdistance
Identification efficiency Gap rateInter- to
intraspecificvariation
Barcoding gap Total scoreBLAST1 Nearest
distancesITS2 8 12 8 8 8 4 48ITS 6 28 22 0 0 6 62psbA-trnH 6 26 18 0 2 2 54rbcL 4 12 14 4 6 8 48matK 4 14 24 4 4 2 52COI 2 6 10 6 0 6 30lowastThe total score of six parameters was set by 10 30 30 10 10 and 10 in order Identification efficiency based on two methods was set by 30 score because of itsimportance for identification
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
10 Evidence-Based Complementary and Alternative Medicine
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
Thanks are due to the National Natural Science Foun-dation of China (nos 81274013 8130069 and 81473315)and the National Science and Technology Major Projectsfor ldquoMajor New Drugs Innovation and Developmentrdquo (no2011BAI07B01)
References
[1] Y Kuo W Tsai S Loke T Wu and W Chiou ldquoAstragalusmembranaceus flavonoids (AMF) ameliorate chronic fatiguesyndrome induced by food intake restriction plus forced swim-mingrdquo Journal of Ethnopharmacology vol 122 no 1 pp 28ndash342009
[2] W C S Cho and K N Leung ldquoIn vitro and in vivoimmunomodulating and immunorestorative effects of Astra-galus membranaceusrdquo Journal of Ethnopharmacology vol 113no 1 pp 132ndash141 2007
[3] T T X Dong X Q Ma C Clarke et al ldquoPhylogeny ofAstragalus in China molecular evidence f rom the DNAsequences of 5S rRNA spacer ITS and 18S rRNArdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6709ndash67142003
[4] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPreparativeisolation and purification of two isoflavones from Astragalusmembranaceus Bge var mongholicus (Bge) Hsiao by high-speed counter-current chromatographyrdquo Journal of Chromatog-raphy A vol 992 no 1-2 pp 193ndash197 2003
[5] X Ma P Tu Y Chen T Zhang Y Wei and Y Ito ldquoPrepar-ative isolation and purification of isoflavan and pterocarpanglycosides fromAstragalusmembranaceusBge varmongholicus(Bge) Hsiao by high-speed counter-current chromatographyrdquoJournal of Chromatography A vol 1023 no 2 pp 311ndash315 2004
[6] P Y Yip andH S Kwan ldquoMolecular identification of Astragalusmembranaceus at the species and locality levelsrdquo Journal ofEthnopharmacology vol 106 no 2 pp 222ndash229 2006
[7] Y Z Zhao ldquoInvestigation the source and distribution of RadixAstraglirdquo Chinese Traditional and Herbal Drugs vol 35 no 10pp 1189ndash1190 2004
[8] Y H Zhang LM Zhang X B Liu et al ldquoStudy onmorpholog-ical andmicroscopic identification for different producing areasof Radix Astragalirdquo Journal of Chinese Medicinal Materials vol36 no 10 pp 1602ndash1604 2013
[9] L Wei and F T Zeng ldquoUsing thin-layer chromatography andultra-violet spectroscopy to identify Radix Astragali and itsadulterantsrdquo Journal of Chinese Medicinal Materials vol 16 no12 pp 14ndash17 1993
[10] G Li H Zhao Y Liu et al ldquoStudy on Chinese herb astragalusmembranceus by FTIR fingerprintrdquo Spectroscopy and SpectralAnalysis vol 30 no 6 pp 1493ndash1497 2010
[11] X Q Ma Q Shi J A Duan T T X Dong and K W K TsimldquoChemical analysis of Radix Astragali (Huangqi) in Chinaa comparison with its adulterants and seasonal variationsrdquoJournal of Agricultural and Food Chemistry vol 50 no 17 pp4861ndash4866 2002
[12] H J Na J Y Um S C Kim et al ldquoMolecular discrimination ofmedicinal Astragali radix by RAPD analysisrdquo Immunopharma-cology and Immunotoxicology vol 26 no 2 pp 265ndash272 2004
[13] L X Duan T L Chen M Li et al ldquoUse of the metabolomicsapproach to characterize chinese medicinal material HuangqirdquoMolecular Plant vol 5 no 2 pp 376ndash386 2012
[14] 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
[15] P D N Hebert E H Penton J M Burns D H Janzen andWHallwachs ldquoTen species in one DNA barcoding reveals crypticspecies in the neotropical skipper butterflyAstraptes fulgeratorrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 41 pp 14812ndash14817 2004
[16] S H Zheng X Jiang L B Wu Z H Wang and L F HuangldquoChemical and genetic discrimination of cistanches herba basedon UPLC-QTOFMS and DNA barcodingrdquo PLoS ONE vol 9no 5 Article ID e98061 2014
[17] L F Huang S H Zheng L B Wu X Jiang and S LChen ldquoEcotypes of Cistanche deserticolabased on chemicalcomponent and molecular traitsrdquo Scientia Sinica Vitae vol 44no 3 pp 318ndash328 2014
[18] X Q Ma J A Duan D Y Zhu T T X Dong and K W KTsim ldquoSpecies identification of Radix Astragali (Huangqi) byDNA sequence of its 5S-rRNA spacer domainrdquo Phytochemistryvol 54 no 4 pp 363ndash368 2000
[19] G Chen X L Wang W S Wong X D Liu B Xia and N LildquoApplication of 31015840 Untranslated Region (UTR) sequence-basedamplified polymorphism analysis in the rapid authentication ofRadix astragalirdquo Journal of Agricultural and FoodChemistry vol53 no 22 pp 8551ndash8556 2005
[20] J Liu H-B Chen B-L Gou Z-Z Zhao Z-T Liang and TYi ldquoStudy of the relationship between genetics and geographyin determining the quality of Astragali Radixrdquo Biological andPharmaceutical Bulletin vol 34 no 9 pp 1404ndash1412 2011
[21] Z H Cui Y Li Q J Yuan L Zhou and M Li ldquoMolecularidentification of Astragali Radix and its adulterants by ITSsequencesrdquo China Journal of Chinese Materia Medica vol 37no 24 pp 3773ndash3776 2012
[22] T Gao H Yao X Y Ma Y J Zhu and J Y Song ldquoIdentificationof Astragalus plants in China using the region ITS2rdquo WorldScience and TechnologyModernization of Traditional ChineseMedicine and Materia Medica vol 12 no 2 pp 222ndash227 2010
[23] H-Y Guo W-W Wang N Yang et al ldquoDNA barcoding pro-vides distinction between Radix Astragali and its adulterantsrdquoScience China Life Sciences vol 53 no 8 pp 992ndash999 2010
[24] CBOL Plant Working Group ldquoA DNA barcode for land plantsrdquoProceedings of the National Academy of Sciences of United Statesof America vol 106 no 31 pp 12794ndash12797 2009
[25] S L Chen H Yao J P Han et al ldquoValidation of the ITS2 regionas a novelDNAbarcode for identifyingmedicinal plant speciesrdquoPLoS ONE vol 5 no 1 Article ID e8613 2010
[26] W J Kress K J Wurdack E A Zimmer L A Weigt and DH Janzen ldquoUse of DNA barcodes to identify flowering plantsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 23 pp 8369ndash8374 2005
[27] A Keller T Schleicher J Schultz T Muller T Dandekar andM Wolf ldquo58S-28S rRNA interaction and HMM-based ITS2annotationrdquo Gene vol 430 no 1-2 pp 50ndash57 2009
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
Evidence-Based Complementary and Alternative Medicine 11
[28] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6 molecular evoluationaay genetics analysis version60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash2729 2013
[29] C P Meyer and G Paulay ldquoDNA barcoding error rates basedon comprehensive samplingrdquo PLoS Biology vol 3 no 12 articlee422 2005
[30] H A Ross S Murugan and W L S Li ldquoTesting the reliabilityof genetic methods of species identification via simulationrdquoSystematic Biology vol 57 no 2 pp 216ndash230 2008
[31] D A Morrison ldquoIncreasing the efficiency of searches for themaximum likelihood tree in a phylogenetic analysis of up to150 nucleotide sequencesrdquo Systematic Biology vol 56 no 6 pp988ndash1010 2007
[32] Y P Zhang M K Nie S Y Shi et al ldquoIntegration of mag-netic solid phase fishing and off-line two-dimensional high-performance liquid chromatographydiode array detectormassspectrometry for screening and identification of human serumalbumin binders from Radix Astragalirdquo Food Chemistry vol146 no 1 pp 56ndash64 2014
[33] X H Liu L G Zhao J Liang et al ldquoComponent analysisand structure identification of active substances for anti-gastriculcer effects in Radix Astragali by liquid chromatography andtandem mass spectrometryrdquo Journal of Chromatography B vol960 no 1 pp 43ndash51 2014
[34] C Chu H-X Cai M-T Ren et al ldquoCharacterization of novelastragalosidemalonates fromRadix Astragali byHPLCwith ESIquadrupole TOFMSrdquo Journal of Separation Science vol 33 no4-5 pp 570ndash581 2010
[35] J Fu L F Huang H T Zhang S H Yang and S LChen ldquoStructural features of a polysaccharide from Astragalusmembranaceus (Fisch) Bge var mongholicus (Bge) HsiaordquoJournal of Asian Natural Products Research vol 15 no 6 pp687ndash692 2013
[36] X Huang Y Liu F Song Z Liu and S Liu ldquoStudies on prin-cipal components and antioxidant activity of different RadixAstragali samples using high-performance liquid chromatogra-phyelectrospray ionization multiple-stage tandem mass spec-trometryrdquo Talanta vol 78 no 3 pp 1090ndash1101 2009
[37] A Nalbantsoy T Nesil O Yilmaz-Dilsiz G Aksu S Khan andE Bedir ldquoEvaluation of the immunomodulatory properties inmice and in vitro anti-inflammatory activity of cycloartane typesaponins from Astragalus speciesrdquo Journal of Ethnopharmacol-ogy vol 139 no 2 pp 574ndash581 2012
[38] W L Xiao T J Motley U J Unachukwu et al ldquoChemical andgenetic assessment of variability in commercial Radix Astragali(Astragalus spp) by ion trap LC-MS and nuclear ribosomalDNA barcoding sequence analysesrdquo Journal of Agricultural andFood Chemistry vol 59 no 5 pp 1548ndash1556 2011
