Molecular biological methods using Random Amplifica-tion of Polymorphic DNA (RAPD),1) Sequence Character-ized Amplified Region (SCAR) markers2), Restriction Frag-ment Length Polymorphisms (RFLP)3) and Amplified Frag-ment Length Polymorphisms (AFLP)4) are frequently employed to analyze the genetic variation between species orindividual organisms. Herbal medicinal products vary incomposition and properties, unlike conventional pharmaceu-tical products. Correct identification and quality control ofthe starting material is, therefore, an essential prerequisite forensuring their quality, safety and efficacy.

Most regulatory guidelines and pharmacopoeias recom-mend macroscopic and microscopic evaluation and chemicalprofiling of plant materials for purposes of quality controland standardization. Such evaluation is based on comparingvisual parameters and microscopic properties with standardreference material. However, these criteria are subjective, andsubstitutes or adulterants may closely resemble the genuinematerial. Chemical profiling methods such as Thin LayerChromatograph (TLC) and High Performance Thin LayerChromatograph (HPTLC) are routinely used for qualitativedetermination of herb medicines. Nevertheless the use ofchromatographic techniques and marker compounds to stan-dardize botanical preparations has limitations because of thevaried sources and chemical complexity of such prepara-tions. In particular, many extrinsic factors such as methods ofcultivation, harvesting, drying and storing may affect the ulti-mate chemical profile of a given herb.5) Moreover since manyassociated agents and bonding agents are mixed with herbalextracts at 90—100 °C for 2—4 h, it is difficult to identify theplants involved from the final products (extracts, capsules,liquids) by TLC.

The analysis of genomic DNA, on the other hand, has theadvantages of being applicable to all parts of a plant and not

being affected by conditions of culture.6) Genetic analysis hasbeen frequently used to accurately identifying the origin ofherbal medicines. Medicinal products whose origins areoften controversial, such as Angelica gigas root, Saposh-nikovia root, Coix seed, Citrus unshiu peel, Angelica decur-siva radix, Magnolia bark, Polygonatum rhizome, and Bu-pleurum root, have been intensively studied and other herbalmedicines have also been analyzed by RAPD.6)

Generally, DNA is unstable to heat treatment and de-stroyed at temperatures over 75 °C. Hence, in the case ofherbal medicine extracts that are prepared at a temperature inexcess of 90 °C for two hours, most of the DNA should bemodified or damaged even though some DNA may remain.Therefore, it has been considered impossible to trace the ori-gin of plant extracts by molecular biological methods such asPCR and there have been few if any attempts to use PCR toidentify the origin of herbal medicine preparations. However,as a result of the progress in molecular biological methods,PCR approaches for detecting Genetically Modified Organ-isms (GMO) have recently been developed.7) In GMO Prod-ucts, only specific genes of bean and corn were amplified byPCR, and thereby this result verified the existence of GMO.PCR methodology has been used to identify GMOs not onlyin raw materials but also in manufactured food products thathave been treated at 122 °C for 12 min and at 132 °C for 16 s.7)

We previously extracted, and amplified by RAPD, genomicDNA from the delegate Ginseng of each country: Panax ginseng (Korean), Panax notoginseng (BURK) F. H. CHENG

(Chinese), Panax japonicus (Japanese), and Panax quinque-folius L. (American). We identified OPA 5 (Operon randomprimer type A 5) that reacts specifically with the Panaxspecies of each country.8) We sequenced the 359 bp fragmentamplified by OPA 5 and identified a Ginseng marker primer

April 2005 Biol. Pharm. Bull. 28(4) 671—676 (2005) 671

∗ To whom correspondence should be addressed. e-mail: hongjink@cau.ac.kr © 2005 Pharmaceutical Society of Japan

Identification of Panax Species in the Herbal Medicine Preparations UsingGradient PCR Method

Young Hun SHIM,a Chan Dong PARK,c Do Hoon KIM,c Jung Hee CHO,c Myung Hwan CHOb, and

Hong Jin KIM*,a

a College of Pharmacy, Chung Ang University; Seoul 156–756, Korea: b Department of Biology, Konkuk University; Seoul143–701, Korea: and c Korea Foood & Drug Administration; Seoul 122–704, Korea.Received December 1, 2004; accepted January 19, 2005

In order to identify the existence of Panax species in herbal medicine preparations, the Ginseng specificmarker primer was selected and created based on the sequence of Korean ginseng DNA fragment, 359 bp. Thegradient PCR was performed on 40 types of the herbal medicines including the 7 types of Araliaceae that are inthe same family with the Panax ginseng using the created Ginseng maker primer. As result, Panax notoginseng(Chinese), Panax japonicus (Japanese) and Panax quinquefolius (American), along with Panax ginseng (Korean)were the only ones amplified. However, in the case of Atractylodes lancea, one of the herbal medicines not catego-rized as Panax species, the DNA was prominently amplified by the Ginseng marker primer. The sequence of theamplified DNA of Atractylodes lancea was identified, resulting in enabling the differentiation from the Panaxspecies by the Restriction Fragment Length Polymorphisms (RFLP) method. In addition, the results of the gradi-ent PCR performed on the herbal medicine preparations that consists of Panax ginseng showed that 290 bp sizeof the original DNA fragments of Panax ginseng was amplified on the herbal medicine preparations containingPanax ginseng. Therefore, these results suggest a possibility of creating a new testing method for identifying spe-cific herb medicines using the gradient PCR, a molecular biological method not only on Panax ginseng, but alsoon other herbal medicines and herbal medicine preparations.

Key words Panax species; PCR; herbal medicine; Ginseng maker primer

(SIM2) that specifically amplified that fragment from theDNA of Panax species.

In this study, we show that gradient PCR using the SIM2primer can uniquely identify Panax species in herbal medi-cines, and herbal medicinal preparations containing diversecomponents.

MATERIALS AND METHODS

Plant Materials The roots of six types of Panax speciessuch as the Panax Ginseng C. A. MEYER (Korean), Panaxnotoginseng (BURK) F. H. CHENG (Chinese), Panax japonica(Japanese), and Panax quinquefolium L. (American, Cana-dian) were used in this experiment. Among these, the Panaxnotoginseng (Chinese: circulating product in the marketplace) had an obscure origin, but was compared to otherPanax species. In the case of the Panax quinquefolius(American), the verified standard samples were provided touse in the experiment by the Korea ginseng & Tobacco research institute. The Panax japonicus (Japanese) were usedin the experiment after an expert confirming those bought inJapan. Panax species were identified by discrimination specialist—Professor Chang Soo Yook (Kyung Hee Univer-sity). The Panax species of each country used in this experi-ment were grinded finely and filtered by a size 100 sieve, andonly the finest powder was used. In addition, for the herbalmedicine preparations, 14 items including the Bak-Ho-Ga-Insam-Tang (白虎加人蔘湯), a resource containing Panaxginseng, and 16 items including Eun-Kyo-San (銀翹散), a resource without ginseng, all of which being available in themarketplace, were bought from Korean pharmacy (BoryungPharmacy, Seoul) and used in the experiment.

DNA Purification In order to isolate DNA in herbalmedicinal preparations, the preparations were grinded intopowder. 5—10 g of each sample powder was extracted in 100 ml of distillated water for 15 min by ultra-sonication andcentrifuged for three times. The DNA in the dried pellet wasisolated using CTAB buffer method.8,9)

Cloning of the DNA Fragment of Panax ginseng Ampli-fied by OPA-5 and Its Sequencing The DNA fragment(359 bp) of Panax ginseng amplified by Operon randomprimer Type A5 (OPA5) was cloned using PCR-TOPOcloning kit (Invitrogen, U.S.A.) and its sequences were iden-tified using automatic DNA sequencing method.8)

Ginseng Marker Primer Three ginseng marker primers(SIM2, SIMGS, SIMSP) based on the known Panax ginsenggene region8) were synthesized using “Primer3” primer syn-thesizing program. SIMSP primer (Forward: 5�-AGGGGTC-TTGCTATAGCGGAAC-3�, Reverse: 5�-AGTCTTAATTTC-ATATTTTCGTATG-3�), SIMGS primer (Forward: 5�-CTA-TAGCGGAACACGAGGGA-3�, Reverse: 5�-AGTTCGCC-ACCAACTGTAGC-3�) and SIM2 primer (Forward: 5�-CT-ATAGCGGAACACGAGGGA-3�, Reverse: 5�-ATACCAAG-CGCTCGCTAATG-3�) were synthesized for the amplifica-tion of DNA fragment 359 bp, 210 bp and 290 bp of Panaxginseng, respectively (Fig. 2).

Gradient PCR on Herbal Medicinal Preparations Con-taining Ginseng Species PCR was performed in herbalmedicine preparations using Ginseng marker primer (SIM2).The final 20 m l of the volume were used for the reactionusing PCR pre-mix kit for the gradient PCR. The initial cycle

was 5 min at 94 °C for predenaturation (1st step), and fol-lowed by 35 cycles of 1 min at 94 °C for denaturation (2nd step), reacted 30 s at 50—60 °C of 12 steps (3rd step)and 2 min at 72 °C for extension. The gradient PCR annealing temperatures of 12 steps are ①50.0 °C, ②50.3 °C, ③ 50.9 °C, ④ 51.7 °C, ⑤ 52.8 °C, ⑥ 54.3 °C, ⑦ 56.0 °C, ⑧57.4 °C, ⑨58.5 °C, ⑩59.3 °C, ⑪59.8 °C, ⑫60.0 °C. Theamplified DNA fragments were separated on 1.5% agarosegel and visualized with ethidium bromide staining.

RESULTS AND DISCUSSION

Cloning and Sequencing of OPA5 Product The Panaxginseng DNA fragment (359 bp) that were amplified by theOPA5 primer were cloned into the TA cloning vector and theamplified DNA region of the Panax species was sequenced.The homology of the identified sequence region on Panaxspecies was searched in Gene bank and the result indicates a91% homology with the tobacco chloroplast DNA (Fig. 1).In addition, the identified sequence of Panax ginseng DNAfragments (359 bp) are generally the DNA region existing inthe chloroplast of plants, also exhibiting a 90% homologywith the chloroplast of a plant known other than the Tobacco(Accession Number X12745 M28017),『Atropa BelladonnaLinne』(Accession Number AJ316582) (Fig. 1). The verifiedDNA fragments of the Panax ginseng were searched for themost suitable primer compositing location by utilizing theprimer 3 program, and three types of Ginseng markerprimers (SIMGS, SIM2, and SIMSP) were synthesized.

Preparation of Ginseng Maker Primer The SIMSPprimer was designed to amplify the entire 359 bp Panax ginseng DNA fragment (Fig. 2), the SIMGS primer to amplify a shorter region of about 210 bp, and the SIM2primer to amplify about 290 bp (Fig. 2). Panax ginseng be-longs to the Araliaceae. To test the reliability of theseprimers we extracted DNA from 7 varied Araliaceae, i.e.,Acanthopanax divaricatus, Acanthopanax senticosus, Araliaelata, Acanthopanax sessiliflorum, Acanthopanax senticosus,Acanthopanax gracillstylus, and Acanthopanax japonicus(Japanese) as well as 40 types of medicinal plants, and amplified them using 3 of the three Ginseng marker primers.The 290 bp Panax ginseng fragment was specifically ampli-fied from Panax giseng by the SIM2 primer (Figs. 3a, b), andthe DNAs extracted from the Bupleurum falcatum andAtractylodes rhizome also generated a small amount of prod-uct (Fig. 3a). No PCR product was obtained with well-known medicinal plants such as Glycyrrhiza glabra, Cin-namomum cassia, and Angelica gigas (Fig. 3b). However,Atractylodes rhizome appeared a strong DNA band unlikeBupleurum falcatum. In order to discriminate between thePCR product of Panax ginseng (A) and that of Atractylodesrhizome (C) we analyzed the sequences of the two DNA frag-ment They turned out to be 99% homologous, but we found aHpyCH4 IV restriction enzyme site in the 1% of Atractylodesrhizome DNA not homologous with P. ginseng (Fig. 4).Therefore, the products amplified by SIM2 from Panax gin-seng and Atractylodes rhizome can be distinguished by RFLPanalysis (Fig. 5).

Identification of Panax ginseng in Herbal MedicinePreparations with Ginseng Marker Primer (SIM2) Theextract was made from the mixed medicinal plants according

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to the prescription by abstracting for 3 h in 90—100 °C asdoes pharmaceutical companies. The DNA was isolated fromthe extract and amplified with the Ginseng marker primer(SIM2). The prescriptions used in this experiment were thePanax ginseng Single-Taste decocted medicine and thePanax ginseng Compound decocted medicine called Sok-Moung-Tang (續命湯) (Life-Continuing decocted medicine).Between these, Sok-Moung-Tang (續命湯) was decocted intwo respective pots; one with the original prescribed amountof Panax ginseng and the other negative of any Panax gin-seng whatsoever. The two were compared to each other bythe gradient PCR method. As a result, in the Panax ginsengSingle-Taste decocted medicine that has only Panax ginsengas the ingredient, and in the Panax ginseng Compound decocted medicine (Sok-Moung-Tang (續命湯) the 290 bpDNA fragments were amplified at 50—60 °C, while in theSok-Moung-Tang (續命湯) without the Panax ginseng notamplified (Fig. 6). In addition, in the extract granulations ofprescriptions such as the currently marketed Bak-Ho-Ga-Insam-Tang (白虎加人蔘湯) etc., the DNA was amplifiedusing the gradient PCR as the same conditions of above. Asresult, all of the 14 herbal medicine preparations containingPanax ginseng , i.e., Ga-Mi-Gue-Bi-Tang (加味歸脾湯), Bo-Jeng-Ex-Gi-Tang (補中 氣湯), Dae-Gun-Chung-Tang (大建中湯 ), Shib-Jun-Dae-Bo-Tang (十全大補湯 ), Lee-Chung-

Tang (理中湯), Insam-Pae-Dok-Tang (人蔘敗毒湯), Youk-Goon-Za-Tang (六君子湯), Bak-Ho-Ga-Insam-Tang (白虎加人蔘湯), So-Poong-San ( 風散), Chun-Sung-Whan (天醒丸), Maecmoondong-Tang (麥門冬湯), Sok-Moung-Tang (續命湯), Siho-Ga-Yongol-Moryu-Tang (柴胡加龍骨蠣湯), andSa-Gun-Za-Tang (四君子湯), revealed the amplification of290 bp Panax species DNA fragments (data not shown). Inaddition, there are 16 herbal medicine preparations non-con-taining the Panax ginseng including Kwak-Hyang-Jung-Gi-San ( 香正氣散), Dae-Chung-Yong-Tang (大 龍湯), Dae-Siho-Tang (大柴胡湯), Do-Haek-Seng-Gi-Tang (桃核承氣湯),Dangki-Jakyak-Tang (當歸芍藥湯), Wal-Bi-Ga-Chool-Tang(越婢加朮湯), On-Chung-Em ( 飮), Daehwang-Mokdan-phi-Tang (大 牧丹皮湯), Eun-Kyo-San (銀翹散), Galgun-Tang ( 根湯 ), Ma-Haeng-Gam-Suk-Tang ( 杏甘石湯 ),Siho-Gaegi-Gunkang-Tang (柴胡桂枝薑乾湯), Kwak-Hyang-Jung-Gi-San ( 香 正 氣 散 ), Bangpoong-Tong-Sung-San (防風通 散), Ga-Mi-So-Yo-San (加味逍遙散), An-Jung-San(安中散), and Gaegi-Ga-Yongoal-Moryo-Tang (桂枝加龍骨牡蠣湯). Among them, 12 herbal medicine preparations exclud-ing Bangpoong-Tong-Sung-San (防風通 散), An-Jung-San(安中散 ), Dae-Siho-Tang (大柴胡湯 ), and Siho-Gaegi-Gunkang-Tang (柴胡桂枝薑乾湯) (Fig. 7b) showed no signalsof the amplified 290 bp Panax species DNA fragment. Thus,Bangpoong-Tong-Sung-San (防風通 散 ), Dae-Siho-Tang(大柴胡湯), Siho-Gaegi-Gunkang-Tang (柴胡桂枝薑乾湯),An-Jung-San (安中散), and others are Panax ginseng non-consisting and still showed the amplified 290 bp Panaxspecies DNA fragments (Fig. 8). In the case of the Panaxginseng non-containing medicine preparation, when theDNA was amplified using the gradient PCR method in 1—3specific temperature range among 12 steps of temperaturerange, the amplified band could be a false-positive. However,in the herbal medicine preparations containing Panax gin-

April 2005 673

Fig. 2. Schimatic Diagrams of Panax ginseng Marker Primers

Fig. 1. Comparision of Tobacco Chloroplast DNA and Panax ginseng DNA Fragment

The DNA sequence of tabacco chloroplast was obtained in Gene bank (1092735151–5039–28257014553.BLASTQ4).

seng the 290 bp Panax species DNA fragments were ampli-fied in broad annealing temperature ranges. Therefore, thefalse-positive reaction was discriminated by the annealingtemperature range of the gradient PCR method. From theidentification method of Panax ginseng using the GradientPCR method, the most suitable annealing temperature for theSIM2 primer used as a Ginseng marker was 57 °C. On thegradient PCR temperature range between 50 and 60 °C, 12steps of the annealing temperature were determined based onthis range. Therefore, as a result to the gradient PCR, the290 bp DNA fragments of the Panax ginseng were amplifiedequivalently overall in 50—60 °C (Fig. 7a), while the Panaxginseng 290 bp DNA fragments not amplified on the non-containing resource (Fig. 7b). Morever, when the 290 bpDNA fragments were amplified only in a specific tempera-ture, the amplification result of Panax ginseng non-contain-ing medicine preparations was interpreted as a false-positive(Fig. 8).

The gradient PCR analysis was used to identify Panax gin-seng in herbal medicine preparations using Ginseng markerprimer (SIM2). In the samples of some herbal medicinepreparations the Panax ginseng DNA fragments were not

amplified because of the DNA sample concentration andPCR conditions. The longer the annealing time of the PCRwas, the less the specification of the amplification was (datanot shown). The DNA was amplified most sensitively by theSIMGS among the Ginseng marker primers but the medici-nal plants other than the Panax species were also amplifiedfrequently (data not shown). The SIMSP was synthesized tobe able to amplify the largest DNA fragment (359 bp) amongthe Ginseng marker primer (Fig. 2), but had the problem ofamplifying only the DNA of the Panax species and lost thesensitivity to the herbal medicine preparations containingPanax ginseng. SIM2 primer used in this experiment wassynthesized to amplify the DNA fragment (290 bp). ThePanax ginseng specific DNA fragments were amplified pre-cisely in the herbal medicines and herbal medicine prepara-tions. Therefore, the SIM2 primer was chosen for the ampli-fication in this experiment. Because it was difficult to mea-sure the Panax ginseng DNA concentration among the totalDNA concentration extracted from herbal medicine prepara-tion, it was impossible to establish the fixed PCR reactionconditions. To overcome the problems of the PCR condition,the gradient PCR method was performed in this experimentby changing the annealing temperature in a certain thermo-static range.

The established Ginseng marker primer (SIM2) was usedto perform PCR on 40 types of medicinal plants includingthe 7 types of Araliaceae currently used as medication, andas a result, the DNA fragments of Bupleurum falcatum andAtractylodes rhizome were amplified. However, in the case ofBupleurum falcatum, the amplified DNA fragment was notreproducible and the intensity of the amplified band is alsoextremely low (Fig. 3a). Therefore, Panax ginseng was easilydiscriminated from the Bupleurum falcatum by the gradientPCR method using the SIM2 primer. A portion of theAtractylodes rhizome gene was prominently amplified and itwas undefinable from the Panax ginseng with only the PCRresults. However, the Panax species were differentiated bythe RFLP method (Fig. 4). In addition, the herb medicinecomponents were mixed and extracted in 90—100 °C for 3 h as same condition of the pharmaceutical companies man-ufacturing process. The DNA of the Panax species was am-plified using the gradient PCR to distinguish the Panaxginseng consisting from the Panax ginseng non-consistingamong the extract granulate currently circulating.

The results of the gradient PCR indicate that for the Panaxginseng consisting items, the 290 bp original DNA fragmentsof the Panax ginseng were amplified evenly in all 12 steps oftemperature (Fig. 7a), while for the Panax ginseng non-con-taining items the DNA not amplified (Fig. 7b). In addition, inthe result of the gradient PCR, the 290 bp DNA fragments inthe preparations not containing the Panax ginseng that wereamplified in a specific temperature of only 1—3 steps amongthe 12 steps was considered as a false-positive reaction (Fig. 8). Therefore, Panax ginseng consisting items could beeasily distinguished from the Panax ginseng non-consistingitems by the number of the amplified bands of Panax speciesDNA fragments. The one possibility of the DNA amplifica-tion result in the Panax ginseng non-consisting items is thatthe partial damage and mutation occur during the DNA extraction so that the non-specific DNA was amplified by theGinseng marker primer. It is presumed that there are many

674 Vol. 28, No. 4

Fig. 3. PCR Analysis on Herbal Medicinal Plants Using Ginseng MarkerPrimer (SIM2)

The amplified products were separated on agarose gel. *: 1 kb DNA ladder marker,(a) 1: P. japonicus, 2: P. quinquefolius (American), 3: P. quinquefolius (Canadian), 4: P.quinquefolius (American), 5: Atractylodes japonica, 6: Pinellia ternata, 7: Bupleu-rum falcatum, 8: Perilla frutescens, 9: Paeonia lactiflora, 10: Atractylodes lancea, 11: Cnidium officinale, 12: Alisma orientale, 13: Schizandra chinensis, 14: Scutellariabaicalensis, 15: Astragalus membranaceus, 16: Citrus unshiu, 17: Gardenia jasmi-noides, 18: Magnolia ovobata, 19: Tribulus terrestris (underline represents positive forthe amplification) and (b) 1: P. ginseng, 2: P. ginseng (Chinese), 3: P. notoginseng, 4: Acanthopanax sessiliflorum, 5: Acanthopanax senticosus, 6: Fatsia japonica, 7: Cni-dium officinale, 8: Zingiber officinale, 9: Asiasarum heterotropoides, 10: Lonicerajaponica, 11: Platycodon grandiflorum, 12: Glycyrrhiza glabra, 13: Cinnamomum cas-sia, 14: Angelica gigas, 15: FRheum palmatum, 16: Liriope platyphylla, 17: Mentha arvensis, 18: Poria cocos, 19: Saposhnikovia divaricata (underline represents positivefor the amplification).

medicinal plants that have an analogous gene to the Panaxspecies and could react to Ginseng marker primer among the540 species of the medicinal plants that were not able to beestablished by this experiment. Therefore, it is consideredthat if the study on Panax ginseng genes is extensively con-tinued it is possible to find a new Ginseng marker primer thatcan respond to only Panax species, and that the commercialutilization of the such items as the Panax species identifica-tion kit would be possible.

It is possible to establish a scientific identification methodof herbal medicinal plants and their preparations overcomingthe limits of the TLC method currently utilized in the identi-fication methods for the herbal medicine preparations. Theherbal medicine preparations can especially be affected by

the spots, color, and processing length through accessoryagents and holding agents added along with the main compo-nents, and the more main substances there are, the more similar the color and processing length are shown. If it ispossible to overcome the disadvantages of the mechanicalidentification tests and to develop novel molecular biologicalmethods such as advanced PCR method, it could be a revolu-tional testing method of the herbal medicine preparationsidentification test. In addition, it could be utilized as the examiner of the other high-priced herbal medicines with issues of origins being criticized, starting with this paper as atriggering point. If the study on the identification test ofherbal medicine preparations using such methods as theRAPD and gradient PCR is continued, it is assumed to beable to be applied on all other herbal medicine preparationsin future.

April 2005 675

Fig. 5. RFLP Analysis of HpyCH4 IV on the DNA Fragments for PanaxSpecies and Atractylodes lancea Amplified by SIM2 Primer

The amplified products were separated on agarose gel. *: PCR marker, 1: P. ginsengDNA fragment amplified by SIM2 primer, 2: P. ginseng, 3: P. ginseng (Chinese), 4: P. notoginseng, 5: P. japonicus, 6: P. quinquefolius (American), 7: P. quinquefolius(Canadian), 8: P. quinquefolius (American), 9: Atractylodes lancea, 10: Bupleurum falcatum. In lane 10 20–fold more DNA amounts were loaded on agarose gel than lane1 to 9.

Fig. 6. Gradient PCR Analysis for Sok-Myung-Tang (Extract with Manual:Not Contained Panax ginseng)

The amplified products were separated on agarose gel. *: 1 kb DNA ladder marker,annealing temp. 50—60 °C, 12 steps (1: 50.0 °C, 2: 50.3 °C, 3: 50.9 °C,4: 51.7 °C, 5: 52.8 °C, 6: 54.3 °C, 7: 56.0 °C, 8: 57.4 °C, 9: 58.5 °C, 10: 59.3 °C, 11: 59.8 °C, 12: 60.0 °C).

Fig. 4. Comparision of the Sequences for Panax ginseng, Panax quinquefolium (American) and Atractylodes lancea

A: Panax ginseng (Korean), B: Panax quinquefolius (American), C: Atractylodes lancea.

The results of this study suggest a possibility that to iden-tify the genetical characteristics of respective herbal medi-cine makes possible to develop specific marker primers thatreact to certain herbal medicine, presenting a possibility ofdeveloping a new identification method for many herbalmedicines at once during herbal medicine preparationsthrough the gradient PCR methods. If the marker primer fora certain herbal medicine is continuously developed in future, it is presumed that there shall be great achievementson reaching a new level of quality control for herbal medici-nal preparations by creating items such as identification DNAchips.

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Fig. 7. Gradient PCR Analysis for (a) Siho-Ga-Yongol-Moryu-Tang (Extract Granulation: Contained Panax ginseng) and (b) Kwak-Hyang-Jung-Gi-San (Extract Granulation: Not Contained Panax ginseng)

The amplified products were separated on agarose gel. *: 1 kb DNA ladder marker,annealing temp. 50—60 °C, 12 steps (1: 50.0 °C, 2: 50.3 °C, 3: 50.9 °C, 4: 51.7 °C, 5: 52.8 °C, 6: 54.3 °C, 7: 56.0 °C, 8: 57.4 °C, 9: 58.5 °C, 10: 59.3 °C, 11: 59.8 °C, 12: 60.0 °C).

Fig. 8. Gradient PCR Analysis for Bang-Poong-Tong-Sung-San (ExtractGranulation: Not Contained Panax ginseng)

The amplified products were separated on agarose gel. *: 1 kb DNA ladder marker,annealing temp. 50—60 °C, 12 steps (1: 50.0 °C, 2: 50.3 °C, 3: 50.9 °C, 4: 51.7 °C, 5: 52.8 °C, 6: 54.3 °C, 7: 56.0 °C, 8: 57.4 °C, 9: 58.5 °C, 10: 59.3 °C, 11: 59.8 °C,12 : 60.0 °C).