Genetic Characterization and Phytochemical Analysis of Wild and Cultivated Populations of...

Genetic Characterization and Phytochemical Analysis of Wild and CultivatedPopulations of Scutellaria baicalensis

by Su Su, Chang-Ming He, Lin-Chu Li, Jia-Kuan Chen, and Tong-Shui Zhou*

Research Center of Natural Products, School of Life Sciences, Fudan University, Shanghai 200433,P. R. China (phone/fax: þ86-21-65642206; e-mail: [email protected])

Scutellaria baicalensis was collected from four wild and four cultivated populations from differentlocations in China. Forty-two samples were analyzed using random amplification of polymorphic DNA(RAPD) techniques for genetic profiling, and high performance liquid chromatography (HPLC)techniques to determine the flavonoid content. The selected 23 RAPD primers yielded a total of838 clear and reproducible bands of which 237 were found to be polymorphic. The wild populationexhibited higher polymorphism than that of the cultivated population. The dendrogram generated by theUPGMA method via Nei9s genetic distance revealed three distinct genotypes from the cultivatedpopulations and several branches from the wild populations. The contents of baicalin and wogonoside indried roots of the samples ranged from (w/w) 8.63 to 17.84%, and from 1.99 to 4.21%, respectively,whereas their aglycones, baicalein and wogonin, were within the range of only 0.04–0.23%. The totalcontent of the four flavonoids varied from 9.45 to 26.24%. Comparatively, the cultivated populationscontained much higher levels of baicalin and total flavonoids than those in the wild populations. Theresults from genetic characterization and phytochemical analysis demonstrated that the quality variationof this drug was mainly determined by extrinsic environmental or agricultural factors, rather than bygenetic differences. Our findings can be used for the commercial production and germplasm managementof this medicinal plant.

Introduction. – Scutellaria baicalensis Georgi is a plant commonly used intraditional Chinese medicine that was first recorded in Shen Nong Ben Cao Jing in ca.100 bc. As one of the most important heat-clearing and dampness-drying drugs, roots ofthis herb (Radix Scutellariae ; Huangqin) have been widely used in the treatment ofhepatitis, jaundice, tumor, leukemia, hyperlipaemia, arteriosclerosis, diarrhea, andinflammatory diseases [1– 3]. Previous phytochemical research revealed that the mainbioactivities of this herb are attributed to baicalin and other flavone glucuronideanalogues [4– 6] (Fig. 1). These Scutellaria flavonoids have received worldwideattention in recent years due to their various interesting pharmacological effectsincluding hepatoprotection, neuroprotection, anti-inflammation, anti-cancer, anti-HIV,

CHEMISTRY & BIODIVERSITY – Vol. 5 (2008) 1353

H 2008 Verlag Helvetica Chimica Acta AG, ZJrich

Fig. 1. The main bioactive flavonoids in roots of Scutellaria baicalensis

and anti-hepatitis B virus activities [7 –10]. The levels of these constituents are used aschemical markers for the quality control and standardization of Huangqin [6] [11 – 13].

As a perennial herb of the family Labiatae, S. baicalensis is widely distributed inNorth China, Russia, Korea, and Japan [14]. The wild resources from Gansu, Shanxi,Inner Mongolia, Hebei, and Shandong provinces of China were the main traditionalorigins of this drug. In particular, the wild product from an area around Chi-Feng inInner Mongolia to Cheng-De of Hebei province (Fig. 2) was famous for its high quality,and was known as MRehe Huangqin9 [1]. In recent decades, cultivated materials haveoverwhelmingly become the major commercial supply, owing to a rapid expansion inthe global markets combined with a sharp decline of wild resources. The germplasm ofthe presently large-scale cultivated S. baicalensis was directly domesticated from localwild resources by farmers during the past 30 years. Thus, the genotypes cultivated indifferent ranges might have different wild origins and could be substantially diversifiedfrom each other. The frequent dispersal of the germplasm among different cultivationregions further increases the complexity of genotypes of the cultivated S. baicalensis.The great heterogeneity of the germplasm has been proposed as the main cause of thequality variation in the commercial product and furthermore, has jeopardized thesustainable utilization of this plant [15].

Genetic characterization of the germplasm using various molecular markers hasbeen recognized as one of the most important approaches for establishing GACP

Fig. 2. The main producing areas of Scutellaria baicalensis in China and the sampling sites of wild (WC,LH, CD, PQ) and cultivated (CF, AG, JN, JX) populations in the study. Abbreviations: WC: Wei-Chang;LH: Long-Hua; CD: Cheng-De; PQ: Ping-Quan; CF: Chi-Feng; AG: An-Guo; JN: Jiao-Nan; JX: Ju-

Xian.

CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)1354

(Good Agricultural and Collection Practices) techniques of medicinal plants [16] [17].In addition, genetic profiling based on molecular techniques also plays an increasinglysignificant role in the management and sustainable utilization of plant genetic resources[18] [19]. The germplasm heterogeneity of wild and cultivated populations of S.baicalensis has been shown by random amplified polymorphic DNA (RAPD)molecular markers [20] [21], and the quality variation with reference to differentgeographical origins among wild and cultivated root samples has been investigated byHPLC [22– 25]. However, there is no published research concerning the intraspecificgenetic variation and its relationships with the secondary metabolite contents, or theauthentic genotype and the wild origins of the cultivated populations.

In this article, genetic characterization of the wild and cultivated populations of S.baicalensis from the main producing areas in China was carried out by using RAPDmolecular markers. The variations in contents of baicalin, wogonoside, baicalein, andwogonin, the four major bioactive flavonoids of this herb (Fig. 1), were analyzed byHPLC. The aims of the study were as follows: 1) to obtain information on theintraspecific genetic variation among the wild and cultivated populations of S.baicalensis ; 2) to determine the authentic genotype and the wild origin of the cultivatedpopulations; 3) to reveal the possible relationships between the secondary metabolitecontents and the genetic characterization of the species; and 4) to provide basic geneticand biochemical knowledge for protection and sustainable utilization of thiseconomically important medicinal plant.

Results. – In the RAPD profiling, 23 primers that produced clear and reproduciblebands were selected for the amplification of all samples. The selected primersgenerated an average of 4.7 of total and 4.0 of polymorphic bands per primer (Table 1).The size of the DNA fragments ranged from 100 to 2500 bp. The maximum number offragments (8 bands) was found from primer S28, whereas the least number (1 band)was produced by primer S13 (Table 1). From a total of 838 bands, 237 polymorphicbands (37.2%) were scored from 42 individuals of the wild and cultivated populations.


Table 1. Nucleotide Sequence of RAPD Primers and Their PCR Products in This Study

Primer Sequence Tba) Npb) Primer Sequence Tba) Npb)

S1 GTTTCGCTCC 6 5 S21 CAGGCCCTTC 3 2S4 GGACTGGAGT 4 3 S22 TGCCGAGCTG 7 6S5 TGCGCCCTTC 7 7 S24 AATCGGGCG 7 6S6 TGCTCTGCCC 2 2 S25 AGGGGTCTTG 3 2S7 GGTGACGCAG 5 4 S27 GAAACGGGTG 5 4S11 GTAGACCCGT 4 2 S28 GTGACGTAGG 8 8S12 CCTTGCGCA 6 6 S29 GGGTAACGCC 4 4S13 TTCCCCCGCT 1 1 S30 GTGATCGCAG 7 5S15 GGAGGGTGTT 5 3 S32 TCGGCGATAG 5 4S16 TTTGCCCGGA 2 1 S35 TTCCGAACCC 4 3S37 GACCGCTTGT 4 4 S36 AGCCAGCGAA 2 2S39 CAAACGTCGG 7 7 Total 108 91

a) Tb: Total bands. b) Np: Number of polymorphic bands.

Comparatively, the wild population exhibited much higher polymorphism (152/341,44.6%) than that of the cultivated population (85/297, 28.6%; Table 2). Examples of theRAPD patterns generated by primers S21 and S27 are shown in Fig. 3.

Based on the observed RAPD data (present/absent, 1/0), Nei9s (1978) unbiasedgenetic distances were calculated, and an UPGMA dendrogram was generated todemonstrate the phylogenetic relationships among all populations and accessions in thestudy [26]. The result showed that, apart from accessions from populations WC and PQ,individuals belonging to one population were usually grouped together, while thegenetic divergence among different populations was supported by high bootstrapvalues (>40%; Fig. 4). Ten individuals from PQ, a wild population from Hebeiprovince, were separated into five branches, exhibiting the most genetic diversity. Four


Table 2. Geographical Location of the Studied Populations and Their Genetic Differentiation Parametersas Assessed by RAPD Molecular Markers

Population Collection site Coordinates Ssa) Tbb) Pnc) Ppd)

CD Cheng-De, Hebei Province N 116835’; E 41848’ 3 80 29 36.3PQ Ping-Quan, Hebei Province N 118846’; E 40857’ 10 100 59 59.0WC Wei-chang, Hebei Province N 117844’; E 41859’ 4 85 40 47.1LH Long-Hua, Hebei Province N 117845’; E 41859’ 4 76 24 31.6

Total wild 21 341 152 44.6

CF Chi-Feng, Inner Mongolia N 118846’; E 42806’ 8 93 41 44.1AG An-Guo, Hebei Province N 115818’; E 38822’ 4 63 6 9.5JN Jiao-Nan, Shandong Province N 119852’; E 35828’ 4 75 30 40.0JX Ju-Xian, Shandong Province N 118840’; E 35824’ 5 66 8 12.1

Total cultivated 21 297 85 28.6

Total 42 838 237 37.2

a) Ss: Sample size. b) Tb: Total bands. c) Pn: Polymorphic number. d) Pp: Polymorphic percentage [%].

Fig. 3. RAPD Amplification products generated by primers S21 and S27. a) Products of S21: Lanes 1–4are samples from LH population; Lanes 5–8 are samples from AG population. b) Products of S27: Lanes

1–5 are samples from PQ population; Lanes 6–8 are samples from LH population.

individuals from WC, another wild population from the same geographical range, weresplit into two groups. JN and JX, two cultivated populations from Shandong province,were grouped together with a high bootstrap value (99%), while the cultivatedpopulation of AG from Hebei province was genetically closely related to two wildpopulations (LH and WC) from the same province. The close relationship between thecultivated populations from Shandong (JN and JX) and Hebei (AG) was supported bya high bootstrap value (81%), while the cultivated population from Inner Mongolia(CF) was isolated from the other three cultivated populations.

The optimized HPLC method resulted in a reasonable resolution for assessingcontents of baicalin and wogonoside along with their respective aglycones, baicaleinand wogonin, in roots of this species (Fig. 5). These flavonoid compounds were furtheridentified by 1) comparing the retention times of the peaks with those of the standardseluted under the same conditions, and 2) spiking the sample with stock standardsolutions. The peaks representing flavonoids in the samples were distinct. Thecalibration curves for each authentic standard exhibited good linearity (r¼0.9999). Arecovery experiment was carried out to evaluate the accuracy of the method. Theaverage recoveries of the tested compounds were 99.7% (baicalin), 100.5% (wogono-side), 101.3% (baicalein), and 100.2% (wogonin) (n ¼ 5). All the relative standard

Fig. 4. Dendrogram generated by UPGMA method via Nei3s genetic distance based on RAPD data.Statistical support (data shown in %) of branches was evaluated with 1000 bootstrap resamples. Supportvalues >40% are indicated. Branches with lower support values (<40%) are regarded as one single

branch.


deviation (RSD) values calculated from peak areas in the tests of precision,repeatability and recovery of the method were within 3.0% (n¼5; Table 3).

Fig. 6 shows the results of the HPLC analysis of individual and total flavonoidcontents, displayed as mean values (%, w/w), and their total amount in individuals ofeach population. The results demonstrated that flavonoids in roots of this speciesmainly exist as glycosides, with baicalin as the dominant component. The contents of


Fig. 5. Typical HPLC chromatograms of pure standards mixture (1–4) (a) and sample (b) for assessingflavonoid contents in Scutellaria baicalensis. 1, Baicalin; 2, wogonoside; 3, baicalein; 4, wogonin.

Table 3. Calibration Data and Accuracy, Precision, and Repeatability of HPLC Method for QuantitativeAssessment of Active Flavonoids in Roots of Scutellaria baicalensis (n¼5)

Compounds tR [min] Regressionequationa)

Linear range[mg/ml]

Accuracyb)Recovery (RSD)

Precision(RSD)b)c)

Repeatability(RSD)b)c)

Baicalin 14.5 y¼64xþ90 9.1–332.5 99.7 (2.0) 0.92 0.79Wogonoside 21.2 y¼57xþ12 3.0–115.8 100.5 (1.3) 0.87 1.33Baicalein 27.9 y¼148xþ74 0.3–128.8 101.3 (1.2) 1.50 2.49Wogonin 37.0 y¼121xþ13 0.2–185.7 100.2 (2.4) 1.83 2.71

a) r¼0.9999 for all equations. b) Data are shown in %. c) Data are calculated from peak area.

baicalin and wogonoside in the accessions ranged from 8.63 to 17.84% and from 1.99 to4.21%, respectively. Only minor amounts of aglycones were detected in the samples,with average values ranging from 0.04 to 0.23% for each constituent. The total contentsof these four flavonoids in individuals of the studied samples ranged from 9.45 to26.24%. Comparatively, the cultivated populations contained much higher contents offlavonoids than those of the wild population. The average contents of baicalin and totalflavonoids in four cultivated populations were 15.26 and 19.42%, respectively, whilethese values in the four wild populations were only 9.5 and 11.96%. Remarkably, threecultivated populations from Hebei (population AG) and Shandong (populations JNand JX) contained 22.26, 19.39, and 20.02% total flavonoids, and 17.84, 15.95, and15.57% baicalin. These values were much higher than those of CF, a cultivatedpopulation from Inner Mongolia, which only contained 16.6% total flavonoids and11.76% baicalin.

Discussion. – The RAPD approach has been previously employed by differentauthors to elucidate the genetic heterogeneity within the cultivated and the wildpopulations of this species. Their results also showed that a high genetic variabilityexisted within both the cultivated [20] and the wild [21] populations. However, neitherthe distinct genotype classification nor the exact origin of the cultivated germplasmshave been reported due to the complexity of the detected RAPD polymorphisms, andthe lack of bootstrap support of branches in the dendrograms. In our investigation, wecompared genetic characteristics and elucidated relationships between the cultivatedand the wild populations. The average RAPD polymorphic percentage within the


Fig. 6. Flavonoid contents in roots of Scutellaria baicalensis assessed by HPLC analysis. Each valuerepresents the mean�SE of individuals within each population. For abbreviations of population names,

refer to Fig. 2.

cultivated populations was lower than those within the wild populations, implying arelatively homogenous status of the germplasm within each cultivated population. Thiswas supported by the UPGMA dendrogram which showed that accessions from thewild populations were phylogenetically much more diversified from each other thanthose from the cultivated populations. The higher genetic distances among thecultivated populations implied that the genotypes in the different cultivation areas werediversified from each other.

According to the UPGMA dendrogram, three distinct genotypes with highbootstrap support could be classified among the cultivated populations of S. baicalensis.The close correlation between the genotype classification and the geographicallocalization of populations suggested that these different cultivated genotypes werepossibly derived from their respective local wild resources. Our finding exactlyreflected the current cultivation status and the known domestication history of this drugin China. Indeed, the cultivated germplasms were directly domesticated from localresources by farmers in the different cultivation regions. There was no deliberateselection, and, therefore, populations differed substantially from place to place [15].The cultivated population from An-Guo of Hebei province was identified as genotypeA together with two wild populations from Long-Hua and Wei-Chang from the sameprovince, clearly suggesting that the wild resources around Long-Hua and Wei-Changwere the origin of this genotype. Genotype B was formed by two cultivated populationsfrom Ju-Xian and Jiao-Nan of Shandong province, but its origin was complicated. Afield investigation demonstrated that Ju-Xian, the most important cultivation countythat produced ca. 80% of the annual production of this drug, was not only an exchangecentre of the commercial product, but was also a dispersal center for seeds andseedlings of this plant. Therefore, various genotypes from other provinces wereintroduced into this site, in addition to the local domesticated germplasm. Furthermore,certain new genotypes might have generated as a consequence of hybridizing betweenthe original different genotypes. Nevertheless, the close phylogenetic relationshipsbetween genotypes A and B suggested that these two genotypes were geneticallysimilar and might share the same wild ancestry from Hebei province. Genotype Cwas composed of a single cultivated population from Chi-Feng in Inner Mongolia.The substantial isolation of genotype C from genotype A and B in the dendrogramimplied that this genotype was probably newly domesticated from the local wildresources of Inner Mongolia. This hypothesis was also supported by HPLC analysisof flavonoid contents, which showed that the three cultivated populations in geno-types A and B contained much higher baicalin and total flavonoids than those ingenotype C.

Accessions of the wild populations from Hebei province were clustered into severalbranches in the dendrogram, suggesting that the wild resources in this historicallyauthentic producing area of Rehe Huang-Qin have abundant S. baicalensis genotypes.Until now, only few genotypes have been domesticated. Our results show that moregenotypes are exploitable for cultivation and breeding from this gene pool in the future.Therefore, great efforts should be made to protect the wild resources of S. baicalensis inthis area.

Many reports in the literature have demonstrated that the contents of bioactiveflavonoids in roots of S. baicalensis varied depending on their geographical origin


[6] [22] [23]. Some other researchers have indicated that the cultivated Huangqinusually contains higher contents of baicalin and wogonoside, etc., than those from wildresources [24] [25]. The possible causes of these variations, i.e., either as a direct resultof genetic differences (intrinsic factors) or as a consequence of the impact of differentenvironmental or agricultural conditions (extrinsic factors), have not been discussed inpublished reports. In our research, the flavonoid variations among the populations werenot consistent with the classification of the genotypes, suggesting that differences in thedrugs9 quality could result from extrinsic rather than intrinsic factors. The averagecontents of baicalin and total flavonoids in three cultivated populations from genotypesA and B (AG, JN, and JX) were 15.45 and 20.56% (w/w), respectively, while thesevalues in two wild populations belonging to genotype A were only 9.37 and 11.63%. Theamount of active flavonoids in the cultivated populations increased nearly 43% overthat of the wild ancestry of the same genotype. Generally, the average content of totalflavonoids in the cultivated populations was 38% higher than that in the wildpopulations. Our finding highlighted that environmental or agricultural factors mightplay a very important role in the regulation of flavonoid biosynthesis in roots of S.baicalensis.

Conclusions. – Our results demonstrated that great genetic variations exist in wildand cultivated populations of S. baicalensis. At least three genotypes could be identifiedfrom the presently large-scale cultivated populations. The wild resources in Hebeiprovince have many genotypes, and, therefore, there is a priority to protect thesepopulations. Despite the distinct genetic differences, the variation in quality of the drugwas mainly influenced and determined by environmental or agricultural factors. Ourfindings provide useful information for the development of GACP techniques, as wellas the protection and sustainable utilization of genetic resources of this medicinal plant.The genotype classifications and their relationships with the secondary metabolitecontents need to be further investigated by using other molecular techniques and moreextensive sampling. Another challenging and interesting task in future studies isdetermining the regulatory mechanisms by which environmental or agriculturalconditions affect flavonoid biosynthesis in roots of this plant.

This work was supported by grants from the Shanghai Commission of Science and Technology (GrantNo. 03DZ19547 and 03DZ19532).

Experimental Part

1. Plant Materials. A total of 42 fresh individuals of S. baicalensis were collected in September andOctober, 2000. Samples were selected from four wild populations from Hebei province and fourcultivated populations from Inner Mongolia, Hebei, and Shandong provinces (Fig. 2 and Table 1). Thegeographical coordinates of each sampling site were recorded by portable GPS. Samples were collectedat a spatial distance of 30 m. The fresh leaves from each individual were placed in a plastic bag containingsilica gel as desiccant. The corresponding roots of the individual were wrapped in bibulous paper, takenback to the laboratory, and dried immediately to constant weight at 508. All samples of leaves and rootswere stored at �208 until analysis.

2. DNA Profiling. Template DNAs were extracted using the high salt-low pH method. Dried leaves(0.2 g) were ground in liquid N2 and then extracted with 600 ml of extraction buffer (100 mm AcONa;50 mm EDTA, pH 7.5; 500 mm NaCl; 2% PVP; 1.4% SDS). The extract was precipitated by an equal


volume of AcOK solvent (pH 4.8) and then partitioned with an equal volume of aq. phenol/CHCl3/isoamyl alcohol 25 : 24 : 1. The DNA template obtained by precipitating with �208 aq. i-PrOH (70%) wasdissolved in 30 ml of TE (1% Tris · HCl; 0.2% EDTA, pH 8.0) and quantified by UV spectrophotometry.The resuspended DNA was then diluted in sterile distilled H2O to 50 ng/ml for RAPD analysis. Twenty-three random primers were selected to assay genetic variation in the samples, based on replicableproducts and relatively high polymorphisms. The reaction was carried out in a 50-ml volume containing1�buffer, 200 mm each of dATP, dGTP, dCTP, and dTTP, 0.5 mm random primer, 50 ng of genomic DNAand 1.25 U Taq polymerase (TaKaRa Inc.). The PCR was performed in a PTC100 thermocycler (MJResearch Inc., Watertown, MA). A denaturation period of 5 min at 948 was followed by 45 cycles of 1 minat 368 and 2 min at 728, and then 10 min at 728 for final extension. An 8.0 ml aliquot of the amplificationproducts was separated by electrophoresis on a 1.5% agarose gel in 1�TBE buffer, and the DNAfingerprints were photographed by an automatic imaging system.

3. Chemical Analysis. Each dried root sample was powdered individually and 300 mg of powder wastaken from each sample. Extraction was carried out twice, each with 30 ml of MeOH/H2O 3 : 1 in anultrasonic generator for 30 min. The combined MeOH extract was filtered and evaporated to dryness invacuo. The viscous residue was dissolved in 100 ml of MeOH and filtered through a 0.45-mm Nylonsyringe filter (Millex-HN, Ireland) before injection for HPLC analysis. High-purity (> 99%) baicalin,wogonoside, baicalein, and wogonin (Tauto Biotech, Shanghai, P. R. China) were used as standards forquant. analysis of flavonoids. The standard curves were calibrated by using the linear least-squaresregression equations derived from the peak areas of five injections. The system suitability test wasperformed by making five replicate injections of the standard solution. The relative standard deviations(RSD) of these properties were used as indicators of system suitability (Table 3). The HPLC analysis wasperformed on an Agilent 1100 series system with a G1311A Quatpump, a G1314A variable wavelengthdetector, and a Chemstation (Rev. A. 10. 02; Agilent Technologies, D-Waldbronn). The mobile phase wasa gradient of MeOH (A) and H2O/H3PO4 (99.9: 0.1 (v/v)) (B). The gradient was as follows: 0–42 min,45–69% eluent A. Flow rate was 1.0 ml/min. Sample injection volume was 20 ml. The flavonoids wereseparated using a Zorbax SB-C18 column (4.6�250 mm, 5 mm), with a security guard column,Phenomenex C18 ODS (4�3.0 mm). The absorbance was recorded at 280 nm.

4. Statistical Analysis. The amplified DNA polymorphic fragments were scored as binary presence (1)or absence (0), and the data matrix of the RAPD phenotypes was assembled for further analysis.Distance estimation and UPGMA tree were generated with the PHYLIP program (version 3.63) byusing Nei9s unbiased genetic distance. Statistical support of the branches was evaluated with 1000bootstrap samples [26].

REFERENCES

[1] Editorial Board of China Herbals, MChina Herbals (Vol. 2)9, Shanghai Science & TechnicalPublisher, Shanghai, 1998, p. 1682.

[2] J.-M. Huang, C.-J. Wang, F.-P. Chou, T.-H. Tseng, Y.-S. Hsieh, J.-D. Hsu, C.-Y. Chu, Arch. Toxicol.2005, 79, 102.

[3] D. Y. Zhang, J. Wu, F. Ye, L. Xue, S. Jiang, J. Yi, W. Zhang, H. Wei, M. Sung, W. Wang, X. Li, CancerRes. 2003, 63, 4037.

[4] K. Nishikawa, H. Furukawa, T. Fujioka, H. Fujii, K. Mihashi, K. Shimomura, K. Ishimaru,Phytochemistry 1999, 52, 885.

[5] Y. Zhou, M. Hirotani, T. Yoshikawa, T. Furuya, Phytochemistry 1997, 44, 83.[6] H. Bochorakova, H. Paulova, J. Slanina, P. Musil, E. Taborska, Phytother. Res. 2003, 17, 640.[7] Y. Zhao, H. Li, Z. Gao, G. Gong, H. Xu, Eur. J. Pharmacol. 2006, 536, 192.[8] Y.-Z. Shang, H. Miao, J.-J. Cheng, J.-M. Qi, Biol. Pharm. Bull. 2006, 29, 805.[9] H. J. Heo, D.-O. Kim, S. J. Choi, D. H. Shin, C. Y. Lee, J. Agric. Food Chem. 2004, 52, 4128.

[10] M.-H. Cha, I.-C. Kim, B.-H. Lee, Y. Yoon, J. Med. Food. 2006, 9, 145.


[11] Chinese Pharmacopoeia Committee, Ministry of Public Health, People9s Republic of China,MPharmacopoeia of the People9s Republic of China9, Chemical Industry Press, Beijing, 2005, Vol. 1,p. 211.

[12] F. Ye, H. Wang, S. Liang, J. Wu, J. Shao, X. Cheng, Y. Tu, D. Zhang, Nutr. Cancer 2004, 49, 217.[13] Y. Y. Zhang, H. Y. Don, Y. Z. Guo, H. Ageta, Y. Harigaya, M. Onda, K. Hashimoto, Y. Ikeya, M.

Okada, M. Maruno, Biomed. Chromatogr. 1998, 12, 31.[14] Editorial Board of Flora, MFlora of People9s Republic of China, Vol. 65 (part 2)9. Science Press,

Beijing, 1977, p. 194.[15] Q. Yang, Y. Bai, Q.-L. Chen, Y. Zhang, Y.-H. Chen, W.-Q. Wang, Lishizhen Med. Mater. Med. Res.

2006, 17, 1159.[16] World Health Organization, MWHO guidelines on good agricultural and collection practices

(GACP) for medicinal plants9, WHO, Marketing & Dissemination, Geneva, 2004.[17] T. T. X. Dong, X. Q. Ma, C. Clarke, Z. H. Song, Z. N. Ji, C. K. Lo, K. W. K. Tsim, J. Agric. Food

Chem. 2003, 51, 6709.[18] K. Ohtsubo, S. Nakamura, J. Agric. Food Chem. 2007, 55, 1501.[19] J. Raune, A. Sonnino, MThe role of biotechnology in exploring and protecting agricultural genetic

resources9, FAO of the United Nations, Rome, 2006.[20] A.-J. Shao, X. Li, L.-Q. Huang, S.-F. Lin, J. Chen, China J. Chin. Mater. Med. 2006, 310, 452.[21] X.-F. Feng, S.-L. Hu, B.-L. Guo, J.-S. Li, Y.-N. Yan, World Sci. Technol.: Modern Tradit. Chin. Med.

Mater. Med. 2002, 4, 38.[22] X.-N. Zhou, B. Yang, China J. Chin. Mater. Med. 2006, 31, 47.[23] S.-H. Song, Y. Zhang, J.-Z. Wang, China J. Chin. Mater. Med. 2006, 31, 598.[24] S. Su, Y. Wang, J.-M. Yan, G.-Q. Li, J.-K. Chen, T.-S. Zhou, J. Fudan Univ. (Nat. Sci.) 2002, 41, 714.[25] H.-B. Li, Y. Jiang, F. Chen, J. Chromatogr., B 2004, 812, 277.[26] J. Felsenstein, Cladistics 1989, 5, 164.

Received July 29, 2007


Genetic Characterization and Phytochemical Analysis of Wild and Cultivated Populations of...

Documents

Transcript of Genetic Characterization and Phytochemical Analysis of Wild and Cultivated Populations of...