| Objective:As the main parts of Research on Ecologic Suitability and Proper Division of Production Places for Arctium Lappa (Grant No.2006BAI09B02-6), which was affiliated to the National Key Technology R&D Program and Quality Criteria Revise of Furctus Arctii and Furctus Arctii Praeparata (Grant No. YD101, YD102) for Chinese Pharmacopeia and the important content of Research on Standardization and Share of Arctium Lappa Germplasm Resource (Grant No.2005DKA21004), which was affiliated to the project Standardization and Share of Pharmaceutical Plants'Germplasm Resources funded by the Ministry of Science and Technology, the present study included resource investigation, ecologic factor analysis, quality evaluation, analysis based on ecologic suitability and TCMGIS and quality criteria revise of Furctus Arctii and Furctus Arctii Praeparata as well. By these means, breeding mothods, normalized cultivation procedure and quality evalution of Arctium lappa were carefully studied.Method and Result:Arctium lappa resource investigation and samples collecting were carried out through coherent standard. GPS meter was used to measure longitude and latitude of the collection areas. The obtained Arctium lappa germplasm resource was stored and sent to State Warehouse for Germplasm Resource. Therefore, the content related to Arctium lappa in Information Sharing System of Chinese Pharmaceutical Plants'Germplasm Resource was fulfilled. The corresponding ecologic factors such as humidity, sunshine and annual precipitation etc. were extracted from TCMGIS database. Multicriteria analysis of Arctium lappa soil samples was adopted to evaluate endophyte and soil microorganism. Data such as germination percentage,1,000-grain-weight, chromosome number, karyotype and ITS sequence were measured. By HPLC, the index components such as Arctiin were evaluated. Gray correlation analysis method was used to sort qulity of Arctium lappa germplasm resource and thus the influence to Furctus Arctii by ecologic and germplasm factors were discussed. Through statistical analysis combined with the conclusion drawn from on-the-spot investigation and ecologic suitability analysis, division of production places for Arctium Lappa and protocol for Guandali normalized cultivation were established. Quality criteria of Furctus Arctii in Chinese Pharmacopoeia (2005 edition) were revised. The new criteria of Furctus Arctii and Furctus Arctii Praeparata were established, which were also recorded in Chinese Pharmacopoeia of 2010 edition.Conclusion:By evaluating Arctium lappa germplasm resource in 51 regions from 12 provinces, we found that its market price was relatively low, wild Arctium lappa was the main resource for Fructus Arctii and the cultivated Arctium lappa was rare in herbal markets. Of all wild Arctium lappa species, Guandali possessed the largest deposit, which widely distributed in Northeast China. Arctium lappa was cultivated in Shandong and Jiangsu provinces and its root was exported to Japan, Korea and other southeast Asian countries as healthy vegetable. Dudali which was largely cultivated in Tongxiang, Zhejiang province and recorded in many documents because of its high quality was now almost absent in abovementioned places due to fast economy development of this region and its low price.Soil fungi in Arctium lappa rhizosphere includes blue mold, Mucor, Fusarium, Trichoderma etc. The toxic effect of Fusarium F130 and F131 toward Arctium lappa was found to be most obvious. Through the methods of morphology determination and ITS sequence analysis, F130 and F131 were identified as Fusarium solani.48 days later after inoculating tender seedling of Arctium lappa with Fusarium F131, the amount of Fusarium add up to 5.8×104cfu/g. By biological control experiment, we found 3512A could effectively colonize in Arctium lappa rhizosphere and inhibit Fusarium F131.57 strains of endophyte were isolated and purified from root, stem, leaf, involucre and fruit of Arctium lappa. Dry powder of Fructus Arctii was used as substrate then arctiin and arctigenin in convert incubator were determined by HPLC. As a result,6 strains of endophyte which contributes to convert arctiin were screened. The transformation efficiency of L2 was up to 76.9%. As the transformation product, arctigenin was verified by HPLC-MS. L2 was then identified as Microbacterium oxydans by means of morphous observation, 16SrDNA sequence analysis, physiological and biochemical signature analysis.Comparing 1,000-seed-weight, grain percentage, weight percent, germination vigor, germination rate among different species, Guandali was regarded as the best. Cytology research found the number of Arctium lappa somatic chromosome was 2n=36. As to its spurious breed, the number of somatic chromosome Arctium tomentosum Mill, was 2n=36, Onopordum acanthium L.2n=34, Silybum marianum Gaertn.2n=36, Amorpha fruicosa L. 2n=40. Arctium in China includes 2 species Arctium lappa and A. tomentosum Mill.and their karyotypes are both 2B. The relative length of Arctium lappa chromosome was 2n=36=6L+6M2+16M1+8S and the karyotype formula was K (2n)=2X=36=18m+18sm. The relative length of Arctium tomentosum chromosome was 2n=36=6L+4M2+20M1+6S and the karyotype formula was K(2n)=2X=36=20m+16sm. The asymmetry coefficient of Arctium tomentosum karyotype is 62.01, while that of Arctium lappa is 62.62. The overall length and long arm overall length of Arctium tomentosum were a little bigger than that of Arctium lappa. The volume of Arctium tomentosum chromosome was 5.03μm3 larger than that of Arctium lappa. Among the 18 pairs of Arctium tomentosum chromosome, m type was 10 pairs (55.56%) and sm type 8 pairs (44.44%), while Arctium lappa 9 (50%) and 9 (50%), separately.By means of molecular biology, we amplified and determined ITS sequence of Arctium lappa from 26 different sampling regions. In the sequence, diversity exists in 14 sites.26 samples were divided into 2 groups according to the phylogenetic tree and thus Arctium lappa was regarded as 2 genotypes. Through SPSS 13.0, there was significant difference between the 2 genotypes as far as the content of arctiin were concerned. Through comparison and phylogenetic tree cluster analysis, ITS sequencing to the 10 samples of Arctium lappa's spurious breed showed such method was reliable to identify the spurious breed and ITS sequence could be used as the standard sequence in molecular identification of Fructus Arctii.Data of 1000-seed weight, contents of arctiin and arctigenin by HPLC showed that Guandali had the best quality. Gray correlation analysis showed the Fructus Arctii sample collected in Meihekou, Jilin province had the best quality. The resource of Guandali was most abundant and the quality of such herbal medicine was also the best. So, Guandali was the famous-region drug for Fructus Arctii.1000-seed weight and arctiin showed significant positive correlation (r=0.774**) by quality correlation analysis. The traditional evaluation criterion was consistent with the contents of index component. Correlation analysis of Arctium lappa's vegetative organs and rhizosphere soil on 12 elements revealed that the content of element Mg and the Fructus Arctii showed significant positive correlation (r=0.419*). The content of element Mg in Fructus Arctii was much higher than that of rhizosphere soil. The average value of Mg in Fructus Arctii was 7553.59μg/g and rhizosphere soil 5923.60 ug/g, which meant Arctium lappa could enrich Mg. The content of element Ca in Arctium lappa's vegetative organs and Fructus Arctii showed significant positive correlation (r=0.446*). The content of element Fe in Arctium lappa's vegetative organs and rhizosphere soil showed significant positive correlation (r=0.407*). The content of element Pb in Fructus Arctii and rhizosphere soil showed significant positive correlation (r=0.476*). The content of elements Na, Cu, Zn, As, Cd, Hg, Mn, K in Fructus Arctii, rhizosphere soil and Arctium lappa's vegetative organs showed no correlation."The medicine is born wonderland." It was reported that the main ecological factors which influenced the Fructus Arctii quality were annual precipitation, the highest temperature and the average temperature in July. Guandali quality evaluation showed that the samples collected in Meihekou, Jilin province and Xiuyan county, Liaoning province had better qualities. The annual precipitation, the highest temperature and the average temperature in July in the above-mentioned places were 724～755 mm,26.9～27.0℃,22.3～22.9℃, respectively. While the main ecological factors in China where it is suitable for the growth of Fructus Arctii were 700～800 mm,25.1～28.0℃and 21.1～24.0℃, respectively.TCMGIS, based on basic geographic information, climate, soil data and the 3rd national Chinese medicin natural resources census, was a rapid analysis system invented by China. Including the subspecies of Guandali and Chuandali, the proper division of production places for Arctium lappa was investigated. Comparing weighting and non-weighting methods, we concluded that adopting the weighting method to calculate data would be better.To guide the choice of the places to plant pharmaceutical plants by research on ecologic suitability and proper division of production places is the basis for sustainable use of the plant resources. The production of the Chinese crude drug may influenced by economic, human and historical factors. Dudali used to be planted in Tongxiang, Zhejiang province. But due to the rapid progress in economy and the exploitation of the wild fields, Dudali has vanished in the area. Through TCMGIS, keeping in mind the local economy, culture and history, we regulated the better proper division of production places for Arctium lappa. According to the GAP regulations, we made the technology protocol for standard cultivation of Liaoning Guandali.Quality criteria research on Fructus Arctii and Furctus Arctii Praeparata was conducted. The content in pharmacopeia (2005 edition) was revised by adding the items of Furctus Arctii Praeparata, determination of water, determination of ash and assay of arctiin, etc. And these items has been included by pharmacopeia (2010 edition).
|
PDF Full Text Request