Study On Guangdong Authentic And Superior Medical Material Pogostemon Cablin By GC-MS Fingerprinting And DNA Molecular Markers Analysis

Objective: Pogostemon cablin (Blanco) Benth., a well-known Chinese herbal medicine and a kind of flavor, belonging to genus Pogostemon in Lamiaceae, is also called Ciruicao or Huoxiang. And its dry stem and leaf are both medical parts for clinical use. Herb patchouli is one of aromatic herbs which is pungent-warm in properties, and attribute to lung, spleen and stomach. It is well accepted that herb patchouli can aromatically transform turbid dampness, harmonizes middle jiao, stop vomiting, release exterior dampness and summer-heat attack. Thus it has been used to remove dampness, relieve summer-heat, exterior syndrome, stop vomiting and stimulate appetite. Moreover, herb patchouli is one of the important components in more than 30 traditional Chinese patent medicines.Pogostemon cablin has been cultivated for more then 1000 years in Southern China, which was introduced from the countries in south-east Asia such as Philippine, Malaysia and India in the Song dynasty. There are distribution in Guangzhou, Zhaoqing, Zhanjiang in Guangdong province, Guangxi province, Fujian province and Taiwan province. Commercial herbs of Pogostemon cablin are divided into Paixiang (cultivated in Guangzhou), Zhaoxiang (cultivated in Zhaoqing), Zhanxiang (cultivated in Zhanjiang) and Nanxiang (cultivated in Hainan), and there are distinct morphological characteristics among samples cultivated in different localities. Generally, Paixiang and Zhaoxiang are treated as high quality unadulterated herb, while Zhanxiang and Nanxiang are just used to extract patchouli oil.Nowadays, Shipai huoxiang is going to be on the edge of extinction because of the rapid citical construction course. Therefore it is very important that we should protect the rare cultivar. In this study, 23 samples collected in different localities are analyzed by chemical techniques and molecular methods, and thus chemical and molecular characteristics of Pogostemon cablin have been found out, which can provide reliable evidences to distinguish Pogostemon cablin and its adulterates, even can provide information of unadulterated samples estimation.Methods: The thesis includes four parts. (1) The first part of the thesis focuses on the establishment of GC/MS analytical methods for the quantification of these two components in Herba Pogostemonis. In GC/MS method, target ion quantification was used after the separation by temperature gradient program. 18 samples of Pogostemon cablin were obtained and used in the studies. The comparisons among different kinds of Herba Pogostemonis from different habitats were made by using the GC/MS methods. (2) The second part of the thesis described the hierarchical clustering analysis based on characteristics of the investigated peaks in GC profiles. The simulative mean chromatogram for the different types of herb patchouli was generated using the Computer Aided Similarity Evaluation System. (3) The reaction of random amplified polymorphism DNA (RAPD) was processed in the third part of the thesis. 23 samples of Pogostemon cablin and 2 more samples P. chinensis C. Y. Wu et Y. C. Huang(PCH)and Pogostemon auricularius(Linn.)Hassk(.PAU)in genus Pogostemon were analyzed by RAPD reaction with 30 random primers, then 6 of 30 primers S42,S43,S224,S227 and OPC-19 were selected to use in the further analysis, and the cluster analysis was processed on the basis of RAPD fingerprinting. (4) In the fourth part, DNA sequencing analysis was applied to clarify the molecular characteristics of P. cablin in different localities. 18S rRNA, matK gene, ITS region and 5S spacer region were sequenced and compared, then cluster analysis was conducted based on all the information of sequences.Results: (1) The results showed that GC-MS coupled with PLE offered a simple, rapid and high sensitive method to analyze components in herb patchouli. And the contents of investigated compounds in herb patchouli,obtained from different places, were high variant. (2) 18 samples of Pogostemon cablin could be divided into three main clusters, patchouliol-type, pogostone-type and the third one between the two chemotypes. The simulative mean chromatogram for the three types of herb patchouli was generated using the Computer Aided Similarity Evaluation System. The fingerprint can help distinguish the substitute or adulterant, and further assess the differences of herb patchouli grown in various areas of China. (3) The RAPD markers of PCH and PAU were much different from P. cablin. There are differences among samples of P. cablin obtained from different localities. The clustering trees based on RAPD markers revealed that PCH and PAU had the lower coefficient with samples of P. cablin. Among 23 samples of P. cablin, G2,G3,G5,G19,G20,G21,G22,G23,G24,G26,G27,G28, G29,G30,G31 and G32 are assembled as one group, and G8,G9,G10,G11,G12,G13 and G16 are assembled as another group. (4) The length of 18S rRNA, matK gene, ITS region and 5S spacer region are 1809 bp, 1216 bp, 824 or 825bp, 212bp, respectively. The results revealed that there are base variation in different samples. There is only a base convertion of A and C at the position of 680 in the sequence of 18S rRNA. 15 base variations were found at the position of 51, 57, 506, 595, 636, 639, 676, 721, 722, 1158, 1161, 1198, 1199, 1214 and 1216 in the sequence of matK gene. 20 base variations are found in the sequence of ITS region, and 18 in ITS1 region, 2 in ITS2 region. These base variations are at the position of 38, 52, 79, 82, 157, 199, 205, 222, 224, 251, 291, 292, 373, 392, 405, 415, 437, 469, 520 and 669, respectively. The sequences of 5S spacer region are all similar among the 23 samples. The results showed a good correlation between genotype and distribution of Pogostemon cablin by the clustering tree analysis: The genetic distance of samples cultivated in the same localities is much nearer, so these samples are getting together; The samples which cultivated in close localities are assembled as neighbor groups. Such as the samples in Guangzhou and in Gaoyao, Leizhou and Suixi, Xuwen and Hainan, as well; All 5 tissue culture samples are always getting together, and have nearer genetic distance with G5, which reveals that the origin of their parents are possibly the kind of G5. DNA sequencing analysis could provide useful molecular information for germplasm evaluation of the plant species verification.Conclusion: GC-MS fingerprinting, RAPD fingerprinting and DNA sequencing were applied in this study. Among 23 samples of Pogostemon cablin, 5 samples are cultivated in Guangzhou (G19, G29, G30, G31, G32), 4 in Gaoyao (G2, G3, G27, G28), 2 in Wuchuan (G16, G26), 2 in Suixi (G10, G11), 2 in Leizhou (G8, G9), 1 in Xuwen (G13), 1 in Hainan (G12).The contents of ten compounds includingβ-patchoulene, trans-caryophyllene,α-guaiene, seychellene,β-guaiene,δ-guaiene, spathulenol, patchouli acohol, cyclododecane, and pogostone in Herba Pogostemonis, collected from eighteen places, were identified and determined by using gas chromatography-mass spectrometry (GC-MS) coupled with pressurized liquid extraction (PLE).Hierarchical clustering analysis based on characteristics of 10 investigated peaks in GC profiles showed that 18 samples could be divided into three main clusters, patchouliol-type, pogostone-type and the third one between the two chemotypes. The fingerprint can help distinguish the substitute or adulterant, and further assess the differences of herb patchouli grown in various areas of China.Samples in different localities have different RAPD markers and different genetic sequences, moreover, there are a good correlation between genotype and distribution of samples. Based on the clustering trees, samples G2, G3, G27, G28 have nearer genetic relation to G19, etc, this discovery well confirms the traditional viewpoint that the quality of Zhaoxiang is close to Paixiang. Besides, the genotype of samples G16, G26 is also nearer to these above samples, and morphological similarities have been observed, which maybe caused by personal choice of germplasm resources.In this study, inherent characteristics of Pogostemon cablin in different localities have been elucidated applying GC-MS fingerprint analysis, together with RAPD fingerprint and DNA sequencing analysis. In further research, the pharmacodynamic research and clinical use of Pogostemon cablin in different localities are to be necessarily studied, so as to establish the better evaluating system of germplasm resources of Pogostemon cablin.