Investigation, Molecular Identification And Systematic Relationship Of Ku Ding Cha Plant Resources

Ku-Ding-Cha is a bitter beverage tea consumed widely in south China as an alternative to green tea and also used as herbal medicine in folk and minority nationality regions. According to acient medical documents, Ku-Ding-Cha is used for clearing heat, helping producing saliva and quenching a thirst, promoting digestion and reducing phlegm, relieving sore-throat and sore-larynx, curing dysentery and so on. Recently Ku-Ding-Cha draws people's attention because of its pharmacological effects such as antiatheroscloresis, reducing blood pressure, regulating blood sugar level and antidiabetics, however, a review of plant species, crude drug identification, chemical composition, pharmacological effects and clinical application of Ku-Ding-Cha researches shows that the reported plant resources of Ku-Ding-Cha are complicated, including various plants from different families and genera, which is a typical homonym phenomenon. Different original plants of Ku-Ding-Cha differ from each other in chemical composition, pharmacological and clinical effects especially between plants of different family and genus. So, it is meaningful to make clear the practically used original plants of Ku-Ding-Cha, finding a consistently identification method and clarifying the genetic relationship of different original plants for further research and development of Ku-Ding-Cha medicinal plant resources.Herbal textual research on the name, original plants and medicinal effects and ethnopharmacological research through district plant data, field investigation and specimens study were carried out to make clear the original plant and traditional usage of Ku-Ding-Cha. The results showed that Ku-Ding-Cha had been widely used in the south China for a long history using as herbal tea for clearing heat, antiphlogosis and relieving pains and curing dizzy, tinnitus, dentalgia, eye diseases, pharyngitis, hepatitis and dysentery. Morphological characteristics and geographical distribution of Ilex kudingcha is best match for the description of the ancient herbals. With regard to ethnopharmacological research, different original plants have some common medicinal efficacies together with particular curative effects.Investigation was conducted on the Ku-Ding-Cha commodities collected from different places. The main producing areas, the growing conditions, the resources distribution, and botanical characteristics of the original plants were observed. Combining with literatures and floras on Ku-Ding-Cha, the species of Ku-Ding-Cha original plants and their using area were summarized. Morphological and pharmacognostic descriptions can be used as simple identification method for original plants and crude drugs of "Ku Ding Cha".Main plant resources of Ku-Ding-Cha are Ligustrum and Ilex plants. However, according to the original plants reported in the literature (according to the literatures on its original plants) and the application of local materials, Ku Ding Cha involves different sources of a variety of plant families and genera, which often have similar morphological characteristics with Ku Ding Cha. In the present study, the chloroplast rbcL gene was used for DNA molecular identification of Kudingcha resources from different plant families and genera and distinguish these different sources effectively at the genus level. Phylogenetic analysis was carried out with MP method. Different plants of the same genus form monophyletic groups with well support rate and can be effectively identified.Ligustrum species, usually called "privet," have long been cultivated in many areas of the world as hedge plants and street trees. Among these species, L. sinense, L. lucidum, L. japonicum and L. robustum are described as invasive species in some areas. The leaves and twigs of L. robustum subsp. chinense and its relatives are used as Ku Ding Cha in southwestern China. As Ligustrum species are difficult to discriminate using morphological characters, a rapid and accurate molecular identification method is necessary. Four potential DNA barcodes, nuclear ribosomal ITS and three chloroplast DNA regions (rbcL, matK and psbA-trnH), were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information followed by psbA-trnH. matK. and rbcL. All the sequences of the four regions correctly identified the species to the genus level using BLAST alignment. At the species level, the discriminating power was 36.8%(rhcL).38.9%(matK).77.8%(psbA-trnH) and 80%(ITS) based on NJ trees. Using character-based and MP tree methods together, the discriminating ability of psbA-trnH increased to 88.9%. With the ITS, all species could be differentiated when combining the NJ tree method with character-based or MP tree methods.Holly plants are widely distributed in the world, including well-known yerba mate in Latin America. Many Ilex species in China are used as traditional medicinal plants and Ilex species are the mainstream resoures of Ku Ding Cha. Molecular identification of 27 Ilex species including different Ku Ding Cha species, traditional Chinese medicinal plants andâ… . paraguarensis used in latin America andâ… . vomitoria used in North America based on nuclear internal transcribed spacer (ITS) and 5.8S rDNA region. Using special primers for Ilex, ITS and 5.8S rDNA sequences were amplified and sequenced. The alignment can provide enough information to distinguish all species experiments, suggesting that this identification method is rapid and effective for Ilex.Ku Ding Cha is a class of homonym plant resources, and its genetic relationships are different between different species. According to pharmacophylogeny, plants with close genetic relationship are similar in systematic classification, chemical constituents and pharmacological effect. Phylogenetic tree was constructed based on molecular systematics method to show different relationships of the original plants. The combination of molecular systematic studies and existing evidence of chemical constituents and pharmacological effects, genetic relationship of Ku Ding Cha original plants were discussed to provide basis for the further research and development of Ku Ding Cha.