Systematic Identification And Comprehensive Evaluation Research On A Specific Tea Plant Of Yuanbao Mountain In Guangxi

Camellia is a genus of more species and systematically original in Theaceae,which is all produced in Asia.And China is the distribution center of the genus,with more than 80%of the species.Located in the southern part of China,Guangxi is a climate zone with a transition from tropical to subtropical zone.And it has a unique geographical and ecological environment,which is one of the secondary origin centers of tea trees.Its territory is rich in tea germplasm resources,including some of its specific resources.According to the survey,a wild tea resource is distributed in Yuanbao Mountain,which is more than 1000 meters above sea level in Antai Township,Rongshui County,Liuzhou City,northwestern Guangxi.It is called“original tea”by the local people.Its leaves have glossy features and there are many leaf hairs in its buds and leaves.The“original tea”is obviously different from other local tea tree?Camellia sinensis?resource types in terms of leaf size,leaf shape,leaf color and germination ability,etc.For a long time,local people and businesses have been accustomed to making them into baked green tea for drinking.Its tea infusion color is light green and bright,and the aroma is with clear floral fragrance and lasting long.And the taste is fresh,mellow and sweet.Its main components were tested by Tea Research Institute of Guilin in Guangxi.And results showed that the content of tea polyphenols in the mixed samples was very high,reaching more than 40%,which was higher than the national black tea rare varieties resource standard.Previously,the members of the research team conducted a preliminary study on the resources.The results showed that its contents of tea polyphenols and theobromine were high,but the content of caffeine was extremely low,and it contained certain characteristic unknown components,which have shown good development potential and utilization value in specific species breeding and deep processing of tea,etc.Based on the result,a systematic comparative study was conducted by using a combination of morphological taxonomy,anatomy,molecular biology and chemical taxonomy in order to further correctly understand,protect and utilize the resource and to identify its attribution.So as to understand the chemical composition of the resource more in-depth and comprehensively,UPLC-ESI-Q-TOF/MS and GC×GC-TOF/MS analysis techniques were used to analyze the basic compound composition of the effective active components of the resource.Based on the characteristic of high content of tea polyphenols and low content of catechins of the resource,the alkaloids and major polyphenols were separated and identified by using a combination of column chromatography,preparative high performance liquid chromatography and high-speed countercurrent chromatography and spectral techniques such as mass spectrometry?MS?,ultraviolet spectroscopy?UV?,infrared spectroscopy?IR?and nuclear magnetic resonance?NMR?.In order to preliminarily analyze the biological activity of the resource,the antioxidant activity of the fresh leaf fixed sample,the black tea process sample and the green tea process sample were evaluated by DPPH,FRAP and ABTS.And the antioxidant mechanism and material basis of the resources were preliminarily discussed through the quantitative analysis of the antioxidant components of the tested samples.On the basis,a comprehensive toxicological safety evaluation of the resource was carried out by oral acute toxicity test,mouse bone marrow micronucleus test,Ames test and 30-day chronic feeding subacute toxicity test.The main research results showed that:1.Classification and identification of tea plants in Yuanbao MountainOn the basis of comparative differences of the morphology and anatomical structure between the Yuanbao Mountain tea plant and its related species——Camellia costata Hu et S.Y.Liang and Camellia yungkiangensis H.T.Chang by using analytical techniques such as morphological taxonomy,anatomy,molecular biology,and chemical taxonomy,genetic clustering was conducted by using molecular marker technology.And then difference in chemical composition between the resource and its related species was systematically analyzed and compared.Then on this basis,Principal Component Analysis and Chemical Cluster Analysis were used to explore the phylogenetic relationship between the resource and its related species.Then,all chemical compositions of the resource and its related species were analyzed by UPLC-ESI-Q-TOF/MS technology.And differences among each resource were analyzed by bioinformatics analysis method.And thus the taxonomic status of the resource was confirmed by synthesizing the above multi-level information.Results of morphological and anatomical analysis showed that the resource was different from Camellia costata Hu et S.Y.Liang and Camellia yungkiangensis H.T.Chang.And results of molecular biological analysis showed that the tea plants of Yuanbao Mountain and Camellia yungkiangensis H.T.Chang first clustered on the molecular clustering tree diagram,and then merged with Camellia costata Hu et S.Y.Liang into a large category.It was indicated that tea plants of Yuanbao Mountain had higher genetic similarity with Camellia yungkiangensis H.T.Chang than Camellia costata Hu et S.Y.Liang,and the genetic distance between the two was between within species and species and species.The results of chemical taxonomy analysis showed that the chemical composition of tea plants of Yuanbao Mountain was similar to that of Camellia yungkiangensis H.T.Chang,but there were still large differences between the two.Moreover,on the basis of principal component analysis sample distribution map and the cluster analysis tree diagram of chemical composition,the two clustered together and then showed obvious independence.Combining the geographical distribution of the resource and its related species,and synthesizing the above-mentioned morphology,molecular biology and chemical classification results,it was confirmed that the tea plant of Yuanbao Mountain was a unique group formed by the change of altitude and climatic conditions when crossing Yuanbao Mountain in Guangxi and then making it produce more stable variation within the species during the spread of Camellia yungkiangensis H.T.Chang from west to east.Therefore,it was classified as a variant of Camellia yungkiangensis H.T.Chang.According to the International Plant Nomenclature Regulations?St.Louis Regulations?,and the geographical name of the resource,it was named as Yuanbaoshancha.And its Latin name was Camellia yungkiangensis H.T.Chang var.yuanbaoshanica Z.W.Ge,Y.P.Liao et T.L.Chen.2.Systematic analysis of chemical composition of Yuanbao Mountain tea plants based on UPLC-ESI-Q-TOF/MS and GC×GC-TOF/MSIn the study,the chemical constituents of tea plant of Yuanbao Mountain was comprehensively analyzed by UPLC-ESI-Q-TOF/MS technology and GC×GC-TOF/MS technology.104 kinds of compounds were identified in negative ion mode using UPLC-ESI-Q-TOF/MS technology,including 21 kinds of phenolic acids,23 kinds of dimeric catechins,15 kinds of flavan-3-ols,2 kinds of amino acids,37 kinds of flavone Glycosides,3 kinds of Carbohydrates,2 kinds of Organic Acids and 1 kinds of Nucleosides.Among them,except for Serine,Glutamic acid and catechin,the other 94 were reported for the first time in this resource.The aroma components of leaves of Yuanbao Mountain tea plants were analyzed by GC×GC-TOF/MS technique,and 383 kinds of aroma compounds were identified,including 22 types of aroma compounds such as alcohol,enol,alkene,amine,alkane,aldehydes,olefine aldehyde,ethers,esters,lactones,ene esters,ketone,ketenes,phenols,organic acids,sulfocompound,nitrogen heterocyclic,oxyheterocyclic compounds,aromatic hydrocarbon,alkyne,anhydride,cyanide.Among them,the number of aromatic hydrocarbon compounds was the highest,reaching 39,followed by the number of oxygen heterocyclic compounds,32.The number of other types of compounds including esters,alcohol,aldehydes,alkane,olefine aldehyde,ketones,enol,alkene,ketenes,ethers,nitrogen heterocyclic,organic acids,sulfocompound,ene esters,phenols,amine,cyanide,lactones,alkyne and anhydride respectively were 28,28,27,26,24,24,22,22,22,17,13,11,11,9,9,7,5,3,2 and 2.The relative content of ether compound was the highest?25.19%?,followed by aldehyde?15.78%?.The relative contents of other types were below 10%,and that of organic acids,phenols,cyanide,anhydride,alkyne and lactones were the lowest,all below1%.The chemical composition of the leaves of Yuanbao Mountain tea plants were comprehensively analyzed and identified in this study,which can provide a strong basis and preliminary research basis for further research on its pharmacological health effects and better development and application.3.Isolation and identification of main alkaloids and polyphenols of tea plants of Yuanbao MountainIn order to find out the chemical composition of the main alkaloids and polyphenols in this resource,the column chromatography,preparative high performance liquid chromatography and high speed countercurrent chromatography were used to isolate and identify these compounds in this study based on the characteristic of high content of tea polyphenols and low content of catechins of the resource.And 10 polyphenolic compound monomers with purity greater than 90%and 1 alkaloid compound monomer were obtained.By mass spectrometry?MS?,ultraviolet spectroscopy?UV?,infrared spectroscopy?IR?,nuclear magnetic resonance?NMR?and other spectroscopic techniques,it was confirmed that 10 polyphenolic compounds were:1,3-di-O-galloyl-?-D-glucopyranose,Catechin,1-O-galloyl-4,6-?S?-hexahydroxydibenzoyl-?-D-glucopyranose,1,3-di-O-galloyl-4,6-?S?-hex ahydroxydibenzoyl-?-D-glucopyranose,1,4,6-tri-O-galloyl-?-D-glucopyranose,1,3,4,6-tetra-O-galloyl-?-D-glucopyranose,Epicatechin gallate,3-O-galloyl proanthocyanidin B1,Isoquercitrin,Astragalin.And the alkaloid compound was theobromine.It can be seen from the peak area of the high-performance liquid chromatography of 10 polyphenolic compounds that their contents were high in the resources,so they can be judged to be the most important polyphenolic compounds in the resource.4.Study on the antioxidant activity of Tea Plants of Yuanbao Mountain in VitroIn order to preliminarily analyze the biological activity of Yuanbao Mountain tea plants,the antioxidant activities of the fresh leaf fixing samples,black tea process samples and green tea process samples of this resource were evaluated by DPPH,FRAP and ABTS,using fresh leaf fixing samples,black tea process samples and green tea process samples of Jiuwanshandachashu?Camellia sinensis?as contrast.Results showed that in the activity of removing DPPH free radicals and ABTS free radicals,the green tea process samples,fresh leaf fixing samples and black tea process samples of Yuanbao Mountain tea plant was similar to that of the green tea process samples,fresh leaf fixing samples and black tea process samples of Jiuwanshandachashu?Camellia sinensis?respectively.In the FRAP iron ion reduction ability,the green tea process samples of Yuanbao Mountain tea plant had the strongest activity,followed by the fresh leaf fixing samples and the green tea process samples of Jiuwanshandachashu?Camellia sinensis?;and the antioxidant activity of black tea process samples of Jiuwanshandachashu?Camellia sinensis?was lowest in all samples.The IC₅₀0 values of the three antioxidant indicators showed that the DPPH IC₅₀0 values of the fresh leaf fixing samples,the black tea process samples and green tea process samples of Yuanbao Mountain tea plants were all smaller than the ABTS IC₅₀0 value and the FRAP IC₅₀0 value,and the FRAP IC₅₀0 value was the largest in all of them.It indicated that the ability of removing free radicals of Yuanbao Mountain tea plants was stronger than that of iron ions,and the ability of removing liposoluble free radical?DPPH?free radicals was significantly higher than that of removing water-soluble free radicals?FRAP?.The antioxidant activity and main chemical components of each sample were analyzed by Pearson correlation analysis.Results showed that the content of tea polyphenols,flavonoids and catechins were significantly correlated with the antioxidant activity of the samples,which indicated that these components were the most important antioxidants in tea.But the other compounds such as free amino acids and alkaloids had no significant correlation with the antioxidant activity of the samples.The above results indicated that tea plants of Yuanbao Mountain had strong antioxidant activity in vitro,and the main material basis of their antioxidants was high content of tea polyphenols.Therefore,the polyphenols extracted from tea plants of Yuanbao Mountain can be used as antioxidants and had potential application value and prospects in the food and medical industries.5.Toxicological safety evaluation of tea plants of Yuanbao MountainTaking the fresh leaf fixing samples and black tea process samples of Yuanbao Mountain tea plant as materials,then referring to the food toxicology safety evaluation procedure,systematic evaluation of the toxicological safety of the resource was studied through oral acute toxicity test,mouse bone marrow cell micronucleus test,Ames test and 30days chronic feeding subacute Toxicity test.Results showed that the LD₅₀0 of Yuanbao Mountain tea plant?fresh leaf fixing sample extracts?for male and female animals was 5.123g/kg.bw?4.323 g/kg.bw?6.185 g/kg.bw?;and that of Yuanbao Mountain tea plant?black tea process sample extracts?for male and female animals was 7.573 g/kg.bw?6.425 g/kg.bw to8.635 g/kg.bw?.According to the food toxicity grading standard,the LD50 of the two aqueous extract samples were both greater than 5000 mg/kg.bw,so it can be determined that the two aqueous extract samples were practically non-toxic.In addition,according to the extraction rate of the water extract of the sample,it can be seen that the LD50 of the fresh leaf fixing sample of Yuanbao Mountain tea plant and the black tea process sample were 13.341g/kg.bw and 25.413 g/kg.bw,respectively.And they can be respectively judged as being practically non-toxic and non-toxic according to the food toxicity grading standards.Moreover,the Ames test and the mouse bone marrow cell micronucleus test results were all negative.When the test samples were orally administered to rats for 30 days by oral gavage at 0.87 g/kg.bw,1.74 g/kg.bw,3.47 g/kg.bw,among these indicators such as the weight gain,food intake,food utilization rate,hematological index,blood biochemical index,wet weight of the organ,and the dirty/body ratio,most of the indicators of each dose group of the test sample had no obvious dose-response relationship and the values fluctuated within the normal range during the test,except that individual indicators were significantly different in each dose group compared with the negative control group.And gross anatomical observation and histopathological examination showed no pathological changes in the test samples that were toxic for SD rats for 30 days.Therefore,from the above test results,it can be determined that tea plants of Yuanbao Mountain were non-toxic foods according to the food toxicological safety evaluation criteria.And as a drinking food,it was safe and non-toxic in a scientific and reasonable dosage range.