| Background:Guang Chenpi,a kind of national geographic indication product with homology of medicine and food.The genuine product is the dried and mature peel of Citrus reticulata‘Chach’,which has rich chemical composition and medicinal values such as regulating qi and invigorating spleen,stopping vomiting in stomach,dampness and reducing phlegm.It has been widely used in clinical treatment and non-staple food processing and development.According to the records in“Ben Cao Feng Yuan”,“Citri Reticulatae Pericarpium,acrid-bitter tasty,warm in nature,produced in Xinhui in eastern Guangdong,the older,the better”.As one of the first lingnan medicinal herbs under legislative protection in Guangdong province,the quality of Citri Reticulatae Pericarpium is better produced in Xinhui district in Jiangmen,Guangdong,and the traditional Chinese medicine theory believes that the properties of Citri Reticulatae Pericarpium after aging are more in line with the pharmacological needs of traditional Chinese medicine.At present,the industry about Guang Chenpi is in the stage of rapid development.The planting area of its base plant,Citrus reticulata‘Chach’,is as high as 100,000 mu in Xinhui district,Guangdong province,and its annual market output value has exceeded 10 billion yuan.However,up to now,there are still limitations in the unified standardization of the germplasm variety protection and breeding,the standardized planting of Citrus reticulata‘Chach’tree,the processing technology of Citrus reticulata‘Chach’fruit and the aging technology of Citrus reticulata‘Chach’peel.It is difficult to effectively guarantee the quality of Guang Chenpi from the whole industrial chain process.In order to better promote the healthy development of the industry about Guang Chenpi,this paper took Citrus reticulata‘Chach’as the main research object,and first made a supplement to the chemical basis of Guang Chenpi.Combined with volatile oil,flavonoids,organic acids,limonin and their antioxidant activities,the effects of different storage environments and storage containers on the quality of Guang Chenpi were studied to provide scientific basis for the selection of storage conditions in the process of industrialization.In addition,DNA barcoding genetic stability analysis and variation detection were carried out for Citrus reticulata‘Chach’,which provided a scientific basis for its excellent germplasm breeding and a new idea for its quality control,and promoted the sustainable development of Mandarins industry in the future.Aims:（1）The chemical constituents of Citri Reticulatae Pericarpium from different varieties were analyzed comprehensively and the differences of chemical constituents between Guang Chenpi and Chenpi were compared.（2）To study the dynamic changes of chemical components in different storage environments and containers.（3）DNA barcoding genetic stability analysis and mutation locus detection were performed on Citrus reticulata‘Chach’of different propagation methods,tree ages,varieties and rootstocks.Methods:（1）Combined with supercritical CO2fluid extraction and UHPLC-Q Exactive Orbitrap-MS,the chemical constituents of 12 varieties of Citri Reticulatae Pericarpium samples（18 batches in total）were identified and analyzed.（2）UV spectrophotometer was used to determine the contents of total flavonoids,total polymethoxyflavonoids,total phenolic acids of Guang Chenpi samples under different storage conditions,and the antioxidant activity was determined by DPPH method.（3）High performance liquid chromatography with photo-diode array detector（HPLC-PDA）was used to determine a total of 8 chemical components of Guang Chenpi samples in different storage conditions.（4）The volatile oil was extracted by steam distillation,further separated and identified by gas chromatography-mass spectrometry（GC-MS）.（5）Total DNA was extracted from leaves of 22 batches of Citrus reticulata‘Chach’by plant DNA extraction kit,and PCR amplification products of the four DNA barcodes were identified by agarose gel electrophoresis.The genetic stability of the four DNA barcodes among different Citrus reticulata‘Chach’plants of different propagation methods,tree ages,varieties and rootstocks was investigated by ABI 3730XL genetic analyzer.In addition,DNBSEQ-T7 gene sequencer was used to sequence the c DNA library and analyze the mutation sites.Results:（1）A total of 57 chemical components were identified from Citri Reticulatae Pericarpium samples of different varieties,of which 36 chemical components were detected in Citri Reticulatae Pericarpium extracts for the first time,and coumarin components were detected for the first time.In addition,a total of 42components were identified in Guang Chenpi,including 9 PMFs,3 other flavonoids,10 organic acids,3 coumarins,4 other esters,3 aldehydes,4 terpenoids,2 limonin and4 other compounds.Among them,10 organic acids were identified from Citrus reticulata‘Chach’,while about 5 were identified from other varieties.（2）During one year storage in different environments,the contents of total flavonoids and hesperidin showed a decreasing trend,and the contents of low temperature and low humidity group decreased more.Ferulic acid content showed an increasing trend in different storage environments,and ferulic acid content increased 7 times in high temperature and high humidity group than that in other conditions,and its antioxidant activity was enhanced,which was speculated to be related to enzyme catalysis induced under appropriate high temperature and high humidity conditions.Statistical analysis results showed that there was no significant difference in the volatile oil content of Guang Chenpi samples during one year storage in four different environments（P>0.05）,but the relative content of limonene decreased at high temperature and constant humidity,high temperature and high humidity and low temperature and low humidity.Under the condition of high temperature and high humidity,carvacrol and methyl2-（methylamino）benzoate relative content were significantly decreased.However,diethyl phthalate was not detected in the first 6 months,and its content increased significantly after 6 months,suggesting that the characteristic component methyl2-（methylamino）benzoate underwent substance transformation.（3）In different container storage process under different temperature,the degradation speed of the total content of flavonoids and hesperidin content in high temperature is 2 times in normal temperature.The contents of total flavonoids and hesperidin of Guang Chenpi samples stored in PE sealed bag were the least in both high temperature and normal temperature.The content of ferulic acid increased slightly,and the antioxidant capacity of the sample stored in PE bag and gunny-sack at high temperature was enhanced.In addition,the analysis result showed that volatile oil content of Guang Chenpi samples under different temperatures in four different container had no significant difference（p>0.05）,but the alkene content decreased,aldehydes,alkanes component content rise,and esters,alcohols components present change rule in different temperature and container.The relative content of methyl2-（methylamino）benzoate in gunny-sack decreased,while the relative content of linalool in tin pot increased obviously.（4）Homologous analysis results showed that the homology of 4 DNA barcodings（ITS2,rbcl,mat K and psb A-trn H）of 22 Citrus reticulata‘Chachi’samples with different propagation methods,tree ages,varieties and rootstocks were 100.00%,99.97%,99.99%and 99.81%,respectively.And 2 SNP sites were identified in the mat K barcode and 1 In Del site was identified in the psb A-trn H barcode.Simultaneously,variant site detection of Citrus reticulata‘Chachi’was analysed by comparing with the reference Citrus reticulata genome,and2652697 SNP sites and 533906 In Del sites were detected from whole-genome resequencing data of 22 samples.Conclusion:（1）SFE method combined with UHPLC-Q-Exactive Orbitrap-MS can quickly and effectively comprehensively identify the chemical components of Citrus Reticulatae Pericarpium extracts from different varieties,and can simultaneously enrich flavonoids,organic acids,coumarins and other esters,limonin and other low-polarity components.Coumarins were detected in Citrus Reticulatae Pericarpium extracts for the first time.In addition,the organic acid composition of Citrus reticulata‘Chachi’was more abundant than that of other varieties.（2）In different storage environments,low temperature and low humidity conditions are not conducive to the aging of Guang Chenpi,while long-term high temperature or high humidity conditions can accelerate the transformation of ferulic acid and other substances in the aging process,but also lead to a significant decrease in the content of hesperidin and methyl 2-（methylamino）benzoate.Therefore,it is believed that there is a scientific basis for the traditional aging of Guang Chenpi under natural conditions,but scientific control of storage temperature and humidity is needed for mass production in order to effectively unify the quality of Guang Chenpi.（3）In long-term storage and aging process,different temperature and storage containers will the change law of dynamic characteristics of the components into different effects,Guang Chenpi of PE bags storage under the condition of high temperature or room temperature can better preservation of the chemical composition,and wide sacks for Guang Chenpi of methyl 2-（methylamino）benzoate,strong aroma of Guang Chenpi stored in tin may be related to the increase of linalool relative content.（4）Four DNA barcodes（ITS2,rbcl,mat K and psb A-trn H）maintained high genetic stability in different propagation modes,tree ages,varieties and rootstocks.At the same time,the discovery of variation in the whole genome resequencing data of 22 Citrus reticulata‘Chachi’samples fully reflected the genetic diversity of Citrus reticulata‘Chachi’under different varieties or propagation modes. |
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