Exploring the aging mechanism of dried tangerine peel based on extensive targeted metabolomics technology and high-throughput sequencing technology
Chenpi is the dried and mature fruit peel of Citrus reticulata Blanco and its cultivated varieties in the Rutaceae family. It has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and is a typical representative of aged traditional Chinese medicine. The name Chenpi first appeared in the “Dietary Therapy Materia Medica”, and literature records that the aging time varies. The “Lei Gong Pao Zhi Lun” states: “The oldest is the most wonderful; The “Daily Materia Medica” records that “aged tangerine peel is even better. The book “Explanation of the Medicinal Properties of Lei Gong’s Cannon Burning” states: “If collected and aged for a long time, many plum blossoms will pass through the summer and the strong qi will be completely eliminated, the temperature will be moderate without the risk of dryness and dispersion, and the qi will flow without the danger of sharp cutting; The “Medicinal Mirror” records: “Chenpi should be aged every other year; The “Yitang Sanshi” records: “The new orange peel has a strong aura and requires extensive production. It takes two to three years for it to be considered superior.
The aging of traditional Chinese medicine refers to the transformation of Chinese medicine from a new drug to an aged medicine after appropriate storage methods, resulting in changes in its performance and efficacy, in order to better meet the clinical needs of traditional Chinese medicine. Throughout history, there have been discussions on the aging of traditional Chinese medicine. The “Shennong Bencao Jing” records that “there are true and false medicines, as well as new and old medicines, and each has its own method”, indicating that “new” and “old” each have their own characteristics and rules of action. This Jing records orange peel but does not mention whether it has aged. The “Annotations on the Classic of Materia Medica of Shennong” first proposed six major aged medicines: “Any wolfberry, citrus fruit, orange peel, pinellia ternata, ephedra, and wuzhuyu that require long-term aging is good. In later generations, medical experts continued to expand the scope of traditional Chinese medicine. The Compendium of Materia Medica states: “Rhubarb, wood thieves, catnip, coriander flowers, locust flowers, etc. are also suitable for aging for a long time, not just for the six types; Until the Qing Dynasty, the book “Bencao Congxin” stated: “It is best to use aged herbs, either to reduce their strength or to remove their fire energy.” It contains nearly 40 flavors of aged herbs. Nowadays, traditional Chinese medicine also emphasizes that certain Chinese medicines should be aged for optimal use. It can be seen that the aging of traditional Chinese medicine is based on the summary of clinical practice. However, there is no in-depth explanation of the aging mechanism in the literature records.

There are many research reports on the aging mechanism of traditional Chinese medicine, such as Wu Zhu Yu, Qi Ai Ye, Ma Huang, Chen Pi, etc., mainly focusing on the comparison of chemical composition and pharmacological effects at different aging times. The quality research is not in-depth, and the conclusions are inconsistent. The research team used the idea of aging and quality related microorganisms in flue-cured tobacco, tea and Baijiu for reference. The previous research found that the growth and metabolism of microorganisms attached to the tangerine peel during aging were closely related to the changes of flavonoids and other active ingredients. It was believed that the metabolism of Aspergillus niger, the dominant microorganism attached to the tangerine peel, promoted the increase of flavonoids. Liu studied the mechanism of microbial metabolism and transformation of active ingredients in aged tangerine peel, and the results showed that microorganisms are involved in the aging process of tangerine peel and can transform compounds in tangerine peel to increase the content of active substances. Furthermore, the diversity of bacteria and fungi on the surface of aged tangerine peel varies with different aging times. It is believed that microorganisms during the aging process of tangerine peel can cause changes in the pharmacological substance basis. A large number of studies have confirmed the correlation between microorganisms and chemical components during the aging process of tangerine peel, but there is no report on the overall analysis of the interaction between attached microorganisms and secondary metabolites in tangerine peel based on metabolomics and high-throughput sequencing. This article is the first to use UPLC-ESI-MS/MS widely targeted metabolomics technology to analyze the secondary metabolites of aged tangerine peel after 1 year and 3 years of storage, comprehensively revealing the secondary metabolites, common metabolites, and differential metabolites in the comparative group samples; Based on high-throughput sequencing technology, the community structure and changes of attached microorganisms in the sample comparison group were comprehensively revealed, and differential microorganisms were screened; Perform correlation analysis between differential metabolites detected by metabolomics and differential microorganisms identified by high-throughput sequencing comparison. The research results provide a theoretical basis for the in-depth study of the aging mechanism of tangerine peel.

Collection of samples. The experimental samples used in this experiment are standard stored tangerine peel from the same origin, batch, and different storage years. Standard warehousing is in-situ storage at the place of origin, with regular seasonal management and recording of temperature and humidity changes. It is a good method for sample traceability and collection, which can avoid the influence of inaccurate environment and year.

Analysis of secondary metabolites. Metabolomics technology can comprehensively and scientifically analyze the overall chemical composition of traditional Chinese medicine, and has been widely used in the quality evaluation of Chinese medicine varieties, such as ginseng, Scutellaria baicalensis, Angelica dahurica, Paeonia lactiflora, and Paeonia lactiflora. This article uses extensive targeted metabolomics techniques to comprehensively detect secondary metabolites in aged tangerine peel after 1 year and 3 years of storage. PCA analysis, OPLS-DA analysis, and heatmap analysis can clearly distinguish samples from different years. By integrating compound retention time and characteristic ions (Q1 and Q3), a total of 386 secondary metabolites were retrieved from the database, including 79 phenolic acids, 143 flavonoids, 4 anthraquinones, 17 lignans and coumarins, 3 tannins, 47 alkaloids, 6 terpenes, and 87 other classes. Compared with traditional high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) techniques, it has the advantage of identifying a large number of compounds and can reflect the overall spectrum of secondary metabolites in dried tangerine peel. To ensure the accuracy of the relative content of each compound, this experiment obtained metabolite mass spectrometry analysis data from different samples, integrated the peak area of all substance mass spectrometry peaks, and integrated and corrected the mass spectrometry peaks of the same metabolite in different samples. This experiment refers to the design methods of clinical and pharmacological experiments. One year of storage of dried tangerine peel was used as the control group, and three years of storage of dried tangerine peel samples was used as the experimental group. Differential metabolites with fold change ≥ 2 or fold change ≤ 0.5 and VIP ≥ 1 were selected. If the difference in metabolites between groups is more than 2 times or less than 0.5 times and VIP ≥ 1, the difference is considered significant. As a result, a total of 79 differential metabolites were screened, among which the compounds with upregulated differences and high relative content ranking can be considered as quality related metabolites of dried tangerine peel. Among the top 20 compounds with differential changes in secondary metabolites of aged tangerine peel after 1 year and 3 years of aging, the up-regulated compounds are: 3,5-dihydroxy-3-methylvaleric acid, 5,7-dihydroxy-1 (3H) – isobenzofuran-one, sinapine, p-hydroxybenzoyl tartaric acid, 3 ‘, 4’, 7-trihydroxyflavone, catechin, limonin, dihydrobaibei alkaloid B, quercetin-O-rutinosin-hexose, and C-hexosyl-aureosin-O-sinapyrylhexose. Among them, catechin, limonin, 3 ‘, 4’, 7-trihydroxyflavone have antibacterial, anti-inflammatory, antioxidant, anticancer and other effects, all of which can be used as potential quality evaluations. Iconic compounds. The treatment of diseases with traditional Chinese medicine has the characteristics of multiple components, targets, and pathways. How to screen the target components for treating diseases among the numerous components is the key to the quality evaluation and quality control of traditional Chinese medicine. Among them, methods such as small molecule compound data prediction, serum medicinal chemistry, and plant metabolomics are widely used. The results of this experiment show that, under the premise of reasonable grouping, the use of UPLC-ESI-MS/MS broad targeted metabolomics technology can efficiently screen for metabolites related to the quality of dried tangerine peel. However, the activity of the screened compounds still needs to be further verified by combining with the traditional functions of dried tangerine peel.
The aging mechanism of tangerine peel. Traditionally, it is believed that aged tangerine peel is good. However, it has been widely reported that during the aging process of tangerine peel, the metabolic transformation of microorganisms affects the chemical components in the peel, thereby promoting the accumulation of active ingredients and achieving the goal of good aged tangerine peel. This result is consistent with the reported increase in total flavonoid content in aged tangerine peel. This article conducts a correlation analysis between the differential metabolites detected by metabolomics and the differential microorganisms identified by high-throughput sequencing. The results also confirm that dominant bacterial genera such as Aspergillus, Penicillium, and Penicillium can significantly affect the active ingredients in dried tangerine peel, consistent with literature reports. The preliminary research of the research group found that during the aging process of dried tangerine peel, the dominant microbial groups attached to the surface are constantly evolving. With the increase of aging time, the dominant microorganisms shift from Zasmidium, Cladosporium, Symmetrospora, and Fusarium to Cladosporium, Aureobasidium, Aspergillus, and Xeromyces. Xeromyces bisporus, a differential microorganism between samples stored for 1 year and 3 years, is a species that is more tolerant to dry mold. Microorganisms that can survive under low water activity conditions can adapt to living environments with extremely low water activity (0.61), which is consistent with the extremely dry condition of aged tangerine peel. At the same time, the experimental results showed that differential microorganisms such as Fusarium, Aspergillus, and Fusarium can simultaneously affect the relative content of various active ingredients and aroma components, such as hesperidin, vanillic acid, puerarin, and acid components. Therefore, comprehensive judgment shows that the differential microorganisms in the samples of dried tangerine peel can promote the relative increase of active ingredients such as hesperidin and other flavonoids, while also changing the relative increase or decrease of aroma components to regulate the odor of dried tangerine peel. Therefore, the scientific connotation of “aged aged tangerine peel is good” may be closely related to the influence of differential microorganisms on the relative content of secondary metabolites.