Study on Flavonoid and Bioactivity Features of the Pericarp of Citri Reticulatae 'Chachi' During Storage

Shejian Liang South China Agricultural University College of Life Sciences Zhijia Wen South China Agricultural University College of Life Sciences Tiexin Tang Zhaoqing Medical College Yufang Liu South China Agricultural University College of Life Sciences Fengliang Dang South China Agricultural University College of Life Sciences Tianxiao Xie South China Agricultural University College of Life Sciences Hong Wu (  wh@scau.edu.cn ) South China Agricultural University College of Life Sciences https://orcid.org/0000-0001-9142-499X


Introduction
The traditional Chinese medicinal material, 'Chenpi' (Citri Reticulatae Pericarpium), is the dry mature pericarp of Citrus reticulata Blanco and its cultivars. According to the cultivation place and cultivar, Citri Reticulatae Pericarpium was divided into two types, 'Chenpi' and 'Guang Chenpi' [1]. 'Guang Chenpi' refers to the Citri Reticulatae Pericarpium cultivated in Xinhui, Guangdong, China, which is the dry, mature pericarp of the cultivar Citrus reticulata Blanco, Citrus reticulata 'Chachi.' The dry, mature pericarp of Citrus reticulata 'Chachi' (PCRC) is of higher quality. Modern phytochemical studies have shown that the chemical constituents of PCRC are avonoids, volatile oils, polysaccharides, and alkaloids [2]. The main active components are avonoids, including avonoid glycosides and polymethoxy avonoids [3]. According to the Chinese Pharmacopoeia, PCRC regulates qi ow to strengthen the spleen and dry dampness to reduce phlegm. It is mainly used to treat abdominal distension, lack of appetite, vomiting, and diarrhea, and coughing with phlegm [1]. Modern pharmacological studies also show that it has antioxidative, antibacterial, antiviral, anti-in ammatory, antitumor, and hypolipidemic effects [4][5][6][7][8][9]. It has a long history that PCRC was used as medicine. PCRC was recorded in a pharmaceutical monograph written approximately 2000 years ago in the Eastern Han Dynasty, "Shennong's classic of materia medica" [10]. Currently, the number of preparations employing PCRC recorded in the Chinese Pharmacopoeia (2020 Edition) is as high as 176 [1]. In addition, PCRC is also the most popular food seasoning or ingredient in China. Among them, the annual market sales of PCRC produced from Xinhui reached 10 billion RMB, ranking rst for three consecutive years in in uence ranking of agricultural products on development forum for the regional agricultural brand of China .
In traditional Chinese medicine, there is the theory of "aging medicine." Aging medicine refers to the medicinal material stored and maintained by speci c methods and then used after aging. The process of storage, maintenance, and aging is the process of changing the properties and e cacy of the medicinal material, making it more suitable to the clinical needs of traditional Chinese medicine [11]. As early as 1500 years ago, in the Southern Dynasties·Liang Dynasty, Hongjing Tao recorded Citri Reticulatae Pericarpium as one of the six aging traditional Chinese medicinal materials, which should be used after aging [12]. Since then, in the Tang, Song, Yuan, Ming, and Qing Dynasties, there have been records that the longer the Citri Reticulatae Pericarpium was stored, the better [12][13][14][15][16][17]. Looking up all the ancient books recording traditional Chinese medicinal materials, the reasons for long storage treatment of aging medicine were not discussed [18]. In recent years, with the help of modern instrumental analysis, researchers have rapidly promoted the development of research on the effect of aging on the active components of Citri Reticulatae Pericarpium. Wang et al. [19] studied the effect of aging on the component accumulation and biological activity of Citri Reticulatae Pericarpium. The results showed that the content of combined polyphenols and avonoids increased signi cantly during the aging process, and the antioxidant activity increased. By means of HPLC, Liu et al [20] showed that the contents of three avonoids, hesperidin, nobiletin, and hesperetin, signi cantly increased as the storage time was prolonged. Zheng et al. [21] quantitatively studied the changes in ve avonoids, hesperidin, nobiletin, 3,5,6,7,8,3',4'-heptamethoxy avone, hesperetin, and 5-hydroxy-6,7,8,3',4'-pentamethoxy avone, in ten batches of PCRC with different storage periods, and the results showed that the contents of ve avonoids tended to increase as storage time increased. Fu et al. [22] determined Citri Reticulatae Pericarpium stored for 36 months by HPLC-dual wavelength detection and found that the content of ve avoids of sinensetin, 4,5,7,8-tetramethoxy avone, nobiletin, hesperetin, and 5-O-demethylnobiletin increased, while the content of hesperidin decreased. In the above results, the characteristics of speci c components in the aging process of PCRC were not consistent with each other and were even contradictory. The reason for the deviation of those results was hard to analyze. However, based on metabonomics, a study indicated that up to 92 avonoids were determined in the pericarp of freshly harvested Citrus reticulata 'Chachi' [23]. Currently, most published works have determined only 3 ~ 5 avonoids to study the change in avonoids in PCRC during aging. It was hard to show the whole pattern. In this study, to comprehensively evaluate the change of avonoids in PCRC during aging, UPLC-MS/MS and widely targeted metabolomics analyses were employed to fully investigate the quantity and composition of avonoids, and the antioxidant potency composite index (APC) was determined to compare the activity. Moreover, the novel coronavirus found at the end of 2019 was named 2019 novel coronavirus or "2019-nCoV" by the World Health Organization (WHO) on January 12, 2020 [24]. As of June 12, 2021, Beijing time, more than 176 million people had been infected with COVID-19, and more than 3.8 million people had died all over the world. Unfortunately, there is currently no speci c drug for COVID-19 in the world. Wu reported that avonoids such as neohesperidin, hesperidin, baicalin, kaempferol 3-O-rutinoside, and rutin from different sources, with antiviral, antibacterial, and anti-in ammatory activities, could effectively interact with some targets of SARS-CoV-2 [24]. Therefore, to study the possible effect of the abundant avonoids in PCRC on the prevention and treatment of COVID-19, further study by means of molecular docking was used to analyze the binding a nity of some important avonoid glycosides and polymethoxy avones at target proteins of SARS-CoV-2. The results in this study showed that the aging process promoted the accumulation of important pharmacologically active compounds, polymethoxy avones, and supplied evidence of active components to the statement of "the longer the medicine was stored the better" for PCRC. The contents of narirutin, neohesperidin, and hesperidin with higher binding a nity with target proteins in SARS-CoV-2 were most abundant in freshly harvested PCRC. The results supplied scienti c data for the quality control, evaluation, and rational utilization of PCRC.

Plant materials
The fruits of Citrus reticulata 'Chachi' were harvested in November 1990November , 2015November , 2016November , 2017November , 2018, and 2019 from Liangmei Farm, Fumei village, Shuangshui town, Xinhui District, Jiangmen City, Guangdong Province. Gatherer was Shu-shen Zhang. All samples were identi ed to be genuine by associate Professor Rong-jing Zhang. After harvest, stains on the surface of the fruits were washed off with clean water. Pericarps were peeled off the fruits, dried under sunlight, and put in sealed bags for storage. The PCRC was stored in a cool and dry place for aging. They were removed and dried under sunlight several times a year.
Sample information is listed in Table 1.

Determination of total avonoid content and total antioxidant activity
Three pieces of PCRC were randomly removed, powdered by a pulverizer, and sieved through a 40 mesh sieve. PCRC powder accurately weighing 0.5 g was transferred into a conical ask, followed by 32 mL of 70% aqueous methanol solution. The mixture was treated with an ultrasonic cleaner (ultrasonic power 100 W, water temperature 60°C) for 1 hour. Then, it was centrifuged at 6000 rpm for 10 min. Supernatant liquid was collected. The extraction was repeated three times. The supernatant liquid of three extractions was combined and diluted to 100 mL in a volumetric ask to give the sample solution. Three replicate sample solutions were prepared separately from every PCRC sample. Determination of total avonoid content adopted from the method of Meyers et al [25].
was slightly adapted. Determination of total antioxidant activity employed a detection kit from Beyotime Biotechnology Co., Ltd. and followed the FRAP and ABTS procedures. The DPPH method referred to the method of Kong et al [26]. with a small adaption.

HPLC Conditions
The sample extracts were analyzed using an LC-ESI-MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 500°C; ion spray voltage (IS) 5500 V; ion source gas I (GSI), gas II(GSII), curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 µmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions were performed with further DP and CE optimization. A speci c set of MRM transitions was monitored for each period according to the metabolites eluted within this period.

Hierarchical Cluster Analysis and Pearson Correlation Coe cients
The HCA (hierarchical cluster analysis) results of samples and metabolites are presented as heatmaps with dendrograms, while Pearson correlation coe cients (PCCs) between samples were calculated by the cor function in R and presented as only heatmaps. Both HCA and PCC were carried out by R package heatmap. For HCA, normalized signal intensities of metabolites (unit variance scaling) are visualized as a color spectrum.

Differential metabolites selected
Signi cantly regulated metabolites between groups were determined by VIP ≥ 1 and absolute Log2FC (fold change) ≥ 1. VIP values were extracted from the OPLS-DA results, which also contained score plots and permutation plots, and were generated using the R package MetaboAnalystR. The data were logtransformed (log2) and mean-centered before OPLS-DA. To avoid over tting, a permutation test (200 permutations) was performed.

KEGG annotation and enrichment analysis
Identi ed metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and annotated metabolites were then mapped to the KEGG pathway database. Pathways with signi cantly regulated metabolites mapped to were then fed into MSEA (metabolite set enrichment analysis), and their signi cance was determined by hypergeometric test p-values.

Multivariate analysis of the metabolites
The ion current strength data of the avonoid metabolites were used to establish a cluster heat map, and the samples were analyzed by principal component analysis (PCA). A cluster heat map showed that three replicate samples from the same groups clustered and indicated that the data were reliable (Fig. 4a). In addition, clustering results of samples from different aging groups showed that six groups of different aging periods rst clustered into two classes. C0, C1, and C2 clustered into one class, and C3, C4, and C29 clustered into the other. According to the length of the aging, they were further divided into four classes: C0 was one class by itself, C1 and C2 were clustered into a class, C3 was one class by itself, and C4 and C29 were clustered into a class. This result indicated that avonoid metabolites of PCRC varied with the length of the aging, and avonoid metabolites of PCRC were more similar as their lengths of the aging were more closed. In Fig. 4b, PC1 and PC2 explained 71.28% of the total difference, and three replicate samples from the same groups were combined. C1 was close to C2. C4 was close to C29. The results were consistent with the cluster heat map.
3.3 Screening and analysis of the differential metabolites between freshly harvested PCRC and aging PCRC   (Fig. 7) and polymethoxy avones (Fig. 8,9) were compared in the form of histograms.
The avonoid glycosides with higher relative contents in PCRC were hesperidin, neohesperidin, naringin, and narirutin. The polymethoxy avones with higher relative contents in PCRC were tangerine and nobiletin. By comparison, it was found that as the aging period extended, four avonoid glycosides, hesperidin, neohesperidin, naringin, and narirutin, decreased, while polymethoxy avones increased.
As shown in Fig. 7, as the aging period was extended, the neohesperidin content decreased. The neohesperidin content of the C3 group was not signi cant; however, the trend generally decreased. The hesperidin content also decreased as the aging period extended. After three years, the change in hesperidin content was not signi cant; however, it was still decreasing. Narirutin content decreased at rst and then increased in the rst two years of storage, slowly decreased after two years, and did not change signi cantly after four years. The change in naringin content was in accordance with the change in narirutin content. Generally, in the aging process, the changes in the contents of the four avonoid glycosides slightly uctuated but eventually signi cantly decreased.
Flavonoid glycosides in PCRC decreased as the aging period extended.
In Fig. 8, the contents of tangerine and nobiletin slightly uctuated; however, the change was not signi cant. Generally, their contents in the groups with shorter aging periods (C0, C1, C2) were lower than those in the groups with longer aging periods (C3, C4, C29), and the change was not signi cant among the C3, C4, C29 groups. The content of 5-hydroxy-6,7,8,3',4'-pentamethoxy avone (demethylnobiletin) uctuated greatly, and the content in the C0, C3, and C29 groups did not change signi cantly and was higher than that in the other groups. The content of 3,5,6,7,8,3',4'-heptamethoxy avone did not change signi cantly from group C0 to group C1, increased gradually from group C1 to group C4, and decreased slightly in groups C4 and C29. The content of 5-hydroxy-6,7,3',4'tetramethoxy avone uctuated and was highest in group C3 and then decreased. The content of 5,7,8,4'-tetramethoxy avone decreased from group C0 to group C1, increased gradually from group C1 to group C3, and slightly decreased from C3 to group C29. The contents of monohydroxy-hexamethoxy avone and 7-hydroxy-3,5,6,8-tetramethoxy avone had similar changing trends, which gradually decreased from group C0 to group C2, sharply increased from group C2 to group C3, and uctuated from group C3 to group C29 with relatively slight changes.
In general, in 15 polymethoxy avones, the content of most of them in PCRC did not have a certain simple trend. However, the contents of polymethoxy avones in groups with shorter aging periods were lower than those in groups with longer aging periods.

Evaluation of total antioxidant activity
To investigate the impact of the change in avonoid content in PCRC during the aging process on the bioactivity, FRAP, ABTS, and DPPH methods were employed to determine the total antioxidant activity of the extract solutions of PCRC in six groups with different aging periods ( Table 3). The antioxidant potency composite index (APC) was used to evaluate the total antioxidant activity. The results were C3 > C4 > C2 > C29 > C0 > C1, and the APCs of group C3 and group C4 were signi cantly higher than those of the other four groups, indicating that they had better antioxidant potency. Wang et al. [19] reported that the total phenols of freshly harvested PCRC had the best antioxidant potency, as tested by the peroxyl radical scavenging capacity (PSC) test. PCRC aged for 1 year and 13 years had the best oxygen radical absorbance capacity (ORAC) test, and PCRC aged for 6 years had the best cellular antioxidant activity (CAA) test.     Naringin and hesperidin had values of -11.80 kcal/mol and 11.60 kcal/mol, respectively.
To compare the potential antiviral activities of PCRC with different aging periods against SARS-CoV-2, the total content of avonoids with lower binding energy than the positive control drug was added up for separate targeting proteins and separate aging periods. Figure 9a shows that the total content of 32 avonoids with lower binding energy than the positive control drug at 3CLpro varied with the aging period. It was found that the content of group C0 was highest. Of the rest of the groups, the content of group C3 was the highest. Figure 9b, 9c, and 9d show the results for the binding target proteins RdRp, PLpro, and spike, respectively. Generally, the total content of avonoids with lower binding energy than the positive control drug was highest in group C0, decreased in the one-year aging process, increased to the second-highest level, and decreased as the aging period extended.  In the latest report, 92 avonoids were detected from the methanol extract solution of PCRC [23], and up to 56 polymethoxy avones were identi ed from PCRC [28]. The marker components of PCRC recorded in the Chinese Pharmacopeia (2020 version, One Sections) were hesperidin, nobiletin, and tangeretin [1].
Studying the main components, hesperidin, nobiletin, and tangeretin, in PCRC were bene cial to the quality evaluation of PCRC [19][20][21][22][23]. However, in the aging process, the contents of polyphenols, avonoids, and particularly polymethoxy avones undergo a series of changes [19,23]. Therefore, investigating the overall change in avonoids in the aging process of PCRC is a prerequisite and guarantees reasonable quality control and standard establishment for PCRC.
In this study, PCRC samples from the same tree (excluding the group of aging period of 29 years), same farm, and stored under the same conditions were used as objects. The total avonoid contents of PCRC from six groups with different aging periods were determined by spectrophotometry. The results showed that with increasing aging time, the total avonoid contents of PCRC decreased at rst, then increased, and then decreased again. Because their molecular structures are more lipophilic, they penetrate bio lms more easily and enhance bioavailabilities. Therefore, the bioactivity of polymethoxy avones was better than that of the relevant structure without methoxylation [29,30]. Polymethoxy avones not only have good antioxidative activity but also have an effect on relieving metabolic syndromes such as hypertriglyceridemia, fatty liver, and insulin resistance. by regulating enteric microorganisms and amino acid metabolism [22,31,32]. They have antitumor effects by inhibiting the growth of tumor cells and inducing the apoptosis of tumor cells [29,30,33]. The results reviewed the aging process to enhance the compound number and total content of the polymethoxy avones in PCRC and further indicated the scienti c meaning of "the longer the aging period is, the better" for PCRC.
However, regarding the antioxidative effect, the potency of PCRC in the groups aged 3 years was better than that in the other 5 groups. The group with the longest aging period of 29 years did not present a signi cant advantage. Its potency was only equal to that of the group with an aging period of 2 years. The results explained the record of an aging period of 2 ~ 3 years in a medical book written by Yuhe Xu in the Qing Dynasty [34].
Currently, COVID-19 is still prevalent around the world. Although vaccines were invented, there is still much uncertainty. New mutated viruses emerged and led to an enhanced ability to spread and continue to deteriorate the epidemic situation in some local areas. Finding active compounds to prevent and/or treat COVID-19 is important and urgent. Some avonoids were reported to interact with some targets of SARS-CoV-2 [27]. Interfering with the spike protein of SARS-CoV-2 and the ACE2 receptor of the host or inhibiting RNA polymerase and important proteases (3CLpro and PLpro) inhibit virus reproduction and might be a potential treatment for COVID-19 [27]. Molecular docking was performed using avonoids from PCRC targeting the spike protein, 3CLpro, PLpro, and RdRp of SARS-CoV-2. It was found that many avonoids from PCRC had better a nity for the target than positive control drugs. The total content of avonoids with lower binding energy than the positive control drug was highest in newly harvested PCRC. This result indicated that newly harvested PCRC would be more appropriate for the preparation to prevent or treat COVID-19. The results are only based on molecular docking. In vivo and in vitro experiments should be further carried out to evaluate the antiviral activity of PCRC avonoids against SARS-CoV-2.

Conclusions
UPLC-MS/MS and metabolomics analysis were employed to analyze the avonoids of PCRC with different aging periods for the rst time. The results showed that the aging process prompted qualitative and quantitative changes in avonoids in PCRC, and polymethoxy avone signi cantly increased in PCRC aged for 3, 4, and 29 years. However, the potential antiviral components against SARS-CoV-2 decreased as the aging period extended. The results of APC experiments showed that PCRC with aging periods of 3 and 4 years presented signi cantly higher antioxidative potency than the other groups, and PCRC with an aging period of 29 years did not present an antioxidative advantage. Therefore, it was recommended to use PCRC with an aging period over 3 years according to the content of bioactive polyemethoxy avones, and for antioxidative use, PCRC with an aging period of 3 ~ 4 years would be better. For use in preparation preventing or treating COVID-19, newly harvested PCRC would be more appropriate. In addition, 5,7,3',4',5'-pentamethoxy dihydro avone and 2'-hydroxy-3,4,5,3'4',6'-hexamethoxychalcone were found from PCRC for the rst time. At the same time, seven avonoids (tectochrysin, apigenin, 2'-hydroxyiso avone,   Volcano diagram of differential metabolites Note: Every point in the volcano diagram represents a metabolite. Horizontal ordinate represents the logarithmic value of quantitative fold change of a certain metabolites between two groups of samples. Vertical coordinate represents VIP value. The greater absolute value of horizontal ordinate indicates the bigger fold change of expression amount between two groups of samples. The greater absolute value of vertical coordinate indicates the expression change is more signi cant and the differential metabolite found by screening is more convincing. The green point indicates the expression of the differential metabolite down-regulated. The red point indicates the expression of the differential metabolite up-regulated. The black point indicates the expression of the differential metabolite is not signi cant.

Figure 6
Venn Diagram of differential avonoid metabolites in ve pair groups for compariso The change of contents of four avonoid glycosides Figure 8 The change of contents of eight polymethoxy avones Figure 9 The change of contents of seven polymethoxy avones