Comparative analysis of chemical constituents in Citri Exocarpium Rubrum, Citri Reticulatae Endocarpium Alba, and Citri Fructus Retinervus

Abstract Citri Exocarpium Rubrum (CER), Citri Reticulatae Endocarpium Alba (CREA), and Citri Fructus Retinervus (CFR) are used as medicine and food, which derive from three different parts of the pericarp of Citrus reticulata Blanco through natural drying. To systematically investigate similarities and differences in phytochemicals about the three herbs, a series of analytic approaches were applied for the qualitative and quantitative analysis of chemical constituents in them. The results indicated a total of 48 volatile compounds were determined representing 99.92% of the total relative content of CER extracts, including 24 alkenes, 11 alcohols, 6 aldehydes, 2 ketones, and 2 phenols, while volatile compounds were not extracted from CREA and CFR. CER was abundant in volatile components that mainly existed in the oil gland. And a total of 32, 35, and 28 nonvolatile compounds were identified from CER, CREA, and CFR extracts, respectively. The total content of flavonoids and phenolic, and hesperidin in CFR was the highest, followed by CREA and CER. Conversely, CER was a rich source of polymethoxyflavones (PMFs), and the total polymethoxyflavone content (TPMFC), the content of nobiletin, 3,5,6,7,8,3′,4′‐heptamethoxyflavone (HMF), tangeretin, and 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone (5‐HPMF) in CREA and CFR were extremely low. Besides, CER and CREA had a higher concentration of synephrine than CFR. The phytochemicals of CER, CREA, and CFR were significantly different, which might provide chemical evidence for the comparative pharmacological activities’ research and rational application of them.


| INTRODUC TI ON
As traditional Chinese medicines (TCMs), Citri Exocarpium Rubrum (CER), Citri Reticulatae Endocarpium Alba (CREA), and Citri Fructus Retinervus (CFR) derive from three different parts of the pericarp of Citrus reticulata Blanco through natural drying. CER, the dried outer pericarp of Citrus reticulata Blanco and its cultivars, known as "Juhong" in China, is widely used to eliminate dampness and phlegm, and other respiratory diseases (Pharmacopoeia CON, 2020). CREA, the dried middle pericarp of Citrus reticulata Blanco and its cultivars, is employed to improve digestion by strengthening spleen function (Chinese Herbalism Editorial Board, 1999) and its Chinese name is "Jubai." CFR, called "Juluo" in China, the endothecium ligamentum of the fruit peel of Citrus reticulata Blanco and its cultivars, is traditionally applied to dredge meridian and promote blood flow (Chinese Herbalism Editorial Board, 1999).
CER, CREA, and CFR have been widely applied in clinical areas to treat different diseases based on the theory of traditional Chinese medicine (TCM), which has been considered to be related to the chemical profiling and the concentration of bioactive constituents.
At present, research on the three TCMs mainly concentrated on their pharmacological activities (Liu et al., 2008;Xiao et al., 2009) [AF]) showed inhibitory effects on acetylcholinesterase and α-glucosidase in a concentration-dependent manner (Guo et al., 2021). Regarding the chemical profiling of them, a few studies focused on flavonoids, such as hesperidin and nobiletin (Zhao et al., 2017). However, there is a lack of research systematically and comparatively reporting on the chemical constituents of the three herbs.
The Citrus reticulata "Chachi" pericarp (CRCP), from Sanjiang of the Xinhui region (Guangdong Province, China), is one cultivar of Citri Reticulatae Pericarpium (CRP) and the main plant materials of CRCP. According to the previous studies (Zheng, Zeng, et al., 2019), CRCP, particularly planted and harvested in the Xinhui region (Guangdong Province, China), is traditionally considered to have superior qualities compared with RCP of other varieties. Therefore, Citrus reticulata "Chachi" was selected as the materials of CER, CREA, and CFR in the study, since it can be used as an excellent source of Citrus reticulata Blanco in clinical application and by-product development.
Owing to the complicated compositions and various natural medicinal plants, a combination of analytical techniques is required to analyze their phytochemical compositions (Choe et al., 2020).
For example, combining the separation ability of chromatography with the qualitative function of mass spectrometry, ultra-high performance liquid chromatography combined with quadrupole-Exactive Orbitrap tandem-mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS/MS) technology is widely used for rapid analysis of plant extracts with higher separation efficiency, higher throughput, faster scanning speed, and higher sensitivity than high performance liquid chromatography-mass spectrometry (HPLC-MS) .

| Sample preparation
Approximately 100 g of CER, CREA, and CFR was used for the extraction of volatile compounds according to the method described in the 2020 edition of Chinese Pharmacopoeia (Pharmacopoeia, 2020).
A 50 μL aliquot of the volatile compounds was dissolved in 950 μL of hexane solvent and filtered through a 0.22μm membrane before GC-MS analysis.
The samples were ground into powder and passed through a 40-mesh sieve. Each dried sample powder (0.2 g) was weighed accurately and was extracted by ultrasonic treatment in a KQ-800KDE instrument (Kunshan Ultrasonic Instruments Co. Ltd) with 20 ml methanol for 30 min at 320 W (40 kHz) and an aliquot of 1 μl of the filtrate was injected for UHPLC-Q-Exactive Orbitrap-MS analysis.
Similarly, each sample powder (0.2 g) was extracted by ultrasonic treatment with 20 ml methanol or ethyl acetate and then filtered to obtain the sample solution. The above methanol extracts were prepared for the determination of the total flavonoid content (TFC) and the total phenolic content (TPC), and the ethyl acetate extracts were prepared for the determination of the total polymethoxyflavone content (TPMFC). Methanol extracts (1 ml) were filtered through a 0.22μm membrane before the HPLC-PDA analysis.

| GC-MS analysis system for volatile components
GC-MS analysis was carried out with an Agilent 7890A gas chromatography system equipped with Agilent DB-5MS Ultra Inert capillary GC column (30 m × 0.25 mm, 0.25 μm) and a 5975C mass spectrometer equipped with a triple-axis detector (Agilent). The heating program settings were as follows: (i) the temperature was set at 60°C first and then increased to 80°C at a rate of 1°C·min −1 for 10 min; (ii) the temperature was ramped up to 250°C at a rate of 5°C·min −1 and to 300°C at 20°C·min −1 for 1 min finally. The other procedures were set as follows: electron impact (EI + ) mode: 70 eV; detector temperature: 270°C; injection volume: 5 μL; injection port temperature: 270°C; high-purity helium flow rate: 1 ml·min −1 ; split ratio: 10:1; scan speed: 0.2 amu·s −1 (from m/z 30 to 550 amu); solvent delay: 4 min. All volatile components were determined by comparing the mass spectra with the NIST08.L database.
The conditions of the MS system were set as follows: the ion spray voltage was maintained at 3.5 kV. The auxiliary gas heater and capillary temperatures were retained at 300 and 320°C, respectively. The sweep gas, auxiliary gas, and sheath gas were, respectively, sustained at 1.7, 3.3, and 10.0 L min −1 . Chemical compound data were gathered from m/z 70 to 1000 Da in the full MS scan mode with a resolution of 70,000.

| UV analysis for the determination of TFC, TPMFC. and TPC
TFC and TPMFC were determined using the external standard method with hesperidin and nobiletin. The methanol extracts for the determination of TFC and the ethyl acetate extracts for the determination of TPMFC were diluted to a proper concentration and the absorbance was measured using a UV 2600 instrument (Shimadzu) at 283 and 330 nm, respectively.
TPC was calculated using the folin phenol colorimetric assay.
The above-mixed solution was allowed to react in the dark at 40°C for 30 min. The absorbance of the mixed solution was measured at 765 nm with gallic acid as a reference standard.

No. t R (min) Compound formula Identified compound Relative percentage (%)
Alkenes

| Analysis of differences in the nonvolatile chemical composition of CER, CREA, and CFR
By optimizing a series of parameters such as the elution gradient of mobile phase and flow rate, better analytical conditions were obtained and were described in "Section 2.5." The total ion chromatogram

| Determination of TFC, TPMFC, and TPC by UV
Flavonoids are considered as the primary bioactive constituents in citrus species, including flavonoid glycosides (flavonoid O-glycoside or flavonoid C-glycoside) and PMFs. As shown in Table 3, CER, CREA, and CFR were abundant in flavonoids, of which the total flavonoid content (TFC) of CFR was the highest (130.08 ± 0.11 mg g −1 ), followed by CREA (104.73 ± 0.00 mg g −1 ) and CER (89.23 ± 0.00 mg g −1 ). PMFs are special flavonoids that are mainly found in citrus. As seen in Table 3, the total polymethoxyflavone content (TPMFC) of CER was 10.78 ± 0.00 mg g −1 , while those of CREA and CFR were extremely low. In addition, the total phenolic content (TPC) of CER, CREA, and CFR, respectively, was 26.73 ± 0.00 mg g −1 , 40.67 ± 0.00 mg g −1 , and 48.47 ± 0.08 mg g −1 .

| Simultaneous determination of six bioactive components by HPLC-PDA
Good linear correlations and the relative standard deviations (RSDs) of repeatability (0.671%-2.595%), precision (0.463%-2.953%), and stability (1.110%-2.449%) were obtained and the recovery was within 97.541%-102.372%, indicating that the HPLC-PDA method is reliable and suitable for CER, CREA, and CFR analyses. The HPLC-PDA chromatograms are shown in Figure 3.

| DISCUSS ION
The work found that CER was highest abundant in volatile components (D-limonene, 71.71%; γ-terpinene, 11.97%) and pharmacological research has demonstrated that D-limonene exhibited anti-inflammatory activity in the prevention and control of respiratory injuries (Santana et al., 2020). Besides, CER was also rich in PMFs (nobiletin, HMF, tangeretin, and 5-HPMF) that are mainly found in citrus. PMFs have been reported to show pharmacological effects on the anti-inflammatory activity (Duan et al., 2017), and a study has mentioned that nobiletin inhibits growth and induces apoptosis in human nasopharyngeal carcinoma (Zheng, Hu, et al., 2019). CER is mainly used for eliminating dampness and phlegm, and other respiratory diseases in the clinical application of Chinese medicine, which may be closely related to its abundant volatile components and PMFs. It was reported that hesperidin exhibits various biological activities in insulin-sensitizing, antioxidant, lipid-lowering (Li and Schluesener, 2017). Besides, synephrine also potentially inhibits the conversion of carbohydrates to lipids (Maldonado et al., 2018). Hesperidin and synephrine can enhance cell energy metabolism, and CREA was abundant in both these compounds, which may be the reason that CREA is employed to improve digestion ability in clinical application. Among the three herbs, CFR was extremely rich in hesperidin, TFC, and TPC.  (Kim et al., 2019). Therefore, the differences in the phytochemicals of CER, CREA, and CFR were further clarified in this study, which might provide the chemical basis for explaining the different pharmacological activities and rational development of them.

ACK N OWLED G M ENTS
This work was primarily supported by the National Key R&D Program

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article.