Evaluation of chemical components in Citri Reticulatae Pericarpium of different cultivars collected from different regions by GC–MS and HPLC

Abstract To discriminate the feasible differences and find potential similarities and relationships of Citri Reticulatae Pericarpium (CRP), this work was accomplished by a comprehensive and reliable method using gas chromatography–mass spectrometer (GC–MS) to analyze the volatile oils and high‐performance liquid chromatography (HPLC) simultaneously to determine the contents of five bioactive flavonoids, namely hesperidin, nobiletin, 3,5,6,7,8,3′,4′‐heptamethoxyflavone, tangeretin, and 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone in 25 batches of CRP samples of 10 cultivars collected from different regions in China. The GC–MS analyses indicated that 98 compounds were successfully identified from the volatile oils obtained and the major constituents of volatile oil are d‐limonene, γ‐terpinene, α‐pinene, linalool, and myrcene. Even 2‐(methylamino) benzoate was found in all cultivar samples harvested at maturation stage. Under the optimal condition, the quantitative analyses of five bioactive flavonoids were successfully performed by HPLC and hierarchical cluster analysis (HCA). Results showed significant differences among cultivars in the contents of five bioactive flavonoids mentioned earlier. The HCA and GC–MS results provided a convenient approach which might be applied for rapid similarity evaluation and also holds the potential for analysis of compounds present in other plants. Therefore, this work obtained offers scientific basis to control quality and develop medicinal value of the medicinal materials in CRP.

Phytochemical studies showed that abundant components are present in CRP, such as flavonoids, alkaloids, phenolic acids, and essential oils (EOs), among which flavonoids were considered to be the primary bioactive components . Moreover, the major components of CRP are dietary flavonoids, which are generally categorized into two groups: flavanone glycosides (primarily hesperidin) and polymethoxylated flavones (PMFs, primarily nobiletin and tangeretin) (Ho & Kuo, 2014;Zeng, Dua, Chen, Li, & Liu, 2017). Currently, hesperidin is used as a chemical reference for quality control of CRP in the Chinese pharmacopeia because of its extremely high concentration (over 3%) (Committee of National Pharmacopoeia, 2015). Besides, EOs are the other principal pharmacological components of CRP (Qin et al., 2013). EOs extracted from plant materials, such as flowers, roots, bark, seeds, fruit peels, and wood, are subtle and aromatic. By report, several methods have been applied for extracting EOs, namely steam distillation (SD) and supercritical fluid extraction-CO 2 (SFE-CO 2 ), and SD as the common method is recorded on Chinese pharmacopeia, while several studies have quantified flavonoids in Citrus herbs using thin-layer chromatography (TLC), HPLC-UV, HPLC-DAD, HPLC-ECD, HPLC-MS, and CE-ECD, primarily focusing on the determination of flavonoids in fruits, juices, or CRP of different Citrus species (Camarda, Di Stefano, Del Bosco, & Schillaci, 2007;Careri, Elviri, Mangia, & Musci, 2000;Liu et al., 2013;Peng, Liu, & Ye, 2006;Sahraoui et al., 2011;Wang & Luo, 1989;Zheng et al., 2009). In recent studies, lots of attention is being paid to flavonoids because of its anticancer, antioxidant, anticonvulsant, and anti-inflammatory properties (Chang et al., 2015;Devi et al., 2015;Dimpfel, 2006;Fu et al., 2017), whereas few on volatile oils which have abundantly valid biological activities on CRP (Duan et al., 2016;Yi, Dong, Liu, Yi, & Zhang, 2015). So far, there were a group of analyses of flavonoids including constituents and contents, and the results indicated obvious differences in CP and GCP (Lin, Li, Ho, & Lo, 2012;Liu et al., 2013;Xing, Zhao, Zhang, & Li, 2017;Zhang et al., 2012;Zheng et al., 2009Zheng et al., , 2013. Modern pharmacology study has demonstrated that volatile oils play critical roles in certain allergy and antitussive, expectorant, and F I G U R E 1 Chemical structure of bioactive flavonoids C1-C5 T A B L E 1 Sample information and the results of the volatile oil extraction (n = 25) anti-inflammatory effects (Wang et al., 2014). In addition, most reports on volatile oils components of Chenpi are focused on the comparison in citrus, such as Citri Reticulatae Pericarpium (CRP) and Citri Reticulatae Pericarpium Viride (CRPV), but few is centered around different cultivars (Chen & Cui, 1998;Gao, 2011;Hu et al., 2014;Mao, Ou, & Wang, 2015;Wang & Li, 2015;Yi et al., 2015). Gas chromatography coupled with mass spectroscopy (GC-MS) and high-performance liquid chromatography (HPLC) with dual wavelength detection have become a reasonable and powerful approach for the qualitative and quantitative analyses in tangerine peels. However, to the best of our knowledge, no such study was yet reported about HPLC method coupled with GC-MS approach to evaluate and discriminate the quality of CRP effectively and comprehensively among different cultivars in order to ensure its superior clinical use.
Thus, the objectives of this study were to develop a comprehensive, accurate, and reliable HPLC method for the simultaneous quantitative determination of five bioactive flavonoids (Figure 1  Must (Chengdu, China) were isolated and purified from "Chachi" CRP by conventional column chromatography, and their structures were identified by EI-MS, 1 HNMR, and 13 C NMR in comparison with the data from the literature. Their purities were determined to be 98% based on HPLC analysis using a peak area normalization method.

| Sample preparation for HPLC analysis
The tested samples were cut into smaller pieces and further ground into powder (100 mesh). Each sample powder (0.5 g) was weighed accurately and extracted with 50 ml methanol using ultrasonicator at room temperature for 30 min at 320 W. After that, the sample was filtered and the 10 ml volume of solution was set at 25 ml volumetric flask. The obtained solution was filtered through a 0.22μm filter membrane, and 20 μl of the filtrate was subjected to HPLC analysis.

| Sample preparation for GC-MS analysis
The peels were manually removed, sun-dried, and stored under dry conditions. Before extraction, the peels were powdered using a mill (Xuyang Equipment Manufacture Company, China) and passed through a 24 mesh sieve. Approximately 200 g of every sample powder we collected were swollen by soaking in 2,000 ml of distilled water for 12 hr prior to hydrodistillation for 5 hr using a standard extractor. Then, the EOs were prepared according to the standard method described in Chinese pharmacopeia (2015 version). The volatile oil was collected in a sterilized glass vial. Water was removed from the volatile oil using anhydrous sodium sulfate. The extraction yield was calculated (in milliliter of oil), was stored at 4°C in the dark, and then dissolved in n-hexane prior to GC-MS analysis. EO yield (g/kg) was calculated with the following formula: EO yield (g/kg) = [mass of EOs obtained (g)/mass of dry matter (kg)].

| Instruments and experimental conditions
GC-MS analysis was performed using an Agilent Technologies 7890A GC coupled with a fused silica capillary column (HP-5MS, 0.25 mm × 30 m, film thickness 0.25 μm) and equipped with a 5975C MS detector (Agilent Technologies, Palo Alto, CA, USA). The column temperature was set at 60°C initially (maintained for 2 min), which was then increased to 80°C at a rate of 1°C/min for 10 min, subsequently raised to 25°C at a rate of 5°C/min, and finally to 300°C at a rate of 20°C/min for 1 min. The inlet temperature was 270°C.
The carrier gas was helium with a constant flow rate of 1.0 ml/min in a split ratio of 10:1 and the injection volume was 5 μl. To the experimental conditions of the mass spectrometer, electron impact (EI+) mass spectra were operated at 70 eV. Injector and detector temperatures were set at 270°C, respectively. Scan at 0.2 scan/s from m/z 30 to 550 amu and solvent was delayed by 4 min. All the data analyses were carried out on a personal computer. The library searches and spectral matching of the resolved pure components were conducted on the NIST (National Institute of Standards and Technology) 08s.L MS database.

| HPLC analysis
The flavonoids were detected at 283 nm and 330 nm by the UV detector using an Agilent 1260 Series HPLC system (Shimadzu Corporation, Japan). The compounds were separated on a Dikma Diamonsil C18 column (4.6 mm × 250 mm i.d., 5 μm) and at a column temperature of 25°C.

| Establishment of calibration curves
To build the calibration curves, the mixed standard stock solution was prepared by dissolving the reference compounds ( for further HPLC analysis. The standard solutions were analyzed in triplicates, and peak areas were used as analytical signal. The calibration curves were constructed by plotting the peak areas versus the concentrations of standards.

| Method validation
The method was validated for linearity, sensitivity, repeatability, and accuracy. The limits of detection (LOD) and limits of quantifica- y and x denote the peak area and corresponding injection concentration (μg/ml), respectively. a and b denote the slope and intercept of the regression line, respectively.
T A B L E 2 Calibration curve data for reference compounds 1-5 (n = 3)

Intraday
Detected a (μg/ml) Accuracy (%) RSD (%) T A B L E 3 Intraday precision of the developed method (n = 6) low levels) of reference compounds were analyzed for six replicates within 1 day. Variations were expressed by the relative standard deviation (RSD) of the data. Recovery was used to evaluate the accuracy of the method. Six different amounts of the standard solutions were added to sample S1, and the recovery was measured in triplicate.
For comparison, unspiked S1 sample was concurrently prepared

| Analytical method validation of HPLC
To establish informative and reliable HPLC condition of tangerine peels, detailed information regarding calibration curves, linear ranges, LOD, and LOQ is displayed in Table 2. For all the examined compounds, all five calibration curves exhibited good linearity (R 2 > .9990), the intraday precisions, repeatability, stability, recovery calculated as relative standard deviation (RSD) were all <3%, and the accuracy ranged from 98.81% to 100.08% which are displayed in Tables 3-5

| Quantitative analysis of samples in HPLC
The developed analytical method was subsequently applied to the simultaneous determination of above five flavonoids in 25 batches of CRP collected from different major citrus-producing regions in China. The contents of oil and five analytical compounds presented as the average value ± SD are summarized in Table 6. The .

| Quality assessment of CRP by HCA
To evaluate the change in similarity of five flavonoids displayed by the 25 CRP samples of different cultivars, an HCA based on the major five flavonoids was performed. The HCA is a multivariate procedure that allows the classification of variables into groups based on Euclidean distances between cases. The dendrogram of the 25 CRP samples is shown in Figure 3. The HCA results demonstrated a better hierarchical classification with all the samples well grouped according to their species. Obviously, as given in Figure 3, CRP samples were apparently classified as one of two groups so that this analysis provided further profound understanding of the distribution of the multiple chemical compounds in CRP. Two major clusters, viz., clusters 1 and 2, can be found in Figure 3. Cluster 1 was composed of 15 samples, while cluster 2 was composed of 10 samples. Although the concentrates of the samples S1-S12 (C. reticulata "Chachi") from Guangdong Province are distinguishing, they could be classified into one cluster. In other words, the chemical ingredients of C. reticulata "Chachi" are similar. It can be seen that cluster 2 includes C. reticulata "Speciosa," C. reticulata "Dahongpao," C. reticulata "Subcompressa," C. reticulata "Erythrosa," C. reticulata "Unshiu," and C. reticulata "Shiyueju," which indicates that the six species possess the similarities and relationships to some extent.
In addition, it can be useful for us to choose the appropriate dried tangerine peels with desired content of components as well as to choose alternate cultivars with more similarity in the absence of a desired variety at the time of requirement.

| Global yields of volatile oil in CRP
In the experiment, EOs were extracted from a total of 15 batches of 10 varieties of dried tangerine peels by using extraction method of EOs in Chinese pharmacopeia (2015 edition). The contents of oil are presented as the average value ± SD (W/W). The EOs extraction yield results acquired among the 25 CRP samples based on a dry weight are present in Table 1.
Obviously, as can be seen that a total of EOs obtained were transparent and oily liquid with a rich smell, whose extraction yield approximately ranges from 0.90 to 67.43 (g/kg). And there existed significant differences in EOs contents and their color, which may be affected by the cultivars and environmental factors, such as storage conditions, time of collection, and preservation. According to Table 1, it is noticeable that C. reticulata "Chachi" which is mainly produced in Xinhui, Guangdong are rich in EOs except for C. reticulata "Shiyueju," even the highest EOs yield reach up to 67.43 g/kg, whereas the lowest is only 0.90 g/kg recorded for the C.
reticulata "Speciosa" picked from Dianjun District, Yichang City, Hubei Province and C. reticulata "Subcompressa" collected from Yongquan Town, Linhai County, Taizhou City, Zhejiang Province, respectively. In addition, the Table 1 showed an obvious difference in the EOs extraction ratio of C. reticulata "Chachi" produced in diverse regions, and found that the extracting rate of C. reticulata "Chachi" producing in Xinhui district, Guangdong Province is higher than other areas in Guangdong Province.
Moreover, it was discovered that the less EOs contents, the deeper the color in this test, and a tentative inference on this result is that the color of EOs may have certain correlation with oil content.

| Typical total ion chromatogram of volatile oil in CRP
GC-MS analysis has been the most popular and powerful assistant technique to identify volatile constituents of EOs. In this study, methanol extracts of CRP samples collected from different citrusproducing areas in China were analyzed by GC-MS analysis under optimal conditions. A total of typical total ion chromatograms (TICs) from CRP samples obtained are shown in Figure 4, which exhibits the differences in TICs of the volatile oils of CRP samples among different cultivars collected from different major citrus-producing areas in China. Apparently, as can be seen, each TIC is completely complex and diverse analytic system. Additionally, from Figure 1, there are significant differences on the peak height of the same retention time in CRP samples. It seems that different peak height is on behalf of the concentration of chemical constituent, and with all the spectra available, the chromatograms of several chief peaks can be easily detected.
Then, the higher peak cluster represents the stronger content of components. Eventually, a library search was carried out for all the peaks using the NIST 08 s.L MS database Figure 5.

| Comparative analysis of volatile components in CRP
Using GC-MS analysis, the quantity of the constituents tested was Sesquiterpenes were elemene, α-farnesene, α-cubebene, F I G U R E 4 Total ion chromatography of volatile oil from CRP. Temperature programmed gas chromatography (TPGC) and electron impact (EI) ion source were used for the analysis of complex samples that we described in the Materials and Methods. Constituents were identified by comparison of authentic compounds with reference spectra in the computer library (NIST 08s.L MS database) and confirmed by comparison of those authentic compounds with data in literature. CRP of (a) Citrus reticulata "Chachi," (b) C. reticulata "Dahongpao," (c) C. reticulata "Subcompressa," (d) C. reticulata "Erythrosa," (e) C. reticulata "Shiyueju," (f) C. reticulata "Kinokuni," (g) C. reticulata "Speciosa," (h) C. reticulata "Tangerina," (i) C. reticulata "Unshiu," and (j) C. reticulata "Ponkan" valencene, α-gurjunene, α-caryophyllene, (+)-aromadendrene, and aristolene. In addition, the fatty acid principally include (Z,Z)-9,12tadecadienoic acid, (Z,Z,Z)-9,12,15-tadecatrienoic acid, octadecanoic acid, and hexadecanoic acid. Immediately following, gas chromatography area normalization method for the determination of the relative content of each component was used. The relative percentage concentrations of the principal chromatographic peak were measured with peak area normalization, which was summarized in Table 8. The types and contents of volatile oil components in part are similar, but they have certain differences in CRP from various regions. The data in Table 8 visibly indicated that the con- EOs (Xie, Yuan, Li, & Tang, 2001;Xu & Zhao, 1995;Xu, Zhao, Zhou, Ding, & Yu, 1994;Zhou et al., 2009). Plentiful of literatures concerning its efficacy in recent years have been published on a wide variety of articles. Moreover, chemical components may lead to different bioactive effects to some extent, especially the main bioactive compounds such as d-limonene, α-pinene, myrcene, and so on. In fact, the amounts of bioactive compounds play more important role in curing some diseases in TCMs used in China. A large and growing body of literature has investigated that the d-limonene plays a significant role in antibacterial action, aroma enhancement function, and antitussive, expectorant, anti-asthmatic, anti-cancer, and other effects (Asamoto et al., 2002;Bezerra, Costa, & Nogueira, 2013;Huang, Sun, Long, & Sun, 2015;Shen, 2002). Significant antimicrobial activity of α-terpineol has been proved (Cosentino et al., 1999). Previous studies have reported α-pinene has antitussive, expectorant, antibacterial, and antifungal effects (Xia & Yu, 2000). There is a large volume of published studies describing the role of linalool has analgesic, anxiolytic, sedative hypnosis, T A B L E 7 (Continued) flavones (PMFs), even the contents of those higher than the traditional famous region drug of GCP (Luo et al., 2017), similarly this experiment also found that the EO extraction yield and main chemical ingredients such as limonene of C. reticulata "Ponkan" and C. reticulata "Shiyueju" have come to the same conclusion.

| CONCLUSION
In this study, a simple and sensitive GC-MS method provided a comprehensive analysis in aroma compositions and HPLC method was de-