# Construction and Chemical Profile on “Activity Fingerprint” of Citri Reticulatae Pericarpium from Different Cultivars Based on HPLC-UV, LC/MS-IT-TOF, and Principal Component Analysis

Authors: Guodong Zheng; Yingxin Chao; Meixia Luo; Bin Xie; Dedong Zhang; Pingjun Hu; Xiujuan Yang; Depo Yang; Minyan Wei
Journal: Evidence-Based Complementary and Alternative Medicine
 (2020)

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## Abstract

Citri Reticulatae Pericarpium (CRP), known as Chenpi (CP) in Chinese, is a medicinal food for health and fitness. In order to find out the characteristic activity chemicals distinguishing various cultivars of CRP and provide a reference for effective development of citrus resources, an “activity fingerprint” of CRP from 21 different cultivars was established based on the evaluation of antitussive and expectorant activities. There were 18 common peaks in the HPLC fingerprint, of which 3 flavonoid glycosides and 14 polymethoxyflavonoids (PMFs) were identified by LC/MS-IT-TOF. Furthermore, five characteristic chemicals were determined and similarity calculation with principal component analysis (PCA) was combined together to compare the similarity and difference among various cultivars. The results showed that some other cultivars were also similar toC. reticulata “Chachi” except for C. reticulata “Tangerina” and C. reticulata “Dahongpao” recorded in Chinese Pharmacopoeia. Most importantly, the peels of C. reticulata “Shiyueju,” C. reticulata “Ponkan,” C. reticulata “Tribute,” and C. reticulata “Bayueju,” traditionally rarely used for medicinal food, were highly similar to that of C. reticulata “Chachi” and rich in bioactive flavonoids, which can be considered the effective medicinal resources of CRP.


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## Body

## 1. Introduction

Citri Reticulatae Pericarpium (CRP), also known as Chenpi (CP) in Chinese, the dry mature peel ofCitrus reticulata Blanco or its cultivated varieties [1], has been traditionally used as both nutritional food and herbal medicine for clinic treatment of cough with phlegm, vomiting and diarrhea, and stomach ache [2]. There are four cultivars of CRP officially listed in Chinese Pharmacopoeia (2015 version), such as C. reticulata “Chachi,” C. reticulata “Dahongpao,” C. reticulata “Tangerina,” and C. reticulata “Unshiu.” Among them, the desiccative ripe peel of Citrus reticulata “Chachi,” called Guang Chenpi (GCP) in Chinese and produced chiefly in Xinhui (Guangdong, China), is well known as a traditional genuine herb with high quality. However, there are no more uniform quality standards on CRP application and other nonlisted cultivars were often sold at best quality prices, resulting in a confusing market with uneven quality of CRP. Thus, it is necessary to establish a scientific method and find the characteristic activity chemicals distinguishing various cultivars of CRP for quality control.High-performance liquid chromatography (HPLC) fingerprint analysis has recently been applied as a quality evaluation method with high specificity, stability, reproducibility, and integrity because a large number of samples could be well analyzed by describing the similarities and differences through similarity results [3, 4]. Although it is a good assumption to evaluate and compare a great number of CRP samples through the HPLC fingerprint, previous studies mainly focused on a single cultivar of C. reticulata “Chachi” from different places of Guangdong Province or four recorded cultivars [5], while other nonrecorded but common cultivars have not been taken into consideration. It is more reliable to find out characteristics distinguishing varieties to protect the health of the CRP market when adopting as many different cultivars as possible for quality comparison. In addition, the HPLC fingerprint is just a kind of qualitative analysis and the compounds corresponding to its characteristic peak have not been profiled [6], so it is of significance to further study the characteristic components in combination with other identification and quantification techniques.Flavonoids have been proved to be the main chemical components in CRP, extracted mostly from methanol, and distributed into two types: flavonoid glycosides and polymethoxyflavonoids (PMFs). And recently, pharmacological studies reported for individual flavonoids described anti-inflammatory [7, 8], antitumor [9, 10], antibacterial [11, 12], and antioxidant activities [13, 14] and cardiovascular protection [15, 16]. However, there are few reports about the pharmacological activity of the mixed CRP extract so that the HPLC fingerprint has not yet been proved to relate to active compounds. Moreover, it is uncertain whether the methanol extract of CRP (MECRP) has antitussive and expectorant activities. The antitussive and expectorant activity evaluation of the MECRP played an indispensable part of “activity fingerprint” for systematical quality control.In this study, 88 samples including 21 different cultivars were used for quality analysis. The MECRP was firstly prepared on the animal models for antitussive and expectorant activity evaluation. HPLC and HPLC combined with ion-trap and time-of-flight mass spectrometry (LC/MS-IT-TOF) were then applied to clarify the characteristic chemicals corresponding to each common peak in the fingerprint and determine the content of five main flavonoids. Finally, similarity calculation and principal component analysis (PCA) were combined to compare the similarity and difference among various cultivars. Overall, this effective method of “activity fingerprint” would not only contribute to establishing an excellent quality evaluation system of CRP to find out the characteristic activity chemicals distinguishing various cultivars but also provide a reference for the effective development of citrus resources.


## 2. Materials and Methods

### 2.1. Plant and Animal Materials

Eighty-eight batches of samples including 21 different cultivars were gathered from different major citrus-producing regions in China (Table1). The sample S1 (Lot no. 20141016) is chosen as reference crude herb for the activity assay and method validation with the qualification of China’s protected geographical indication product, provided by the natural planting base in Luokeng Town by Xinbaotang Company. The voucher specimens, appraised by Prof. Bo Wu, have been deposited at the Laboratory of Pharmacognosy, Guangzhou Medical University, Guangdong Province, China.Table 1
Similarity evaluation and contents of five compounds of CRP collected from different regions in China.
No.
Cultivars
Specific origins
Harvest time
Storage (years)
Similarity
Average similarity
Contents of five flavonoids (mg/g,n = 3)a,b
1
2
3
4
5
S1
C. reticulata “Chachi” from the Xinhui region
Luokeng, Xinhui, Guangdong
2014.10.16
4
0.992
0.994
32.24 ± 0.01
3.52 ± 0.00
0.34 ± 0.00
2.43 ± 0.00
0.26 ± 0.00
S2
Yinhuwan, Xinhui, Guangdong
2014.10.14
4
0.999
29.62 ± 0.02
3.72 ± 0.00
0.35 ± 0.00
2.39 ± 0.00
0.28 ± 0.00
S3
Sanjiang, Xinhui, Guangdong
2014.11.18
4
0.998
27.59 ± 0.05
3.27 ± 0.01
0.27 ± 0.00
2.14 ± 0.00
0.22 ± 0.00
S4
Xiaogang, Xinhui, Guangdong
2014.10.24
4
0.994
28.67 ± 0.01
4.31 ± 0.00
0.38 ± 0.00
2.82 ± 0.00
0.26 ± 0.00
S5
Tianlu, Xinhui, Guangdong
2014.11.22
4
0.995
38.15 ± 0.02
3.82 ± 0.00
0.39 ± 0.00
2.41 ± 0.00
0.28 ± 0.00
S6
Yanan, Xinhui, Guangdong
2014.10.01
4
0.993
38.51 ± 0.03
5.03 ± 0.00
0.44 ± 0.00
3.14 ± 0.00
0.47 ± 0.00
S7
Siqian, Xinhui, Guangdong
2014.11.22
4
0.996
26.72 ± 0.03
2.63 ± 0.00
0.18 ± 0.00
1.73 ± 0.00
0.21 ± 0.00
S8
Shenlv, Xinhui, Guangdong
2015.10.11
3
0.991
18.14 ± 0.00
2.84 ± 0.00
0.28 ± 0.00
1.76 ± 0.00
0.16 ± 0.00
S9
Shuangshui, Xinhui, Guangdong
2014.11.02
4
0.992
21.38 ± 0.01
3.56 ± 0.00
0.37 ± 0.00
2.18 ± 0.00
0.24 ± 0.00
S10
Chakeng, Xinhui, Guangdong
2014.11.25
4
0.997
30.27 ± 0.02
2.27 ± 0.00
0.19 ± 0.00
1.67 ± 0.00
0.19 ± 0.00
S11
Nantan, Xinhui, Guangdong
2014.10.20
4
0.991
30.10 ± 0.01
3.92 ± 0.00
0.22 ± 0.00
3.18 ± 0.00
0.25 ± 0.00
S12
Qibao, Xinhui, Guangdong
2014.11.16
4
0.993
24.33 ± 0.01
2.90 ± 0.00
0.26 ± 0.00
2.16 ± 0.00
0.28 ± 0.00
S13
C. reticulata “Chachi” from other regions
Kaiping, Jiangmen, Guangdong
2014.09.26
4
0.974
0.974
43.09 ± 0.03
4.45 ± 0.00
0.34 ± 0.00
3.10 ± 0.00
0.36 ± 0.00
S14
Heshan, Jiangmen, Guangdong
2014.11.17
4
0.991
33.75 ± 0.03
3.30 ± 0.00
0.28 ± 0.00
2.70 ± 0.00
0.30 ± 0.00
S15
Gaoyao, Zhaoqing, Guangdong
2014.10.21
4
0.975
28.56 ± 0.01
2.93 ± 0.00
0.22 ± 0.00
2.75 ± 0.00
0.27 ± 0.00
S16
Gaoyao, Zhaoqing, Guangdong
2014.11.23
4
0.968
57.51 ± 0.02
2.67 ± 0.00
0.18 ± 0.00
2.66 ± 0.00
0.30 ± 0.00
S17
Wengyuanbazi, Shaoguan, Guangdong
2014.11.20
4
0.971
34.69 ± 0.01
3.86 ± 0.00
0.30 ± 0.00
2.78 ± 0.00
0.35 ± 0.00
S18
Longmen, Huizhou, Guangdong
2014.10.15
4
0.977
39.32 ± 0.01
2.91 ± 0.00
0.31 ± 0.00
2.40 ± 0.00
0.31 ± 0.00
S19
Longcheng, Huizhou, Guangdong
2014.10.18
4
0.979
36.47 ± 0.00
3.65 ± 0.00
0.23 ± 0.00
2.93 ± 0.00
0.34 ± 0.00
S20
Xinxing, Yunfu, Guangdong
2014.11.06
4
0.969
29.42 ± 0.04
4.57 ± 0.01
0.39 ± 0.00
3.58 ± 0.01
0.45 ± 0.00
S21
Cangwu, Wuzhou, Guangxi
2014.10.17
4
0.963
36.33 ± 0.10
5.25 ± 0.01
0.33 ± 0.00
3.98 ± 0.01
0.36 ± 0.00
S22
Lingshan, Qinzhou, Guangxi
2014.11.19
4
0.975
41.14 ± 0.00
4.07 ± 0.00
0.26 ± 0.00
3.16 ± 0.00
0.41 ± 0.00
S23
C. reticulata “Subcompressa”
Huangyan, Taizhou, Zhejiang
2014.12.10
4
0.924
0.885
40.63 ± 0.00
2.77 ± 0.00
0.48 ± 0.00
2.15 ± 0.00
0.27 ± 0.00
S24
Huangyan, Taizhou, Zhejiang
2014.11.20
4
0.846
53.07 ± 0.02
2.07 ± 0.00
0.34 ± 0.00
1.86 ± 0.00
0.19 ± 0.00
S25
C. reticulata “Wuyueju”
Sihui, Zhaoqing, Guangdong
2014.12.24
4
0.938
0.941
24.98 ± 0.02
3.54 ± 0.00
0.07 ± 0.00
1.38 ± 0.00
0.14 ± 0.00
S26
Deqing, Zhaoqing, Guangdong
2014.12.25
4
0.941
33.49 ± 0.01
4.44 ± 0.00
0.06 ± 0.00
1.73 ± 0.00
0.22 ± 0.00
S27
Sihui, Zhaoqing, Guangdong
2014.12.25
4
0.943
33.13 ± 0.00
4.22 ± 0.00
0.06 ± 0.00
1.68 ± 0.00
0.18 ± 0.00
S28
C. reticulata “Tankan”
Chenghai, Shantou, Guangdong
2015.02.18
3
0.873
0.871
17.81 ± 0.01
4.19 ± 0.00
1.07 ± 0.00
0.68 ± 0.00
0.27 ± 0.00
S29
Zhaoan, Zhangzhou, Fujian
2015.02.20
3
0.876
21.91 ± 0.00
5.55 ± 0.00
1.38 ± 0.00
1.03 ± 0.00
0.32 ± 0.00
S30
Puning, Jieyang, Guangdong
2015.08.15
3
0.866
21.48 ± 0.01
5.13 ± 0.00
1.15 ± 0.00
0.84 ± 0.00
0.31 ± 0.00
S31
C. reticulata “Biangan”
Guiping, Guigang, Guangxi
2014.12.23
4
0.765
0.765
47.57 ± 0.00
1.76 ± 0.00
0.38 ± 0.00
0.69 ± 0.00
0.08 ± 0.00
S32
C. reticulata “Nianju”
Longmen, Huizhou, Guangdong
2015.03.01
3
0.935
0.935
52.46 ± 0.01
5.54 ± 0.00
0.05 ± 0.00
2.21 ± 0.00
0.46 ± 0.00
S33
C. reticulata “Yichangju”
Zhijiang, Yichang, Hubei
2015.11.26
3
0.492
0.518
53.29 ± 0.01
0.18 ± 0.00
0.66 ± 0.00
0.10 ± 0.00
0.05 ± 0.00
S34
Honghu, Jingzhou, Hubei
2014.12.01
4
0.525
56.59 ± 0.08
0.24 ± 0.00
0.47 ± 0.00
0.18 ± 0.00
0.05 ± 0.00
S35
Yiling, Yichang, Hubei
2014.10.24
4
0.556
76.49 ± 0.06
0.62 ± 0.00
0.39 ± 0.00
0.41 ± 0.00
0.06 ± 0.00
S36
Yiling, Yichang, Hubei
2014.10.24
4
0.493
70.20 ± 0.03
0.23 ± 0.00
0.55 ± 0.00
0.13 ± 0.00
0.05 ± 0.00
S37
Dianjun, Yichang, Hubei
2014.11.27
4
0.525
53.08 ± 0.02
0.26 ± 0.00
0.59 ± 0.00
0.10 ± 0.00
0.04 ± 0.00
S38
C. reticulata “Dahongpao”
Chuanshan, Suining, Sichuan
2014.11.16
4
0.934
0.940
54.25 ± 0.02
6.64 ± 0.00
0.15 ± 0.00
2.36 ± 0.00
0.46 ± 0.00
S39
Shuangliu, Chengdu, Sichuan
2014.12.31
4
0.949
43.91 ± 0.09
5.32 ± 0.01
0.07 ± 0.00
2.48 ± 0.00
0.43 ± 0.00
S40
Fengjie, Chongqing, Sichuan
2014.12.17
4
0.929
70.68 ± 0.03
6.16 ± 0.00
0.18 ± 0.00
2.72 ± 0.00
0.45 ± 0.00
S41
Kai, Chongqing, Sichuan
2015.11.26
3
0.948
49.76 ± 0.02
6.79 ± 0.00
0.07 ± 0.00
3.00 ± 0.00
0.52 ± 0.00
S42
Anyue, Ziyang, Sichuan
2015.11.26
3
0.940
45.40 ± 0.03
6.90 ± 0.01
0.22 ± 0.00
2.62 ± 0.00
0.51 ± 0.00
S43
C. reticulata “Shiyueju”
Gaoyao, Zhaoqing, Guangdong
2014.12.25
4
0.958
0.962
54.18 ± 0.02
5.55 ± 0.00
0.09 ± 0.00
4.29 ± 0.00
0.22 ± 0.00
S44
Gaoyao, Zhaoqing, Guangdong
2014.12.24
4
0.962
42.86 ± 0.04
5.45 ± 0.01
0.05 ± 0.00
4.36 ± 0.00
0.20 ± 0.00
S45
Lipu, Guilin, Guangxi
2014.12.26
4
0.963
43.54 ± 0.00
5.23 ± 0.00
0.05 ± 0.00
4.05 ± 0.00
0.16 ± 0.00
S46
Guiping, Guigang, Guangxi
2014.12.24
4
0.957
40.48 ± 0.01
4.12 ± 0.00
0.03 ± 0.00
3.50 ± 0.00
0.11 ± 0.00
S47
Yingde, Qingyuan, Guangdong
2014.12.28
4
0.96
50.18 ± 0.01
5.39 ± 0.00
0.10 ± 0.00
3.69 ± 0.00
0.20 ± 0.00
S48
Yingde, Qingyuan, Guangdong
2014.12.25
4
0.965
38.74 ± 0.00
4.77 ± 0.00
0.10 ± 0.00
3.68 ± 0.00
0.14 ± 0.00
S49
Zengcheng, Guangzhou, Guangdong
2014.12.25
4
0.967
40.40 ± 0.01
5.54 ± 0.00
0.11 ± 0.00
3.73 ± 0.00
0.21 ± 0.00
S50
C. reticulata “Unshiu”
Daxiang, Shaoyang, Hunan
2014.10.28
4
0.473
0.558
65.73 ± 0.06
0.07 ± 0.00
0.49 ± 0.00
0.06 ± 0.00
0.05 ± 0.00
S51
Shigu, Hengyang, Hunan
2014.10.30
4
0.540
61.06 ± 0.05
0.42 ± 0.00
0.60 ± 0.00
0.30 ± 0.00
0.08 ± 0.00
S52
Zhangshu, Yichun, Jiangxi
2014.10.26
4
0.657
56.94 ± 0.05
0.94 ± 0.00
0.47 ± 0.00
0.69 ± 0.00
0.19 ± 0.00
S53
Lingui, Guilin, Guangxi
2014.11.28
4
0.563
57.07 ± 0.01
0.40 ± 0.00
0.69 ± 0.00
0.19 ± 0.00
0.02 ± 0.00
S54
C. reticulata “Xunwuju”
Sanbiao, Xunwu, Jiangxi
2014.11.01
4
0.485
0.489
71.18 ± 0.01
0.18 ± 0.00
0.51 ± 0.00
0.14 ± 0.00
0.09 ± 0.00
S55
Shuiyuan, Xunwu, Jiangx
2014.11.01
4
0.480
61.02 ± 0.02
0.09 ± 0.00
0.36 ± 0.00
0.09 ± 0.00
0.05 ± 0.00
S56
Luoshan, Xunwu, Jiangxi
2014.11.01
4
0.501
64.51 ± 0.09
0.32 ± 0.00
0.92 ± 0.00
0.19 ± 0.00
0.06 ± 0.00
S57
C. reticulata “Suavissima”
Pingyang, Wenzhou, Zhejiang
2014.11.01
4
0.945
0.942
75.54 ± 0.03
9.48 ± 0.02
0.11 ± 0.00
7.35 ± 0.02
1.97 ± 0.01
S58
Lucheng, Wenzhou, Zhejiang
2014.12.28
4
0.942
50.53 ± 0.03
7.30 ± 0.00
0.10 ± 0.00
5.22 ± 0.01
1.33 ± 0.00
S59
Ouhai, Wenzhou, Zhejiang
2014.12.01
4
0.939
60.55 ± 0.02
7.25 ± 0.00
0.09 ± 0.00
5.84 ± 0.00
1.32 ± 0.00
S60
C. reticulata “Qingjiangju”
Qingjiang, Zixing, Hunan
2014.11.30
4
0.485
0.504
77.43 ± 0.04
0.02 ± 0.00
0.41 ± 0.00
0.05 ± 0.00
0.05 ± 0.00
S61
Qingjiang, Zixing, Hunan
2014.10.28
4
0.506
67.26 ± 0.04
0.14 ± 0.00
0.43 ± 0.00
0.09 ± 0.00
0.05 ± 0.00
S62
Sishui, Zixing, Hunan
2014.10.28
4
0.521
75.31 ± 0.03
0.27 ± 0.00
0.69 ± 0.00
0.14 ± 0.00
0.05 ± 0.00
S63
C. reticulata “Tribute”
Deqing, Zhaoqing, Guangdong
2014.11.16
4
0.945
0.949
48.52 ± 0.01
5.59 ± 0.00
0.46 ± 0.00
3.95 ± 0.00
1.18 ± 0.00
S64
Deqing, Zhaoqing, Guangdong
2014.11.04
4
0.953
43.90 ± 0.01
5.65 ± 0.00
0.45 ± 0.00
4.10 ± 0.00
1.15 ± 0.00
S65
Deqing, Zhaoqing, Guangdong
2014.11.30
4
0.949
26.97 ± 0.00
4.46 ± 0.00
0.35 ± 0.00
2.89 ± 0.00
0.88 ± 0.00
S66
C. reticulata “Kinokuni”
Nanfeng, Fuzhou, Jiangxi
2014.10.24
4
0.951
0.953
46.90 ± 0.01
6.02 ± 0.00
0.22 ± 0.00
3.22 ± 0.00
0.78 ± 0.00
S67
Nanfeng, Fuzhou, Jiangxi
2014.11.20
4
0.953
54.85 ± 0.01
9.29 ± 0.00
0.30 ± 0.00
5.06 ± 0.00
1.01 ± 0.00
S68
Ningdu, Ganzhou, Jiangxi
2014.11.28
4
0.945
51.61 ± 0.00
6.40 ± 0.00
0.19 ± 0.00
4.42 ± 0.00
0.73 ± 0.00
S69
Nanfeng, Fuzhou, Jiangxi
2014.10.26
4
0.956
46.97 ± 0.01
7.05 ± 0.00
0.20 ± 0.00
3.58 ± 0.00
0.66 ± 0.00
S70
Ningdu, Ganzhou, Jiangxi
2014.12.03
4
0.961
51.73 ± 0.01
6.40 ± 0.00
0.19 ± 0.00
4.44 ± 0.00
0.74 ± 0.00
S71
C. reticulata “Erythrosa”
Mengquan, Shimen, Hunan
2014.10.29
4
0.571
0.539
80.52 ± 0.10
0.62 ± 0.00
0.40 ± 0.00
0.33 ± 0.00
0.08 ± 0.00
S72
Chujiang, Shimen, Hunan
2014.10.28
4
0.512
65.63 ± 0.05
0.16 ± 0.00
0.36 ± 0.00
0.11 ± 0.00
0.05 ± 0.00
S73
Zaoshi, Shimen, Hunan
2014.10.29
4
0.567
47.05 ± 0.0
0.36 ± 0.00
0.77 ± 0.00
0.19 ± 0.00
0.05 ± 0.00
S74
Xinguan, Shimen, Hunan
2015.10.21
3
0.507
55.50 ± 0.03
0.11 ± 0.00
0.37 ± 0.00
0.08 ± 0.00
0.05 ± 0.00
S75
C. reticulata “Bayueju”
Dongbei, Lianzhou, Guangdong
2014.11.29
4
0.941
0.945
41.15 ± 0.02
6.53 ± 0.01
0.04 ± 0.00
5.28 ± 0.01
0.42 ± 0.00
S76
Dongbei, Lianzhou, Guangdong
2014.11.23
4
0.943
38.62 ± 0.01
4.99 ± 0.00
0.04 ± 0.00
4.33 ± 0.00
0.30 ± 0.00
S77
Lipu, Guilin, Guangxi
2014.10.28
4
0.950
41.50 ± 0.03
5.34 ± 0.00
0.16 ± 0.00
3.24 ± 0.00
0.35 ± 0.00
S78
Sihui, Zhaoqing, Guangdong
2014.10.29
4
0.947
46.37 ± 0.01
7.13 ± 0.00
0.08 ± 0.00
5.13 ± 0.00
0.46 ± 0.00
S79
C. reticulata “Tangerina”
Minhou, Fuzhou, Fujian
2013.01.08
5
0.937
0.943
45.35 ± 0.02
4.29 ± 0.00
0.06 ± 0.00
2.06 ± 0.00
0.34 ± 0.00
S80
Fuqing, Fuzhou, Fujian
2015.12.30
3
0.949
43.92 ± 0.02
6.69 ± 0.00
0.17 ± 0.00
3.17 ± 0.00
0.42 ± 0.00
S81
C. reticulata “Yangshanju”
Chenxi, Wuzhou, Guangxi
2014.11.20
4
0.953
0.950
43.44 ± 0.02
4.38 ± 0.00
0.10 ± 0.00
2.62 ± 0.00
0.30 ± 0.00
S82
Yanan, Xinhui, Guangdong
2014.10.28
4
0.946
42.16 ± 0.02
7.65 ± 0.00
0.35 ± 0.00
3.74 ± 0.00
0.47 ± 0.00
S83
C. reticulata “Linhaiju”
Yongquan, Linhai, Zhejiang
2014.10.27
4
0.494
0.499
70.18 ± 0.06
0.10 ± 0.00
0.40 ± 0.00
0.06 ± 0.00
0.05 ± 0.00
S84
Yongquan, Linhai, Zhejiang
2014.10.29
4
0.503
62.52 ± 0.07
0.07 ± 0.00
0.33 ± 0.00
0.07 ± 0.00
0.05 ± 0.00
S85
C. reticulata “Ponkan”
Pinghe, Zhangzhou, Fujian
2015.02.23
3
0.946
0.957
49.34 ± 0.04
6.05 ± 0.00
0.10 ± 0.00
4.46 ± 0.01
0.92 ± 0.00
S86
Kecheng, Quzhou, Zhejiang
2015.02.17
3
0.967
54.22 ± 0.01
6.70 ± 0.01
0.05 ± 0.00
4.19 ± 0.00
0.94 ± 0.00
S87
Yongchun, Quanzhou, Fujian
2015.02.10
3
0.959
49.07 ± 0.02
5.46 ± 0.00
0.04 ± 0.00
4.27 ± 0.00
0.90 ± 0.00
S88
Dangyang, Yichang, Hubei
2015.02.15
3
0.954
49.23 ± 0.01
5.41 ± 0.00
0.02 ± 0.00
3.65 ± 0.00
0.96 ± 0.00
a1–5 denote the components of hesperidin, nobiletin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, tangeretin, and 5-hydroxy-6,7,8, 3′,4′-pentamethoxyflavone, respectively. bThe content values of five flavonoids are shown as mean ± standard deviation (SD).SPF Kunming mice weighing 18 to 22 g, half male and female, were provided by the Medical Experimental Animal Center of Guangdong Province (License SCXK[Yue] 2013-0002). Animals were fasted for 12 hours before the first and the final administration but given water ad libitum. All the experimental procedures were performed in accordance with the Regulation on the Administration of Laboratory Animals issued by the State Council of China. All animals were killed by cervical dislocation, and all efforts were made to minimize suffering.


### 2.2. Chemicals and Reagents

The positive control drug, dextromethorphan hydrobromide (DH) tablets, for the antitussive activity assay was purchased from Baiyunshan Guanghua (China). The ambroxol hydrochloride (AH) tablets, used for the expectorant activity assay, were obtained from Boehringer Ingelheim (Shanghai, China). The solvents for HPLC and LC/MS-IT-TOF analysis, HPLC-grade acetonitrile and purified water, used after filtration through membranes, were procured from Honeywell (America) and Yibao (China), respectively. Hesperidin (Chengdu Must Bio-Technology, Chengdu, China), nobiletin (Chengdu Must Bio-Technology, Chengdu, China), 3,5,6,7,8,3′,4′-heptamethoxyflavone (Weikeqi, Sichuan, China), tangeretin (Chengdu Must Bio-Technology, Chengdu, China), and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone (Spring & Autumn, Nanjing, China) were all of reference standard grade, with the high purity above 98%. All other reagents were of analytical grade and pure.


### 2.3. Standard Solution and MECRP Preparation

Each reference of the five flavonoids was dissolved together in methanol at an appropriate concentration to obtain the mixed standard solution. Each sample powder was extracted ultrasonically with 100 times of methanol (w/v) for 30 minutes at 320 W [17]. The solvent of the sample S1 was eliminated by a rotary evaporator to obtain the dried MECRP (extraction rate = 31%, g/g), which was dispersed to different concentrations with distilled water for the activity assay. Meanwhile, all the sample solutions with the MECRP were filtered through a 0.22 μm membrane for HPLC-UV and LC/MS-IT-TOF analysis.


### 2.4. Evaluation of Antitussive and Expectorant Activities of MECRP

#### 2.4.1. Antitussive Activity Assay

Fifty Kunming mice were randomly allocated to five groups of half male and half female each: control, MECRP-20, MECRP-40, MECRP-80, and DH-60. The control group and standard group received normal saline (0.9%) and DH (60 mg/kg) daily for 7 continuous days, respectively, while the test groups were administered orally with the MECRP (20, 40, and 80 mg/kg). After final administration for an hour, each animal was put in a sealed glass container and exposed to 25% ammonia water aerosol for 20 s by an ultrasonic nebulizer [18]. The mice were then removed to observe the incubation period of cough and bouts of cough within 3 minutes.


#### 2.4.2. Expectorant Activity Assay

Kunming mice were divided into five groups (n = 10/group): control, MECRP-20, MECRP-40, MECRP-80, and AH-40, and each of the groups was medicated orally continuously for 7 days (1 time/day). The control group and treated groups were administrated based on the method of the antitussive activity assay, whereas the standard group received AH 40 mg/kg orally. Thirty minutes after the final administration, each mouse was injected intraperitoneally with 0.5 mL phenol red solution in saline and killed by cervical dislocation 30 minutes later. The tracheas of the same length were taken from the thyroid cartilage to the trachea branch of the mice and washed ultrasonically with 3 mL NaOH solution (0.03 mol/L) in saline for 10 minutes [19]. Moreover, the absorbance of phenol red in the solution was measured spectrophotometrically at a wavelength of 546 nm, and its content was calculated under the linear regression equation y = 0.1428x + 0.0062 (R2 = 0.9993) (“x” denotes the concentration of phenol red solution and “y” denotes the absorbance).


#### 2.4.3. Statistical Analysis

The results of activity assays were statistically analyzed by SPSS (Version 16.0) and GraphPad Prism software (Version 5.0), which are expressed as mean ± standard deviation (SD). One-way ANOVA was used for the data analysis, followed by the LSD test to compare the differences between control and test groups.P<0.05 was considered statistically significant in this study.


### 2.5. HPLC Fingerprint Analysis

The HPLC condition of the MECRP was performed in a previous method described by Luo et al. [17]. SIL-20A HPLC (Shimadzu, Japan) equipped with an SPD-20A UV detector and a Diamonsil C18 column (250 × 4.6 mm, i.d. 5 μm) was used for HPLC analysis. The mobile phase was solution A (water) and solution B (acetonitrile) with the following gradient program: 0–15 min, 15–40% B; 15–35 min, 40–50% B; 35–40 min, 50–75% B; and 40–50 min, 75–85% B. Other HPLC conditions are shown as follows: flow rate: 1.0 mL/min; column temperature: 25°C; injection volume: 20 μL; and detection wavelength: 283 nm and 330 nm. The chromatograms (330 nm) of C. reticulata “Chachi” samples produced in Xinhui (S1–S12) were firstly imported into the software “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2012)” to establish the GCP fingerprint. With the median method and choosing three chromatographic peaks of hesperidin, nobiletin, and tangeretin for multipoint calibration, a GCP common pattern was generated. Next, the final fingerprint was established according to the representative chromatograms of remaining cultivars (S13–S88) together with the GCP common pattern. The similarity values of different cultivars were finally calculated based on the GCP common pattern.


### 2.6. LC/MS-IT-TOF Analysis

LC/MS-IT-TOF analysis was carried out as previously reported with a minor modification [20]. The HPLC instrument equipped with the ion-trap and time-of-flight mass spectrometer (Shimadzu, Japan) through electrospray ionization (ESI) was used. The compounds of extraction were isolated from the HPLC system with the same condition as in HPLC analysis mentioned above. The electrospray ionization (ESI) source was operated in a positive ionization mode, and its conditions are listed as follows: nitrogen gas flow, 1.5 L/min; dry gas temperature, 200°C; fragmentation voltage, 1.62 kV; nebulizer pressure, 45 psi; and full and dependent scan data acquisition, m/z 100–1000. Ion spectra were recorded from 5 to 40 eV with argon. Data processing was performed using LC/MS Solution 3.60.361 software (Shimadzu), while molecular formula prediction and elemental composition prediction were done using Formula Predictor and Accurate Mass Calculator software, respectively.


### 2.7. Quantitative Determination and PCA

The components of hesperidin and other four PMFs were detected at 283 nm and 330 nm in HPLC, respectively, which were calculated using the external standard method and expressed as mean ± SD. The contents of five flavonoids in all samples were first normalized by SPSS software (Version 16.0), and then principal component analysis was performed.


## 2.1. Plant and Animal Materials

Eighty-eight batches of samples including 21 different cultivars were gathered from different major citrus-producing regions in China (Table1). The sample S1 (Lot no. 20141016) is chosen as reference crude herb for the activity assay and method validation with the qualification of China’s protected geographical indication product, provided by the natural planting base in Luokeng Town by Xinbaotang Company. The voucher specimens, appraised by Prof. Bo Wu, have been deposited at the Laboratory of Pharmacognosy, Guangzhou Medical University, Guangdong Province, China.Table 1
Similarity evaluation and contents of five compounds of CRP collected from different regions in China.
No.
Cultivars
Specific origins
Harvest time
Storage (years)
Similarity
Average similarity
Contents of five flavonoids (mg/g,n = 3)a,b
1
2
3
4
5
S1
C. reticulata “Chachi” from the Xinhui region
Luokeng, Xinhui, Guangdong
2014.10.16
4
0.992
0.994
32.24 ± 0.01
3.52 ± 0.00
0.34 ± 0.00
2.43 ± 0.00
0.26 ± 0.00
S2
Yinhuwan, Xinhui, Guangdong
2014.10.14
4
0.999
29.62 ± 0.02
3.72 ± 0.00
0.35 ± 0.00
2.39 ± 0.00
0.28 ± 0.00
S3
Sanjiang, Xinhui, Guangdong
2014.11.18
4
0.998
27.59 ± 0.05
3.27 ± 0.01
0.27 ± 0.00
2.14 ± 0.00
0.22 ± 0.00
S4
Xiaogang, Xinhui, Guangdong
2014.10.24
4
0.994
28.67 ± 0.01
4.31 ± 0.00
0.38 ± 0.00
2.82 ± 0.00
0.26 ± 0.00
S5
Tianlu, Xinhui, Guangdong
2014.11.22
4
0.995
38.15 ± 0.02
3.82 ± 0.00
0.39 ± 0.00
2.41 ± 0.00
0.28 ± 0.00
S6
Yanan, Xinhui, Guangdong
2014.10.01
4
0.993
38.51 ± 0.03
5.03 ± 0.00
0.44 ± 0.00
3.14 ± 0.00
0.47 ± 0.00
S7
Siqian, Xinhui, Guangdong
2014.11.22
4
0.996
26.72 ± 0.03
2.63 ± 0.00
0.18 ± 0.00
1.73 ± 0.00
0.21 ± 0.00
S8
Shenlv, Xinhui, Guangdong
2015.10.11
3
0.991
18.14 ± 0.00
2.84 ± 0.00
0.28 ± 0.00
1.76 ± 0.00
0.16 ± 0.00
S9
Shuangshui, Xinhui, Guangdong
2014.11.02
4
0.992
21.38 ± 0.01
3.56 ± 0.00
0.37 ± 0.00
2.18 ± 0.00
0.24 ± 0.00
S10
Chakeng, Xinhui, Guangdong
2014.11.25
4
0.997
30.27 ± 0.02
2.27 ± 0.00
0.19 ± 0.00
1.67 ± 0.00
0.19 ± 0.00
S11
Nantan, Xinhui, Guangdong
2014.10.20
4
0.991
30.10 ± 0.01
3.92 ± 0.00
0.22 ± 0.00
3.18 ± 0.00
0.25 ± 0.00
S12
Qibao, Xinhui, Guangdong
2014.11.16
4
0.993
24.33 ± 0.01
2.90 ± 0.00
0.26 ± 0.00
2.16 ± 0.00
0.28 ± 0.00
S13
C. reticulata “Chachi” from other regions
Kaiping, Jiangmen, Guangdong
2014.09.26
4
0.974
0.974
43.09 ± 0.03
4.45 ± 0.00
0.34 ± 0.00
3.10 ± 0.00
0.36 ± 0.00
S14
Heshan, Jiangmen, Guangdong
2014.11.17
4
0.991
33.75 ± 0.03
3.30 ± 0.00
0.28 ± 0.00
2.70 ± 0.00
0.30 ± 0.00
S15
Gaoyao, Zhaoqing, Guangdong
2014.10.21
4
0.975
28.56 ± 0.01
2.93 ± 0.00
0.22 ± 0.00
2.75 ± 0.00
0.27 ± 0.00
S16
Gaoyao, Zhaoqing, Guangdong
2014.11.23
4
0.968
57.51 ± 0.02
2.67 ± 0.00
0.18 ± 0.00
2.66 ± 0.00
0.30 ± 0.00
S17
Wengyuanbazi, Shaoguan, Guangdong
2014.11.20
4
0.971
34.69 ± 0.01
3.86 ± 0.00
0.30 ± 0.00
2.78 ± 0.00
0.35 ± 0.00
S18
Longmen, Huizhou, Guangdong
2014.10.15
4
0.977
39.32 ± 0.01
2.91 ± 0.00
0.31 ± 0.00
2.40 ± 0.00
0.31 ± 0.00
S19
Longcheng, Huizhou, Guangdong
2014.10.18
4
0.979
36.47 ± 0.00
3.65 ± 0.00
0.23 ± 0.00
2.93 ± 0.00
0.34 ± 0.00
S20
Xinxing, Yunfu, Guangdong
2014.11.06
4
0.969
29.42 ± 0.04
4.57 ± 0.01
0.39 ± 0.00
3.58 ± 0.01
0.45 ± 0.00
S21
Cangwu, Wuzhou, Guangxi
2014.10.17
4
0.963
36.33 ± 0.10
5.25 ± 0.01
0.33 ± 0.00
3.98 ± 0.01
0.36 ± 0.00
S22
Lingshan, Qinzhou, Guangxi
2014.11.19
4
0.975
41.14 ± 0.00
4.07 ± 0.00
0.26 ± 0.00
3.16 ± 0.00
0.41 ± 0.00
S23
C. reticulata “Subcompressa”
Huangyan, Taizhou, Zhejiang
2014.12.10
4
0.924
0.885
40.63 ± 0.00
2.77 ± 0.00
0.48 ± 0.00
2.15 ± 0.00
0.27 ± 0.00
S24
Huangyan, Taizhou, Zhejiang
2014.11.20
4
0.846
53.07 ± 0.02
2.07 ± 0.00
0.34 ± 0.00
1.86 ± 0.00
0.19 ± 0.00
S25
C. reticulata “Wuyueju”
Sihui, Zhaoqing, Guangdong
2014.12.24
4
0.938
0.941
24.98 ± 0.02
3.54 ± 0.00
0.07 ± 0.00
1.38 ± 0.00
0.14 ± 0.00
S26
Deqing, Zhaoqing, Guangdong
2014.12.25
4
0.941
33.49 ± 0.01
4.44 ± 0.00
0.06 ± 0.00
1.73 ± 0.00
0.22 ± 0.00
S27
Sihui, Zhaoqing, Guangdong
2014.12.25
4
0.943
33.13 ± 0.00
4.22 ± 0.00
0.06 ± 0.00
1.68 ± 0.00
0.18 ± 0.00
S28
C. reticulata “Tankan”
Chenghai, Shantou, Guangdong
2015.02.18
3
0.873
0.871
17.81 ± 0.01
4.19 ± 0.00
1.07 ± 0.00
0.68 ± 0.00
0.27 ± 0.00
S29
Zhaoan, Zhangzhou, Fujian
2015.02.20
3
0.876
21.91 ± 0.00
5.55 ± 0.00
1.38 ± 0.00
1.03 ± 0.00
0.32 ± 0.00
S30
Puning, Jieyang, Guangdong
2015.08.15
3
0.866
21.48 ± 0.01
5.13 ± 0.00
1.15 ± 0.00
0.84 ± 0.00
0.31 ± 0.00
S31
C. reticulata “Biangan”
Guiping, Guigang, Guangxi
2014.12.23
4
0.765
0.765
47.57 ± 0.00
1.76 ± 0.00
0.38 ± 0.00
0.69 ± 0.00
0.08 ± 0.00
S32
C. reticulata “Nianju”
Longmen, Huizhou, Guangdong
2015.03.01
3
0.935
0.935
52.46 ± 0.01
5.54 ± 0.00
0.05 ± 0.00
2.21 ± 0.00
0.46 ± 0.00
S33
C. reticulata “Yichangju”
Zhijiang, Yichang, Hubei
2015.11.26
3
0.492
0.518
53.29 ± 0.01
0.18 ± 0.00
0.66 ± 0.00
0.10 ± 0.00
0.05 ± 0.00
S34
Honghu, Jingzhou, Hubei
2014.12.01
4
0.525
56.59 ± 0.08
0.24 ± 0.00
0.47 ± 0.00
0.18 ± 0.00
0.05 ± 0.00
S35
Yiling, Yichang, Hubei
2014.10.24
4
0.556
76.49 ± 0.06
0.62 ± 0.00
0.39 ± 0.00
0.41 ± 0.00
0.06 ± 0.00
S36
Yiling, Yichang, Hubei
2014.10.24
4
0.493
70.20 ± 0.03
0.23 ± 0.00
0.55 ± 0.00
0.13 ± 0.00
0.05 ± 0.00
S37
Dianjun, Yichang, Hubei
2014.11.27
4
0.525
53.08 ± 0.02
0.26 ± 0.00
0.59 ± 0.00
0.10 ± 0.00
0.04 ± 0.00
S38
C. reticulata “Dahongpao”
Chuanshan, Suining, Sichuan
2014.11.16
4
0.934
0.940
54.25 ± 0.02
6.64 ± 0.00
0.15 ± 0.00
2.36 ± 0.00
0.46 ± 0.00
S39
Shuangliu, Chengdu, Sichuan
2014.12.31
4
0.949
43.91 ± 0.09
5.32 ± 0.01
0.07 ± 0.00
2.48 ± 0.00
0.43 ± 0.00
S40
Fengjie, Chongqing, Sichuan
2014.12.17
4
0.929
70.68 ± 0.03
6.16 ± 0.00
0.18 ± 0.00
2.72 ± 0.00
0.45 ± 0.00
S41
Kai, Chongqing, Sichuan
2015.11.26
3
0.948
49.76 ± 0.02
6.79 ± 0.00
0.07 ± 0.00
3.00 ± 0.00
0.52 ± 0.00
S42
Anyue, Ziyang, Sichuan
2015.11.26
3
0.940
45.40 ± 0.03
6.90 ± 0.01
0.22 ± 0.00
2.62 ± 0.00
0.51 ± 0.00
S43
C. reticulata “Shiyueju”
Gaoyao, Zhaoqing, Guangdong
2014.12.25
4
0.958
0.962
54.18 ± 0.02
5.55 ± 0.00
0.09 ± 0.00
4.29 ± 0.00
0.22 ± 0.00
S44
Gaoyao, Zhaoqing, Guangdong
2014.12.24
4
0.962
42.86 ± 0.04
5.45 ± 0.01
0.05 ± 0.00
4.36 ± 0.00
0.20 ± 0.00
S45
Lipu, Guilin, Guangxi
2014.12.26
4
0.963
43.54 ± 0.00
5.23 ± 0.00
0.05 ± 0.00
4.05 ± 0.00
0.16 ± 0.00
S46
Guiping, Guigang, Guangxi
2014.12.24
4
0.957
40.48 ± 0.01
4.12 ± 0.00
0.03 ± 0.00
3.50 ± 0.00
0.11 ± 0.00
S47
Yingde, Qingyuan, Guangdong
2014.12.28
4
0.96
50.18 ± 0.01
5.39 ± 0.00
0.10 ± 0.00
3.69 ± 0.00
0.20 ± 0.00
S48
Yingde, Qingyuan, Guangdong
2014.12.25
4
0.965
38.74 ± 0.00
4.77 ± 0.00
0.10 ± 0.00
3.68 ± 0.00
0.14 ± 0.00
S49
Zengcheng, Guangzhou, Guangdong
2014.12.25
4
0.967
40.40 ± 0.01
5.54 ± 0.00
0.11 ± 0.00
3.73 ± 0.00
0.21 ± 0.00
S50
C. reticulata “Unshiu”
Daxiang, Shaoyang, Hunan
2014.10.28
4
0.473
0.558
65.73 ± 0.06
0.07 ± 0.00
0.49 ± 0.00
0.06 ± 0.00
0.05 ± 0.00
S51
Shigu, Hengyang, Hunan
2014.10.30
4
0.540
61.06 ± 0.05
0.42 ± 0.00
0.60 ± 0.00
0.30 ± 0.00
0.08 ± 0.00
S52
Zhangshu, Yichun, Jiangxi
2014.10.26
4
0.657
56.94 ± 0.05
0.94 ± 0.00
0.47 ± 0.00
0.69 ± 0.00
0.19 ± 0.00
S53
Lingui, Guilin, Guangxi
2014.11.28
4
0.563
57.07 ± 0.01
0.40 ± 0.00
0.69 ± 0.00
0.19 ± 0.00
0.02 ± 0.00
S54
C. reticulata “Xunwuju”
Sanbiao, Xunwu, Jiangxi
2014.11.01
4
0.485
0.489
71.18 ± 0.01
0.18 ± 0.00
0.51 ± 0.00
0.14 ± 0.00
0.09 ± 0.00
S55
Shuiyuan, Xunwu, Jiangx
2014.11.01
4
0.480
61.02 ± 0.02
0.09 ± 0.00
0.36 ± 0.00
0.09 ± 0.00
0.05 ± 0.00
S56
Luoshan, Xunwu, Jiangxi
2014.11.01
4
0.501
64.51 ± 0.09
0.32 ± 0.00
0.92 ± 0.00
0.19 ± 0.00
0.06 ± 0.00
S57
C. reticulata “Suavissima”
Pingyang, Wenzhou, Zhejiang
2014.11.01
4
0.945
0.942
75.54 ± 0.03
9.48 ± 0.02
0.11 ± 0.00
7.35 ± 0.02
1.97 ± 0.01
S58
Lucheng, Wenzhou, Zhejiang
2014.12.28
4
0.942
50.53 ± 0.03
7.30 ± 0.00
0.10 ± 0.00
5.22 ± 0.01
1.33 ± 0.00
S59
Ouhai, Wenzhou, Zhejiang
2014.12.01
4
0.939
60.55 ± 0.02
7.25 ± 0.00
0.09 ± 0.00
5.84 ± 0.00
1.32 ± 0.00
S60
C. reticulata “Qingjiangju”
Qingjiang, Zixing, Hunan
2014.11.30
4
0.485
0.504
77.43 ± 0.04
0.02 ± 0.00
0.41 ± 0.00
0.05 ± 0.00
0.05 ± 0.00
S61
Qingjiang, Zixing, Hunan
2014.10.28
4
0.506
67.26 ± 0.04
0.14 ± 0.00
0.43 ± 0.00
0.09 ± 0.00
0.05 ± 0.00
S62
Sishui, Zixing, Hunan
2014.10.28
4
0.521
75.31 ± 0.03
0.27 ± 0.00
0.69 ± 0.00
0.14 ± 0.00
0.05 ± 0.00
S63
C. reticulata “Tribute”
Deqing, Zhaoqing, Guangdong
2014.11.16
4
0.945
0.949
48.52 ± 0.01
5.59 ± 0.00
0.46 ± 0.00
3.95 ± 0.00
1.18 ± 0.00
S64
Deqing, Zhaoqing, Guangdong
2014.11.04
4
0.953
43.90 ± 0.01
5.65 ± 0.00
0.45 ± 0.00
4.10 ± 0.00
1.15 ± 0.00
S65
Deqing, Zhaoqing, Guangdong
2014.11.30
4
0.949
26.97 ± 0.00
4.46 ± 0.00
0.35 ± 0.00
2.89 ± 0.00
0.88 ± 0.00
S66
C. reticulata “Kinokuni”
Nanfeng, Fuzhou, Jiangxi
2014.10.24
4
0.951
0.953
46.90 ± 0.01
6.02 ± 0.00
0.22 ± 0.00
3.22 ± 0.00
0.78 ± 0.00
S67
Nanfeng, Fuzhou, Jiangxi
2014.11.20
4
0.953
54.85 ± 0.01
9.29 ± 0.00
0.30 ± 0.00
5.06 ± 0.00
1.01 ± 0.00
S68
Ningdu, Ganzhou, Jiangxi
2014.11.28
4
0.945
51.61 ± 0.00
6.40 ± 0.00
0.19 ± 0.00
4.42 ± 0.00
0.73 ± 0.00
S69
Nanfeng, Fuzhou, Jiangxi
2014.10.26
4
0.956
46.97 ± 0.01
7.05 ± 0.00
0.20 ± 0.00
3.58 ± 0.00
0.66 ± 0.00
S70
Ningdu, Ganzhou, Jiangxi
2014.12.03
4
0.961
51.73 ± 0.01
6.40 ± 0.00
0.19 ± 0.00
4.44 ± 0.00
0.74 ± 0.00
S71
C. reticulata “Erythrosa”
Mengquan, Shimen, Hunan
2014.10.29
4
0.571
0.539
80.52 ± 0.10
0.62 ± 0.00
0.40 ± 0.00
0.33 ± 0.00
0.08 ± 0.00
S72
Chujiang, Shimen, Hunan
2014.10.28
4
0.512
65.63 ± 0.05
0.16 ± 0.00
0.36 ± 0.00
0.11 ± 0.00
0.05 ± 0.00
S73
Zaoshi, Shimen, Hunan
2014.10.29
4
0.567
47.05 ± 0.0
0.36 ± 0.00
0.77 ± 0.00
0.19 ± 0.00
0.05 ± 0.00
S74
Xinguan, Shimen, Hunan
2015.10.21
3
0.507
55.50 ± 0.03
0.11 ± 0.00
0.37 ± 0.00
0.08 ± 0.00
0.05 ± 0.00
S75
C. reticulata “Bayueju”
Dongbei, Lianzhou, Guangdong
2014.11.29
4
0.941
0.945
41.15 ± 0.02
6.53 ± 0.01
0.04 ± 0.00
5.28 ± 0.01
0.42 ± 0.00
S76
Dongbei, Lianzhou, Guangdong
2014.11.23
4
0.943
38.62 ± 0.01
4.99 ± 0.00
0.04 ± 0.00
4.33 ± 0.00
0.30 ± 0.00
S77
Lipu, Guilin, Guangxi
2014.10.28
4
0.950
41.50 ± 0.03
5.34 ± 0.00
0.16 ± 0.00
3.24 ± 0.00
0.35 ± 0.00
S78
Sihui, Zhaoqing, Guangdong
2014.10.29
4
0.947
46.37 ± 0.01
7.13 ± 0.00
0.08 ± 0.00
5.13 ± 0.00
0.46 ± 0.00
S79
C. reticulata “Tangerina”
Minhou, Fuzhou, Fujian
2013.01.08
5
0.937
0.943
45.35 ± 0.02
4.29 ± 0.00
0.06 ± 0.00
2.06 ± 0.00
0.34 ± 0.00
S80
Fuqing, Fuzhou, Fujian
2015.12.30
3
0.949
43.92 ± 0.02
6.69 ± 0.00
0.17 ± 0.00
3.17 ± 0.00
0.42 ± 0.00
S81
C. reticulata “Yangshanju”
Chenxi, Wuzhou, Guangxi
2014.11.20
4
0.953
0.950
43.44 ± 0.02
4.38 ± 0.00
0.10 ± 0.00
2.62 ± 0.00
0.30 ± 0.00
S82
Yanan, Xinhui, Guangdong
2014.10.28
4
0.946
42.16 ± 0.02
7.65 ± 0.00
0.35 ± 0.00
3.74 ± 0.00
0.47 ± 0.00
S83
C. reticulata “Linhaiju”
Yongquan, Linhai, Zhejiang
2014.10.27
4
0.494
0.499
70.18 ± 0.06
0.10 ± 0.00
0.40 ± 0.00
0.06 ± 0.00
0.05 ± 0.00
S84
Yongquan, Linhai, Zhejiang
2014.10.29
4
0.503
62.52 ± 0.07
0.07 ± 0.00
0.33 ± 0.00
0.07 ± 0.00
0.05 ± 0.00
S85
C. reticulata “Ponkan”
Pinghe, Zhangzhou, Fujian
2015.02.23
3
0.946
0.957
49.34 ± 0.04
6.05 ± 0.00
0.10 ± 0.00
4.46 ± 0.01
0.92 ± 0.00
S86
Kecheng, Quzhou, Zhejiang
2015.02.17
3
0.967
54.22 ± 0.01
6.70 ± 0.01
0.05 ± 0.00
4.19 ± 0.00
0.94 ± 0.00
S87
Yongchun, Quanzhou, Fujian
2015.02.10
3
0.959
49.07 ± 0.02
5.46 ± 0.00
0.04 ± 0.00
4.27 ± 0.00
0.90 ± 0.00
S88
Dangyang, Yichang, Hubei
2015.02.15
3
0.954
49.23 ± 0.01
5.41 ± 0.00
0.02 ± 0.00
3.65 ± 0.00
0.96 ± 0.00
a1–5 denote the components of hesperidin, nobiletin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, tangeretin, and 5-hydroxy-6,7,8, 3′,4′-pentamethoxyflavone, respectively. bThe content values of five flavonoids are shown as mean ± standard deviation (SD).SPF Kunming mice weighing 18 to 22 g, half male and female, were provided by the Medical Experimental Animal Center of Guangdong Province (License SCXK[Yue] 2013-0002). Animals were fasted for 12 hours before the first and the final administration but given water ad libitum. All the experimental procedures were performed in accordance with the Regulation on the Administration of Laboratory Animals issued by the State Council of China. All animals were killed by cervical dislocation, and all efforts were made to minimize suffering.


## 2.2. Chemicals and Reagents

The positive control drug, dextromethorphan hydrobromide (DH) tablets, for the antitussive activity assay was purchased from Baiyunshan Guanghua (China). The ambroxol hydrochloride (AH) tablets, used for the expectorant activity assay, were obtained from Boehringer Ingelheim (Shanghai, China). The solvents for HPLC and LC/MS-IT-TOF analysis, HPLC-grade acetonitrile and purified water, used after filtration through membranes, were procured from Honeywell (America) and Yibao (China), respectively. Hesperidin (Chengdu Must Bio-Technology, Chengdu, China), nobiletin (Chengdu Must Bio-Technology, Chengdu, China), 3,5,6,7,8,3′,4′-heptamethoxyflavone (Weikeqi, Sichuan, China), tangeretin (Chengdu Must Bio-Technology, Chengdu, China), and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone (Spring & Autumn, Nanjing, China) were all of reference standard grade, with the high purity above 98%. All other reagents were of analytical grade and pure.


## 2.3. Standard Solution and MECRP Preparation

Each reference of the five flavonoids was dissolved together in methanol at an appropriate concentration to obtain the mixed standard solution. Each sample powder was extracted ultrasonically with 100 times of methanol (w/v) for 30 minutes at 320 W [17]. The solvent of the sample S1 was eliminated by a rotary evaporator to obtain the dried MECRP (extraction rate = 31%, g/g), which was dispersed to different concentrations with distilled water for the activity assay. Meanwhile, all the sample solutions with the MECRP were filtered through a 0.22 μm membrane for HPLC-UV and LC/MS-IT-TOF analysis.


## 2.4. Evaluation of Antitussive and Expectorant Activities of MECRP

### 2.4.1. Antitussive Activity Assay

Fifty Kunming mice were randomly allocated to five groups of half male and half female each: control, MECRP-20, MECRP-40, MECRP-80, and DH-60. The control group and standard group received normal saline (0.9%) and DH (60 mg/kg) daily for 7 continuous days, respectively, while the test groups were administered orally with the MECRP (20, 40, and 80 mg/kg). After final administration for an hour, each animal was put in a sealed glass container and exposed to 25% ammonia water aerosol for 20 s by an ultrasonic nebulizer [18]. The mice were then removed to observe the incubation period of cough and bouts of cough within 3 minutes.


### 2.4.2. Expectorant Activity Assay

Kunming mice were divided into five groups (n = 10/group): control, MECRP-20, MECRP-40, MECRP-80, and AH-40, and each of the groups was medicated orally continuously for 7 days (1 time/day). The control group and treated groups were administrated based on the method of the antitussive activity assay, whereas the standard group received AH 40 mg/kg orally. Thirty minutes after the final administration, each mouse was injected intraperitoneally with 0.5 mL phenol red solution in saline and killed by cervical dislocation 30 minutes later. The tracheas of the same length were taken from the thyroid cartilage to the trachea branch of the mice and washed ultrasonically with 3 mL NaOH solution (0.03 mol/L) in saline for 10 minutes [19]. Moreover, the absorbance of phenol red in the solution was measured spectrophotometrically at a wavelength of 546 nm, and its content was calculated under the linear regression equation y = 0.1428x + 0.0062 (R2 = 0.9993) (“x” denotes the concentration of phenol red solution and “y” denotes the absorbance).


### 2.4.3. Statistical Analysis

The results of activity assays were statistically analyzed by SPSS (Version 16.0) and GraphPad Prism software (Version 5.0), which are expressed as mean ± standard deviation (SD). One-way ANOVA was used for the data analysis, followed by the LSD test to compare the differences between control and test groups.P<0.05 was considered statistically significant in this study.


## 2.4.1. Antitussive Activity Assay

Fifty Kunming mice were randomly allocated to five groups of half male and half female each: control, MECRP-20, MECRP-40, MECRP-80, and DH-60. The control group and standard group received normal saline (0.9%) and DH (60 mg/kg) daily for 7 continuous days, respectively, while the test groups were administered orally with the MECRP (20, 40, and 80 mg/kg). After final administration for an hour, each animal was put in a sealed glass container and exposed to 25% ammonia water aerosol for 20 s by an ultrasonic nebulizer [18]. The mice were then removed to observe the incubation period of cough and bouts of cough within 3 minutes.


## 2.4.2. Expectorant Activity Assay

Kunming mice were divided into five groups (n = 10/group): control, MECRP-20, MECRP-40, MECRP-80, and AH-40, and each of the groups was medicated orally continuously for 7 days (1 time/day). The control group and treated groups were administrated based on the method of the antitussive activity assay, whereas the standard group received AH 40 mg/kg orally. Thirty minutes after the final administration, each mouse was injected intraperitoneally with 0.5 mL phenol red solution in saline and killed by cervical dislocation 30 minutes later. The tracheas of the same length were taken from the thyroid cartilage to the trachea branch of the mice and washed ultrasonically with 3 mL NaOH solution (0.03 mol/L) in saline for 10 minutes [19]. Moreover, the absorbance of phenol red in the solution was measured spectrophotometrically at a wavelength of 546 nm, and its content was calculated under the linear regression equation y = 0.1428x + 0.0062 (R2 = 0.9993) (“x” denotes the concentration of phenol red solution and “y” denotes the absorbance).


## 2.4.3. Statistical Analysis

The results of activity assays were statistically analyzed by SPSS (Version 16.0) and GraphPad Prism software (Version 5.0), which are expressed as mean ± standard deviation (SD). One-way ANOVA was used for the data analysis, followed by the LSD test to compare the differences between control and test groups.P<0.05 was considered statistically significant in this study.


## 2.5. HPLC Fingerprint Analysis

The HPLC condition of the MECRP was performed in a previous method described by Luo et al. [17]. SIL-20A HPLC (Shimadzu, Japan) equipped with an SPD-20A UV detector and a Diamonsil C18 column (250 × 4.6 mm, i.d. 5 μm) was used for HPLC analysis. The mobile phase was solution A (water) and solution B (acetonitrile) with the following gradient program: 0–15 min, 15–40% B; 15–35 min, 40–50% B; 35–40 min, 50–75% B; and 40–50 min, 75–85% B. Other HPLC conditions are shown as follows: flow rate: 1.0 mL/min; column temperature: 25°C; injection volume: 20 μL; and detection wavelength: 283 nm and 330 nm. The chromatograms (330 nm) of C. reticulata “Chachi” samples produced in Xinhui (S1–S12) were firstly imported into the software “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2012)” to establish the GCP fingerprint. With the median method and choosing three chromatographic peaks of hesperidin, nobiletin, and tangeretin for multipoint calibration, a GCP common pattern was generated. Next, the final fingerprint was established according to the representative chromatograms of remaining cultivars (S13–S88) together with the GCP common pattern. The similarity values of different cultivars were finally calculated based on the GCP common pattern.


## 2.6. LC/MS-IT-TOF Analysis

LC/MS-IT-TOF analysis was carried out as previously reported with a minor modification [20]. The HPLC instrument equipped with the ion-trap and time-of-flight mass spectrometer (Shimadzu, Japan) through electrospray ionization (ESI) was used. The compounds of extraction were isolated from the HPLC system with the same condition as in HPLC analysis mentioned above. The electrospray ionization (ESI) source was operated in a positive ionization mode, and its conditions are listed as follows: nitrogen gas flow, 1.5 L/min; dry gas temperature, 200°C; fragmentation voltage, 1.62 kV; nebulizer pressure, 45 psi; and full and dependent scan data acquisition, m/z 100–1000. Ion spectra were recorded from 5 to 40 eV with argon. Data processing was performed using LC/MS Solution 3.60.361 software (Shimadzu), while molecular formula prediction and elemental composition prediction were done using Formula Predictor and Accurate Mass Calculator software, respectively.


## 2.7. Quantitative Determination and PCA

The components of hesperidin and other four PMFs were detected at 283 nm and 330 nm in HPLC, respectively, which were calculated using the external standard method and expressed as mean ± SD. The contents of five flavonoids in all samples were first normalized by SPSS software (Version 16.0), and then principal component analysis was performed.


## 3. Results and Discussion

### 3.1. Antitussive and Expectorant Activities of MECRP

The effects of the MECRP on ammonia water-irritated cough (Figures1(a) and 1(b)) and tracheal phenol red excretion in mice are depicted in Figure 1(c). Statistical analysis showed significant differences between the control group and treated groups in the incubation period of cough (P<0.05), bouts of cough within 3 minutes (P<0.05), and tracheal phenol red excretion (P<0.05). The test of ammonia water-irritated cough showed that the MECRP significantly prolonged the incubation period of cough and decreased the bouts of coughs in mice, possessing a good ability to relieve cough. In addition, the tracheal phenol red excretion test illustrated that the MECRP could effectively reduce sputum with the increase of the excretion of phenol red in the trachea. It has been well suggested that the MECRP exhibited excellent antitussive and expectorant activities in mice.Figure 1
Effect of the MECRP on ammonia water-irritated cough and tracheal phenol red excretion in mice. (a) Incubation period of cough. (b) Bouts of cough. (c) Tracheal phenol red excretion. All values are expressed as mean ± SD (n = 10). ∗P<0.05 and ∗∗P<0.01 compared to the control group (one-way ANOVA followed by the LSD test).
(a)
(b)
(c)


### 3.2. Similarity Analysis on “Activity Fingerprint”

The GCP fingerprint (Figure2(a)) was firstly established by twelve batches of traditional genuine herbs from Xinhui (S1–S12) due to their high quality. And a GCP common pattern (Figure 2(b)) was further generated through the characteristics of samples S1–S12. There were 18 common peaks determined in the GCP common pattern, of which peaks 3, 9, and 12 were identified as hesperidin, nobiletin, and tangeretin, respectively. According to statistical analysis, the common peak area of each sample (S1–S12) accounted for 97.276%–98.870% (>90%) of total peak areas. And all of the accuracy, repeatability, and stability results showed that the RSD of relative retention time and relative peak area of the 18 common peaks was below 3%, satisfying the demands of fingerprint.Figure 2
(a) GCP fingerprint of samples S1–S12. (b) GCP common pattern R. (c) Fingerprint of representative cultivars of CRP. Peaks 3, 9, and 12 are hesperidin, nobiletin, and tangeretin, respectively.
(a)
(b)
(c)The fingerprint of representative cultivars of CRP was finally established with the GCP common pattern (Figure2(c)). In addition, the similarity evaluation results of 88 samples are shown in Table 1. The similarity values of samples S1–S12 compared to the GCP common pattern were all above 0.990, indicating that the C. reticulata “Chachi” samples from Xinhui were all of good quality uniformly. Moreover, the average similarity of the C. reticulata “Chachi” samples from other areas (S13–S22) was high (0.974), suggesting that there was little characteristic difference among C. reticulata “Chachi” samples. It was also seen that several cultivars of CRP showed high similarity to C. reticulata “Chachi,” such as C. reticulata “Shiyueju,” C. reticulata “Ponkan,” C. reticulata “Kinokuni,” C. reticulata “Yangshanju,” C. reticulata “Tribute,” C. reticulata “Bayueju,” C. reticulata “Suavissima,” C. reticulata “Wuyueju,” and C. reticulata “Nianju,” with the high average values of 0.962, 0.957, 0.953, 0.950, 0.949, 0.945, 0.942, 0.941, and 0.935, respectively. However, the cultivars of C. reticulata “Unshiu,” C. reticulata “Erythrosa,” C. reticulata “Yichangju,” C. reticulata “Qingjiangju,” C. reticulata “Linhaiju,” and C. reticulata “Xunwuju” showed little similarity to C. reticulata “Chachi,” with the low average similarity of 0.558, 0.539, 0.518, 0.504, 0.499, and 0.489, respectively.


### 3.3. Chemical Profiling on “Activity Fingerprint” by LC/MS-IT-TOF

The technology of LC/MS-IT-TOF has been proved to be an effective method for identifying the unknown compounds in materials [21]. The total ion chromatogram (TIC) of CRP sample S1 (a) and mixed standard solution (b) in the positive mode is displayed in Figure 3. After chemical profiling, a total of 17 characteristic compounds, including 3 flavonoid glycosides and 14 PMFs, were identified according to the information of mass fragment ions and retention times, and the detailed information is illustrated in Table 2.Figure 3
(a) Total ion chromatogram (TIC) of CRP in the positive mode. (b) TIC of mixed standard solution in the positive mode. Peaks 3, 9, 11, 12, and 14 are hesperidin, nobiletin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, tangeretin, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone, respectively.
(a)
(b)Table 2
Major chemical compounds of CRP identified by LC/MS-IT-TOF.
Peak no.
RT (min)
[M + H]+ (m/z)
Molecular formula
Fragment ions (m/z)
Identified compounds
2
14.746
581.1854
C27H32O14
563.1743, 545.1634, 401.1057, 339.0855, 315.0814, 273.0756, 263.0544, 245.0433, 231.0385, 219.0280, 195.0291, 179.0287, 153.0179, 135.0564
Naringin
3a
14.995
611.1956
C28H34O15
580.6021, 465.1423, 449.1420, 431.1322, 413.1220, 345.0960, 303.0855, 281.0644, 263.0533, 219.0288, 195.0278, 177.0539, 153.0179
Hesperidin
4
16.162
611.1957
C28H34O15
596.2038, 575.1750, 465.1420, 449.2690, 431.1318, 413.1227, 369.0966, 345.0954, 303.0864, 281.0647, 263.0539, 219.0288, 195.0280, 177.0540, 153.0179
Neohesperidin
5
18.506
595.2011
C28H34O14
449.1152, 433.1460, 397.1286, 379.1211, 365.0712, 287.0888, 256.0431, 224.1293, 195.0290, 183.0582, 165.0598
Poncirin
6
26.814
373.1273
C20H20O7
358.1033, 343.0800, 327.0501, 299.0504, 285.0613, 189.0301, 165.0171, 151.0752
Isosinensetin
7
28.119
389.1224
C20H20O8
374.0996, 360.0797, 359.0761, 341.0662, 331.0804, 328.0944, 313.0592, 211.0154, 193.0088
Monohydroxy-pentamethoxyflavone
8
31.411
373.1265
C20H20O7
357.0960, 343.0837, 339.0865, 329.1018, 327.0533, 313.0688, 312.1002, 297.0766, 269.0820, 181.0395
Sinensetin
9a
36.272
403.1386
C21H22O8
388.1121, 373.0921, 355.0809, 345.0944, 342.1099, 327.0657, 313.0708, 258.0526, 183.0296, 165.0519
Nobiletin
10
37.217
343.1140
C19H18O6
327.0402, 313.0709, 309.0764, 299.0914, 282.0887, 267.0650, 153.0186, 135.0642
Tetramethyl-O-scutellarin
11a
39.931
433.1510
C22H24O9
418.1239, 417.1167, 403.1003, 385.0903, 373.0529, 345.0600, 317.0655, 183.0288, 165.0188
3,5,6,7,8,3′,4′-Heptamethoxyflavone
12a
41.968
373.1281
C20H20O7
358.1050, 343.0812, 328.0579, 325.0725, 312.0993, 297.0761, 283.0589, 211.0256, 183.0292, 135.0442
Tangeretin
13
42.637
375.1458
C19H18O8
345.0606, 327.0507, 299.0558, 227.0550, 211.0613, 196.0361, 183.0287, 167.0343
Dihydroxy-tetramethoxyflavone
14a
44.908
389.1227
C20H20O8
374.0996, 359.0761, 356.0886,343.0440, 341.0662, 331.0804, 328.0944, 327.0862, 298.0473, 197.0092, 165.0758, 151.0520
5-Hydroxy-6,7,8,3′,4′-pentamethoxyflavone
15
45.577
405.1564
C20H20O9
375.1424, 345.1332, 329.1027, 241.0711, 226.0477, 211.0238, 165.0187
Dihydroxy-pentamethoxyflavone
16
46.073
419.1326
C21H22O9
404.1032, 389.0983, 371.0802, 357.0587, 163.0385
Monohydroxy-hexamethoxyflavone
17
47.053
359.1138
C19H18O7
344.0858, 329.0659, 326.1001, 311.0568, 298.0835, 210.1810
5-Hydroxy-6,7,8,4′-tetramethoxyflavone
18
48.439
375.1424
C19H18O8
345.0898, 241.0654, 226.0423, 211.0211, 183.0290
Dihydroxy-tetramethoxyflavone
aConfirmed in comparison with standard compounds.


#### 3.3.1. Identification of Flavonoid Glycosides

Flavonoids are a kind of organic compounds with C6-C3-C6 as the parent nucleus. Among them, flavonoid glycosides are widely present in citrus, including flavone-O-glycosides and flavone-C-glycosides. The flavone-O-glycosides are generally characterized by attaching glycosidic substituents to the flavonoid skeleton via the C-7 hydroxyl group, while the flavone-C-glycosides directly linked glycosidic substituents to the C atoms on the flavone A ring [22].Compound 2 displayed a quasimolecular ion atm/z 581, which formed a fragment ion at m/z 273 due to the loss of terminal Rha-Glc (308 Da). The MS spectrum also showed secondary fragment cracking by aglycon. For instance, the fragment at m/z 153 was produced by RDA cleavage, and the fragment at m/z 245 was formed corresponding to the flavonoid glycoside ion loss of CO (28 Da). Therefore, the compound was considered naringin according to the mass fragmentation information above [23].Compound 3 was putatively identified as hesperidin because of the same mass spectrum and chromatographic characteristics with the reference standard. The high intensity ions atm/z 449 and 303 were observed on the removal of glucose (162 Da) and rhamnosylglucose (308 Da) from the precursor ion, respectively. An extra minor fragment at m/z 153 was yielded by the RDA cleavage of aglycone ion. Compound 4 showed the similar fragment information to compound 3 but was different in the relative abundances, which could be inferred as neohesperidin [24].Compound 5 showed the [M + H]+ ion at m/z 595 in the first-order MS spectrum, suggesting the molecular formula of C28H34O14. A high-intensity ion at m/z 287 after losing neohesperidose (308 Da) was generated. This compound was tentatively inferred as poncirin after the comparison of its mass spectrum data [25].


#### 3.3.2. Identification of PMFs

PMFs are significant flavonoids particularly presented in citrus species, containing multiple methoxyl groups, low polarity, and planar structure [26]. With a basic unit of 222 Da, the molecular weights of PMFs vary depending on the amount of methoxyl and hydroxyl groups [27, 28].Compound 9 was confirmed as nobiletin by comparison to the reference standard with the same fragmentation information. Nobiletin gave an [M + H]+ ion at m/z 403 and yielded fragment ions at m/z 388 ([M + H − CH3]+), 373 ([M + H − 2CH3]+), and 355 ([M + H − 2CH3 − H2O]+). Similarly, compound 11 was deduced as 3,5,6,7,8,3′,4′-heptamethoxyflavone by comparing to the reference standard with the [M + H]+ ion at m/z 433.Compounds 6, 7, 8, 12, 14, and 15 were considered pentamethoxyflavones based on their molecular formula and fragment ions [29]. Comparing with a reference standard allowed us to identify compound 14 as 5-hydroxy-6,7,8,3,3′,4′-pentamethoxyflavone. It exhibited the parent ion [M + H]+ at m/z 389 and the product fragment ion at m/z 374 ([M + H − CH3]+), 359 ([M + H − 2CH3]+), and 341 ([M + H − 2CH3 − H2O]+). Compounds 6 and 8 were known as isosinensetin and sinensetin combining with the mass data of the previous research [30]. Compound 7 was presumed to be monohydroxy-pentamethoxyflavone based on the molecular formula of C20H20O8 and similar fragment of compound 14. Similarly, compound 15 was inferred to be dihydroxy-pentamethoxyflavone differed by the molecular weight of 16 of compound 7.Compound 12 was identified as tangeretin by comparing the retention time and fragmentation information of the reference standard. Moreover, the fragmentation information of compound 17 was similar to that of compound 12 and differed by the molecular weight of 14. Combining with the fragment information previously reported [31], compound 17 was presumed to be 5-hydroxy-6,7,8,4′-tetramethoxyflavone.


### 3.4. Quality Evaluation by Quantitative Determination and PCA

Hesperidin and other four main PMFs selected from 17 characteristic compounds profiled above were determined, and the results are given in Table1. The method validation showed a good linearity (R2 > 0.9990), and the RSDs of accuracy, repeatability, stability, and recovery were all below 3%, indicating that the assay method was feasible.PCA is a well-known analysis method with the purpose of dimensionality reduction to transform multiple indicators into a few comprehensive indicators [32, 33]. Defining the contents of the five flavonoids as five characteristics, PCA was used to determine the correlation among samples from different cultivars in this research. The first two principal components were extracted by PCA (Table 3), accounting for 79.979% of the total variance (PC1 = 59.269% and PC2 = 20.710%). And the variable importance in projection (VIP) values of nobiletin, tangeretin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone were 0.934, 0.965, −0.641, and 0.827, respectively, showing more contribution to PC1, while hesperidin contributed more to PC2 with the VIP value of 0.953. It was indicated that these five compounds were the characteristic chemicals distinguishing various cultivars of CRP; especially, the four PMFs contributed more to PC1, accounting for 59.269% of the total variance.Table 3
Component matrix of PCA.
Components
Variable importance in projection (VIP) values
PC1
PC2
Hesperidin
−0.255
0.953
Nobiletin
0.934
−0.121
3,5,6,7,8,3′,4′-Heptamethoxyflavone
−0.641
−0.247
Tangeretin
0.965
0.011
5-Hydroxy-6,7,8, 3′,4′-pentamethoxyflavone
0.827
0.227
2 components were extracted.As the result of the PCA plot shows (Figure4), 88 samples with 21cultivars were categorized into four groups. Group 1 contained C. reticulata “Unshiu,” C. reticulata “Erythrosa,” C. reticulata “Yichangju,” C. reticulata “Qingjiangju,” C. reticulata “Linhaiju,” C. reticulata “Xunwuju,” and C. reticulata “Biangan,” with the high content of hesperidin and low PMFs. C. reticulata “Tankan” was in group 2 due to its lower content of flavonoids, while C. reticulata “Suavissima” with a higher content of flavonoids was in group 3. Other eleven cultivars were clustered in group 4 with C. reticulata “Chachi,” showing parts of similarities and relationships with the traditional genuine herb. The result of PCA was approximately consistent with that of similarity analysis.Figure 4
PCA results of CRP from different cultivars.Since an LC/MS-IT-TOF was used, 17 characteristic compounds were finally profiled on the 18 common peaks at the “activity fingerprint,” and all of them were identified belonging to flavonoids, suggesting that there may be a potential relationship between the differences among varieties and flavonoid compositions. Then, hesperidin and four main PMFs were selected for quantitative determination and further analysis. And the PCA results confirmed that the differences among varieties were significantly correlated with flavonoids, not only including hesperidin, the single indicator for CRP quality control in Chinese Pharmacopoeia (version 2015) [1], but also including four main PMFs, such as nobiletin, tangeretin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone. Therefore, we could not deny the importance of PMFs in CRP, and it is considerable to add PMFs as indicator components for CRP quality control and identification in Chinese Pharmacopoeia.


## 3.1. Antitussive and Expectorant Activities of MECRP

The effects of the MECRP on ammonia water-irritated cough (Figures1(a) and 1(b)) and tracheal phenol red excretion in mice are depicted in Figure 1(c). Statistical analysis showed significant differences between the control group and treated groups in the incubation period of cough (P<0.05), bouts of cough within 3 minutes (P<0.05), and tracheal phenol red excretion (P<0.05). The test of ammonia water-irritated cough showed that the MECRP significantly prolonged the incubation period of cough and decreased the bouts of coughs in mice, possessing a good ability to relieve cough. In addition, the tracheal phenol red excretion test illustrated that the MECRP could effectively reduce sputum with the increase of the excretion of phenol red in the trachea. It has been well suggested that the MECRP exhibited excellent antitussive and expectorant activities in mice.Figure 1
Effect of the MECRP on ammonia water-irritated cough and tracheal phenol red excretion in mice. (a) Incubation period of cough. (b) Bouts of cough. (c) Tracheal phenol red excretion. All values are expressed as mean ± SD (n = 10). ∗P<0.05 and ∗∗P<0.01 compared to the control group (one-way ANOVA followed by the LSD test).
(a)
(b)
(c)


## 3.2. Similarity Analysis on “Activity Fingerprint”

The GCP fingerprint (Figure2(a)) was firstly established by twelve batches of traditional genuine herbs from Xinhui (S1–S12) due to their high quality. And a GCP common pattern (Figure 2(b)) was further generated through the characteristics of samples S1–S12. There were 18 common peaks determined in the GCP common pattern, of which peaks 3, 9, and 12 were identified as hesperidin, nobiletin, and tangeretin, respectively. According to statistical analysis, the common peak area of each sample (S1–S12) accounted for 97.276%–98.870% (>90%) of total peak areas. And all of the accuracy, repeatability, and stability results showed that the RSD of relative retention time and relative peak area of the 18 common peaks was below 3%, satisfying the demands of fingerprint.Figure 2
(a) GCP fingerprint of samples S1–S12. (b) GCP common pattern R. (c) Fingerprint of representative cultivars of CRP. Peaks 3, 9, and 12 are hesperidin, nobiletin, and tangeretin, respectively.
(a)
(b)
(c)The fingerprint of representative cultivars of CRP was finally established with the GCP common pattern (Figure2(c)). In addition, the similarity evaluation results of 88 samples are shown in Table 1. The similarity values of samples S1–S12 compared to the GCP common pattern were all above 0.990, indicating that the C. reticulata “Chachi” samples from Xinhui were all of good quality uniformly. Moreover, the average similarity of the C. reticulata “Chachi” samples from other areas (S13–S22) was high (0.974), suggesting that there was little characteristic difference among C. reticulata “Chachi” samples. It was also seen that several cultivars of CRP showed high similarity to C. reticulata “Chachi,” such as C. reticulata “Shiyueju,” C. reticulata “Ponkan,” C. reticulata “Kinokuni,” C. reticulata “Yangshanju,” C. reticulata “Tribute,” C. reticulata “Bayueju,” C. reticulata “Suavissima,” C. reticulata “Wuyueju,” and C. reticulata “Nianju,” with the high average values of 0.962, 0.957, 0.953, 0.950, 0.949, 0.945, 0.942, 0.941, and 0.935, respectively. However, the cultivars of C. reticulata “Unshiu,” C. reticulata “Erythrosa,” C. reticulata “Yichangju,” C. reticulata “Qingjiangju,” C. reticulata “Linhaiju,” and C. reticulata “Xunwuju” showed little similarity to C. reticulata “Chachi,” with the low average similarity of 0.558, 0.539, 0.518, 0.504, 0.499, and 0.489, respectively.


## 3.3. Chemical Profiling on “Activity Fingerprint” by LC/MS-IT-TOF

The technology of LC/MS-IT-TOF has been proved to be an effective method for identifying the unknown compounds in materials [21]. The total ion chromatogram (TIC) of CRP sample S1 (a) and mixed standard solution (b) in the positive mode is displayed in Figure 3. After chemical profiling, a total of 17 characteristic compounds, including 3 flavonoid glycosides and 14 PMFs, were identified according to the information of mass fragment ions and retention times, and the detailed information is illustrated in Table 2.Figure 3
(a) Total ion chromatogram (TIC) of CRP in the positive mode. (b) TIC of mixed standard solution in the positive mode. Peaks 3, 9, 11, 12, and 14 are hesperidin, nobiletin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, tangeretin, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone, respectively.
(a)
(b)Table 2
Major chemical compounds of CRP identified by LC/MS-IT-TOF.
Peak no.
RT (min)
[M + H]+ (m/z)
Molecular formula
Fragment ions (m/z)
Identified compounds
2
14.746
581.1854
C27H32O14
563.1743, 545.1634, 401.1057, 339.0855, 315.0814, 273.0756, 263.0544, 245.0433, 231.0385, 219.0280, 195.0291, 179.0287, 153.0179, 135.0564
Naringin
3a
14.995
611.1956
C28H34O15
580.6021, 465.1423, 449.1420, 431.1322, 413.1220, 345.0960, 303.0855, 281.0644, 263.0533, 219.0288, 195.0278, 177.0539, 153.0179
Hesperidin
4
16.162
611.1957
C28H34O15
596.2038, 575.1750, 465.1420, 449.2690, 431.1318, 413.1227, 369.0966, 345.0954, 303.0864, 281.0647, 263.0539, 219.0288, 195.0280, 177.0540, 153.0179
Neohesperidin
5
18.506
595.2011
C28H34O14
449.1152, 433.1460, 397.1286, 379.1211, 365.0712, 287.0888, 256.0431, 224.1293, 195.0290, 183.0582, 165.0598
Poncirin
6
26.814
373.1273
C20H20O7
358.1033, 343.0800, 327.0501, 299.0504, 285.0613, 189.0301, 165.0171, 151.0752
Isosinensetin
7
28.119
389.1224
C20H20O8
374.0996, 360.0797, 359.0761, 341.0662, 331.0804, 328.0944, 313.0592, 211.0154, 193.0088
Monohydroxy-pentamethoxyflavone
8
31.411
373.1265
C20H20O7
357.0960, 343.0837, 339.0865, 329.1018, 327.0533, 313.0688, 312.1002, 297.0766, 269.0820, 181.0395
Sinensetin
9a
36.272
403.1386
C21H22O8
388.1121, 373.0921, 355.0809, 345.0944, 342.1099, 327.0657, 313.0708, 258.0526, 183.0296, 165.0519
Nobiletin
10
37.217
343.1140
C19H18O6
327.0402, 313.0709, 309.0764, 299.0914, 282.0887, 267.0650, 153.0186, 135.0642
Tetramethyl-O-scutellarin
11a
39.931
433.1510
C22H24O9
418.1239, 417.1167, 403.1003, 385.0903, 373.0529, 345.0600, 317.0655, 183.0288, 165.0188
3,5,6,7,8,3′,4′-Heptamethoxyflavone
12a
41.968
373.1281
C20H20O7
358.1050, 343.0812, 328.0579, 325.0725, 312.0993, 297.0761, 283.0589, 211.0256, 183.0292, 135.0442
Tangeretin
13
42.637
375.1458
C19H18O8
345.0606, 327.0507, 299.0558, 227.0550, 211.0613, 196.0361, 183.0287, 167.0343
Dihydroxy-tetramethoxyflavone
14a
44.908
389.1227
C20H20O8
374.0996, 359.0761, 356.0886,343.0440, 341.0662, 331.0804, 328.0944, 327.0862, 298.0473, 197.0092, 165.0758, 151.0520
5-Hydroxy-6,7,8,3′,4′-pentamethoxyflavone
15
45.577
405.1564
C20H20O9
375.1424, 345.1332, 329.1027, 241.0711, 226.0477, 211.0238, 165.0187
Dihydroxy-pentamethoxyflavone
16
46.073
419.1326
C21H22O9
404.1032, 389.0983, 371.0802, 357.0587, 163.0385
Monohydroxy-hexamethoxyflavone
17
47.053
359.1138
C19H18O7
344.0858, 329.0659, 326.1001, 311.0568, 298.0835, 210.1810
5-Hydroxy-6,7,8,4′-tetramethoxyflavone
18
48.439
375.1424
C19H18O8
345.0898, 241.0654, 226.0423, 211.0211, 183.0290
Dihydroxy-tetramethoxyflavone
aConfirmed in comparison with standard compounds.


### 3.3.1. Identification of Flavonoid Glycosides

Flavonoids are a kind of organic compounds with C6-C3-C6 as the parent nucleus. Among them, flavonoid glycosides are widely present in citrus, including flavone-O-glycosides and flavone-C-glycosides. The flavone-O-glycosides are generally characterized by attaching glycosidic substituents to the flavonoid skeleton via the C-7 hydroxyl group, while the flavone-C-glycosides directly linked glycosidic substituents to the C atoms on the flavone A ring [22].Compound 2 displayed a quasimolecular ion atm/z 581, which formed a fragment ion at m/z 273 due to the loss of terminal Rha-Glc (308 Da). The MS spectrum also showed secondary fragment cracking by aglycon. For instance, the fragment at m/z 153 was produced by RDA cleavage, and the fragment at m/z 245 was formed corresponding to the flavonoid glycoside ion loss of CO (28 Da). Therefore, the compound was considered naringin according to the mass fragmentation information above [23].Compound 3 was putatively identified as hesperidin because of the same mass spectrum and chromatographic characteristics with the reference standard. The high intensity ions atm/z 449 and 303 were observed on the removal of glucose (162 Da) and rhamnosylglucose (308 Da) from the precursor ion, respectively. An extra minor fragment at m/z 153 was yielded by the RDA cleavage of aglycone ion. Compound 4 showed the similar fragment information to compound 3 but was different in the relative abundances, which could be inferred as neohesperidin [24].Compound 5 showed the [M + H]+ ion at m/z 595 in the first-order MS spectrum, suggesting the molecular formula of C28H34O14. A high-intensity ion at m/z 287 after losing neohesperidose (308 Da) was generated. This compound was tentatively inferred as poncirin after the comparison of its mass spectrum data [25].


### 3.3.2. Identification of PMFs

PMFs are significant flavonoids particularly presented in citrus species, containing multiple methoxyl groups, low polarity, and planar structure [26]. With a basic unit of 222 Da, the molecular weights of PMFs vary depending on the amount of methoxyl and hydroxyl groups [27, 28].Compound 9 was confirmed as nobiletin by comparison to the reference standard with the same fragmentation information. Nobiletin gave an [M + H]+ ion at m/z 403 and yielded fragment ions at m/z 388 ([M + H − CH3]+), 373 ([M + H − 2CH3]+), and 355 ([M + H − 2CH3 − H2O]+). Similarly, compound 11 was deduced as 3,5,6,7,8,3′,4′-heptamethoxyflavone by comparing to the reference standard with the [M + H]+ ion at m/z 433.Compounds 6, 7, 8, 12, 14, and 15 were considered pentamethoxyflavones based on their molecular formula and fragment ions [29]. Comparing with a reference standard allowed us to identify compound 14 as 5-hydroxy-6,7,8,3,3′,4′-pentamethoxyflavone. It exhibited the parent ion [M + H]+ at m/z 389 and the product fragment ion at m/z 374 ([M + H − CH3]+), 359 ([M + H − 2CH3]+), and 341 ([M + H − 2CH3 − H2O]+). Compounds 6 and 8 were known as isosinensetin and sinensetin combining with the mass data of the previous research [30]. Compound 7 was presumed to be monohydroxy-pentamethoxyflavone based on the molecular formula of C20H20O8 and similar fragment of compound 14. Similarly, compound 15 was inferred to be dihydroxy-pentamethoxyflavone differed by the molecular weight of 16 of compound 7.Compound 12 was identified as tangeretin by comparing the retention time and fragmentation information of the reference standard. Moreover, the fragmentation information of compound 17 was similar to that of compound 12 and differed by the molecular weight of 14. Combining with the fragment information previously reported [31], compound 17 was presumed to be 5-hydroxy-6,7,8,4′-tetramethoxyflavone.


## 3.3.1. Identification of Flavonoid Glycosides

Flavonoids are a kind of organic compounds with C6-C3-C6 as the parent nucleus. Among them, flavonoid glycosides are widely present in citrus, including flavone-O-glycosides and flavone-C-glycosides. The flavone-O-glycosides are generally characterized by attaching glycosidic substituents to the flavonoid skeleton via the C-7 hydroxyl group, while the flavone-C-glycosides directly linked glycosidic substituents to the C atoms on the flavone A ring [22].Compound 2 displayed a quasimolecular ion atm/z 581, which formed a fragment ion at m/z 273 due to the loss of terminal Rha-Glc (308 Da). The MS spectrum also showed secondary fragment cracking by aglycon. For instance, the fragment at m/z 153 was produced by RDA cleavage, and the fragment at m/z 245 was formed corresponding to the flavonoid glycoside ion loss of CO (28 Da). Therefore, the compound was considered naringin according to the mass fragmentation information above [23].Compound 3 was putatively identified as hesperidin because of the same mass spectrum and chromatographic characteristics with the reference standard. The high intensity ions atm/z 449 and 303 were observed on the removal of glucose (162 Da) and rhamnosylglucose (308 Da) from the precursor ion, respectively. An extra minor fragment at m/z 153 was yielded by the RDA cleavage of aglycone ion. Compound 4 showed the similar fragment information to compound 3 but was different in the relative abundances, which could be inferred as neohesperidin [24].Compound 5 showed the [M + H]+ ion at m/z 595 in the first-order MS spectrum, suggesting the molecular formula of C28H34O14. A high-intensity ion at m/z 287 after losing neohesperidose (308 Da) was generated. This compound was tentatively inferred as poncirin after the comparison of its mass spectrum data [25].


## 3.3.2. Identification of PMFs

PMFs are significant flavonoids particularly presented in citrus species, containing multiple methoxyl groups, low polarity, and planar structure [26]. With a basic unit of 222 Da, the molecular weights of PMFs vary depending on the amount of methoxyl and hydroxyl groups [27, 28].Compound 9 was confirmed as nobiletin by comparison to the reference standard with the same fragmentation information. Nobiletin gave an [M + H]+ ion at m/z 403 and yielded fragment ions at m/z 388 ([M + H − CH3]+), 373 ([M + H − 2CH3]+), and 355 ([M + H − 2CH3 − H2O]+). Similarly, compound 11 was deduced as 3,5,6,7,8,3′,4′-heptamethoxyflavone by comparing to the reference standard with the [M + H]+ ion at m/z 433.Compounds 6, 7, 8, 12, 14, and 15 were considered pentamethoxyflavones based on their molecular formula and fragment ions [29]. Comparing with a reference standard allowed us to identify compound 14 as 5-hydroxy-6,7,8,3,3′,4′-pentamethoxyflavone. It exhibited the parent ion [M + H]+ at m/z 389 and the product fragment ion at m/z 374 ([M + H − CH3]+), 359 ([M + H − 2CH3]+), and 341 ([M + H − 2CH3 − H2O]+). Compounds 6 and 8 were known as isosinensetin and sinensetin combining with the mass data of the previous research [30]. Compound 7 was presumed to be monohydroxy-pentamethoxyflavone based on the molecular formula of C20H20O8 and similar fragment of compound 14. Similarly, compound 15 was inferred to be dihydroxy-pentamethoxyflavone differed by the molecular weight of 16 of compound 7.Compound 12 was identified as tangeretin by comparing the retention time and fragmentation information of the reference standard. Moreover, the fragmentation information of compound 17 was similar to that of compound 12 and differed by the molecular weight of 14. Combining with the fragment information previously reported [31], compound 17 was presumed to be 5-hydroxy-6,7,8,4′-tetramethoxyflavone.


## 3.4. Quality Evaluation by Quantitative Determination and PCA

Hesperidin and other four main PMFs selected from 17 characteristic compounds profiled above were determined, and the results are given in Table1. The method validation showed a good linearity (R2 > 0.9990), and the RSDs of accuracy, repeatability, stability, and recovery were all below 3%, indicating that the assay method was feasible.PCA is a well-known analysis method with the purpose of dimensionality reduction to transform multiple indicators into a few comprehensive indicators [32, 33]. Defining the contents of the five flavonoids as five characteristics, PCA was used to determine the correlation among samples from different cultivars in this research. The first two principal components were extracted by PCA (Table 3), accounting for 79.979% of the total variance (PC1 = 59.269% and PC2 = 20.710%). And the variable importance in projection (VIP) values of nobiletin, tangeretin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone were 0.934, 0.965, −0.641, and 0.827, respectively, showing more contribution to PC1, while hesperidin contributed more to PC2 with the VIP value of 0.953. It was indicated that these five compounds were the characteristic chemicals distinguishing various cultivars of CRP; especially, the four PMFs contributed more to PC1, accounting for 59.269% of the total variance.Table 3
Component matrix of PCA.
Components
Variable importance in projection (VIP) values
PC1
PC2
Hesperidin
−0.255
0.953
Nobiletin
0.934
−0.121
3,5,6,7,8,3′,4′-Heptamethoxyflavone
−0.641
−0.247
Tangeretin
0.965
0.011
5-Hydroxy-6,7,8, 3′,4′-pentamethoxyflavone
0.827
0.227
2 components were extracted.As the result of the PCA plot shows (Figure4), 88 samples with 21cultivars were categorized into four groups. Group 1 contained C. reticulata “Unshiu,” C. reticulata “Erythrosa,” C. reticulata “Yichangju,” C. reticulata “Qingjiangju,” C. reticulata “Linhaiju,” C. reticulata “Xunwuju,” and C. reticulata “Biangan,” with the high content of hesperidin and low PMFs. C. reticulata “Tankan” was in group 2 due to its lower content of flavonoids, while C. reticulata “Suavissima” with a higher content of flavonoids was in group 3. Other eleven cultivars were clustered in group 4 with C. reticulata “Chachi,” showing parts of similarities and relationships with the traditional genuine herb. The result of PCA was approximately consistent with that of similarity analysis.Figure 4
PCA results of CRP from different cultivars.Since an LC/MS-IT-TOF was used, 17 characteristic compounds were finally profiled on the 18 common peaks at the “activity fingerprint,” and all of them were identified belonging to flavonoids, suggesting that there may be a potential relationship between the differences among varieties and flavonoid compositions. Then, hesperidin and four main PMFs were selected for quantitative determination and further analysis. And the PCA results confirmed that the differences among varieties were significantly correlated with flavonoids, not only including hesperidin, the single indicator for CRP quality control in Chinese Pharmacopoeia (version 2015) [1], but also including four main PMFs, such as nobiletin, tangeretin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone. Therefore, we could not deny the importance of PMFs in CRP, and it is considerable to add PMFs as indicator components for CRP quality control and identification in Chinese Pharmacopoeia.


## 4. Conclusion

In the present study, an effective “activity fingerprint” on CRP from different cultivars involving antitussive and expectorant activity assays, similarity analysis, LC/MS-IT-TOF analysis, quantitative analysis, and PCA was perfectly established, which was proven to relate to pharmacological activity and conducted to find the characteristic chemicals distinguishing various cultivars of CRP for quality control. From the results above, the recorded cultivars ofC. reticulata “Chachi,” C. reticulata “Tangerina,” and C. reticulata “Dahongpao” were quite similar to each other except for C. reticulata “Unshiu,” which indicated that the selection of the traditional medicinal herb of CRP was rational related to the high content of PMFs. In addition, there are some other cultivars significantly similar to C. reticulata “Chachi,” such as C. reticulata “Kinokuni,” C. reticulata “Yangshanju,” C. reticulata “Wuyueju,” and C. reticulata “Nianju,” and so on. Among them, it was worth noting that the cultivars of C. reticulata “Shiyueju,” C. reticulata “Ponkan,” C. reticulata “Tribute,” and C. reticulata “Bayueju” were rarely used for traditional medicine but exhibited a relatively high content of hesperidin and PMFs, even exceeding that of C. reticulata “Chachi.” Therefore, these four cultivars can be considered effective resources for the application of CRP, which provide a reference for citrus resource development.



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*Source: 4736152-2020-03-03.xml*