Chemical and biological comparison of the fruit extracts of Citrus wilsonii Tanaka and Citrus medica L.

Food Chemistry 173 (2015) 54–60

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Chemical and biological comparison of the fruit extracts of Citrus wilsonii Tanaka and Citrus medica L. Pan Zhao 1, Li Duan 1, Long Guo, Li-Li Dou, Xin Dong, Ping Zhou, Ping Li ⇑, E-Hu Liu ⇑ State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, PR China

a r t i c l e

i n f o

Article history: Received 6 June 2014 Received in revised form 16 September 2014 Accepted 1 October 2014 Available online 13 October 2014 Keywords: Citrus wilsonii Tanaka Citrus medica L. HPLC–QTOF/MS HPLC–DAD Antioxidant activity

a b s t r a c t Citri Fructus (CF), the mature fruit of Citrus wilsonii Tanaka (CWT) or Citrus medica L. (CML), is an important citrus by-product with health promoting and nutritive properties. The present study compares the chemical and biological differences of CWT and CML. Thin layer chromatography and high performance liquid chromatography, coupled with quadrupole time-of-flight tandem mass spectrometry techniques, were employed to compare the chemical profiles of CWT and CML. A total of 25 compounds were identified and the results indicated that there were significant differences in chemical composition between the two CF species. The quantitative results obtained by HPLC coupled with diode array detector method demonstrated that naringin was present in the highest amounts in CWT, whilst nomilin was the most dominant constituent in CML. It was also found that CWT had significantly higher free radicalscavenging activity than CML. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Citri Fructus (CF), the mature fruit of Citrus wilsonii Tanaka (CWT) or Citrus medica L. (CML), is an important citrus by-product exploited by both pharmaceutical and food industries (The State Pharmacopoeia Commission of CF, 2010 edition). It is mainly distributed in Southern China, Taiwan, Vietnam, Laos, Burma and other Southeast Asian countries and is consumed for its dietary and nutritional value in traditional foods and recipes (Chau & Wu, 2006). Phytochemical investigations indicated that flavonoids, coumarins and limonoids were the major bioactive components of CF. Since these compounds bear biological and chemotaxonomic importance, they have attracted considerable interest in the past decades. The chemical analysis of CF not only provides information concerning nutritional value, but also differentiates the species having morphological similarity. In the past decades, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and HPLC coupled to mass spectrometry (HPLC–MS) have now been widely accepted to be the predominant tool for qualitative and quantitative analysis of chemical constituents in botanical products (Chai, Li, & Li, 2005; Chen et al., 2006; Lee et al., 2008; Xu et al., 2012). To date, only one or two constituents, such as

⇑ Corresponding authors. Tel./fax: +86 25 83271379. 1

E-mail addresses: [email protected] (P. Li), [email protected] (E.-H. Liu). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.foodchem.2014.10.010 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

naringin, were used as chemical markers for the quality control of CF (Mao, Li, Gu, Zhu, & Zhong, 2008). However, no research has been conducted to analyse the chemical profiles of CWT and CML, and no attention has been devoted to comparing the biological differences between CWT and CML. In the present study, comparing the chemical and biological differences of CWT and CML, TLC, and HPLC coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC–QTOF/MS) techniques were firstly employed to compare the chemical profiles of CWT and CML. Furthermore, a HPLC coupled with diode array detector (HPLC–DAD) method was utilised to determine the major constituents in the extracts of these two herbs. Also, three different methods (DPPH, FRAP and ABTS) were used to evaluate the antioxidant activity of CWT and CML.

2. Materials and methods 2.1. Reagents The solvents, HPLC grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany), and formic acid with a purity of 96% is of HPLC grade (Tedia, USA). Deionised water (18 MX) was prepared by distilled water through a Milli-Q system (Millipore, Milford, MA, USA). Other reagents and chemicals were of analytical grade. The reference standards of naringin (9), hesperidin (11), meranzin hydrate (12), scoparone (13), 5,7-dimethoxycoumarin (18), limonin (21), nomilin (22), obacunone (23), and

55

‘‘ND’’ means not detected. a Data are represented as the mean ± SD.

Isoimperatorin Obacunone

ND ND ND ND ND ND ND ND ND ND 0.210 ± 0.020 0.365 ± 0.019 0.186 ± 0.018 0.158 ± 0.009 0.274 ± 0.028 0279 ± 0.011 0.230 ± 0.01 0.235 ± 0.033 0.211 ± 0.026 0.240 ± 0.046 3.001 ± 0.101 2.752 ± 0.902 1.933 ± 0.809 2.469 ± 0.732 1.801 ± 0.820 3.230 ± 0.920 1.820 ± 0.102 1.693 ± 0.609 1.717 ± 0.811 1.561 ± 0.928 1.971 ± 0.320 3.141 ± 0.404 2.182 ± 0.681 2.591 ± 0.100 2.532 ± 0.903 3.288 ± 0.645 2.722 ± 0.720 3.819 ± 0.133 3.847 ± 0.950 3.010 ± 0.808

Nomilin Limonin

1.091 ± 0.026 1.341 ± 0.091 1.444 ± 0.059 1.456 ± 0.101 1.169 ± 0.082 2.345 ± 0.072 1.637 ± 0.049 1.830 ± 0.097 1.540 ± 0.044 1.249 ± 0.082 0.457 ± 0.019 0.886 ± 0.028 0.483 ± 0.016 0.587 ± 0.016 0.605 ± 0.027 0.711 ± 0.031 0.653 ± 0.040 0.587 ± 0.033 0.869 ± 0.013 0.572 ± 0.018 ND ND ND ND ND ND ND ND ND ND 0.451 ± 0.021 0.195 ± 0.038 0.232 ± 0.017 0.165 ± 0.019 0.190 ± 0.027 0.271 ± 0.031 0.184 ± 0.021 0.223 ± 0.016 0.194 ± 0.024 0.186 ± 0.011

5,7-Dimethoxycoumarin Meranzin hydrate Hesperidin Naringin

Chaozhou, Guangdong Chaozhou, Guangdong Guilin, Guangxi Huazhou, Guangdong Yulin, Guangxi Yiyang, Hunan Jinhua, Zhejiang Chongqing Changsha, Hunan Jingjiang, Jiangsu Kunming, Yunnan Kunming, Yunnan Chengdou, Sichuan Kunming, Yunnan Kunming, Yunnan Kunming, Yunnan Chengdou, Sichuan Chengdou, Sichuan Kunming, Yunnan Chengdou, Sichuan

Analysis was performed on an Agilent 1260 HPLC system (Agilent Corporation, MA, USA), equipped with vacuum degasser, quaternary gradient pump, auto-sampler and diode array detector, connected to an Agilent Chemstation software. Chromatographic separation was carried out at 20 °C on an Agilent ZorBax SB-C18 column (4.6  50 mm, 1.8 lm). The mobile phase consisted of 0.1% formic acid solution (A) and acetonitrile (B). The gradient program was: 0–5 min, 18–23% B; 5–6 min, 23% B; 6–10 min, 23–50% B; 10–18 min, 50–100% B; and finally, the column was reconditioned with 18% B isocratic for 5 min. The flow-rate was 0.5 ml/ min and the column temperature was 20 °C. The injection volume was 5 ll. The UV detection wavelengths were set at 210 nm, 283 nm and 330 nm, respectively.

Place of collection

2.4. HPLC–DAD quantitative analysis

CWT CWT CWT CWT CWT CWT CWT CWT CWT CWT CML CML CML CML CML CML CML CML CML CML

The standard solution 1 (10 ll) and sample solutions (10 ll) were applied in bands to a suitable thin-layer chromatographic plate (Merck). The plate was then developed with Developing solvent system 1 (ethylacetate:methanol:water = 10:2:2) in a saturated chromatographic chamber. After the solvent front has moved about 4 cm from the origin, the plate was removed from the chamber and air-dried. The plate was further developed in another chamber containing Developing solvent system 2 (methylbenzene:ethylacetate:acetone:formic acid = 8:2:1:0.05). After developing over a path of another 4 cm, the plate was air-dried and sprayed with 5% aluminium chloride solution. The image was captured under UV light (366 nm) after the plate was heated at 105 °C for 1 min.

Table 1 Contents (mg/g, n = 3)a of eight major compounds in CWT and CML collected from different regions.

2.3. TLC analysis

Varieties

2.2.2. Sample preparation The CF powder (0.5 g) was accurately weighted and extracted by ultra-sonator with 25 ml 50% methanol for 30 min. The extraction solution was transferred into a 25 ml volumetric flask, which was filled up to its volume with the same solvent. After centrifuged at 13,000g for 10 min, the supernatant was separated and stored at 4 °C in airtight containers prior to analysis by TLC, HPLC–DAD, HPLC–QTOF/MS and antioxidant assay.

7.424 ± 0.103 4.643 ± 0.097 5.580 ± 0.120 5.141 ± 0.203 4.871 ± 0.086 5.821 ± 0.081 4.291 ± 0.129 3.859 ± 0.329 4.689 ± 0.921 4.748 ± 0.149 ND ND ND ND ND ND ND ND ND ND

2.2.1. Plant materials A total of 20 commercial CF samples (including 10 batches of CWT and 10 batches of CML) were collected from Guangxi, Guangdong, Hunan, Fujian, Sichuan, Jiangsu, Zhejiang and Yunnan Province of China (Table 1). The voucher specimens, identified by Prof. Ping Li from the Department of Pharmacognosy in China Pharmaceutical University, was deposited in the State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China.

ND ND ND ND ND ND ND ND ND ND 1.002 ± 0.039 1.623 ± 0.042 0.853 ± 0.058 0.846 ± 0.019 0.759 ± 0.021 1.732 ± 0.034 1.845 ± 0.083 1.837 ± 0.091 0.923 ± 0.020 1.137 ± 0.036

2.2. Plant materials and extraction

38.802 ± 0.873 49.183 ± 0.653 51.502 ± 0.721 44.891 ± 0.389 50.369 ± 0.498 56.324 ± 1.029 60.324 ± 0.902 56.419 ± 0.603 68.001 ± 0.401 34.180 ± 0.322 0.473 ± 0.089 0.495 ± 0.078 0.440 ± 0.063 0.450 ± 0.071 0.522 ± 0.059 0.613 ± 0.045 0.474 ± 0.083 0.466 ± 0.029 0.438 ± 0.017 0.578 ± 0.075

isoimperatorin (25) were purchased from the National Institute for Control of Biological and Pharmaceutical Products of China. Standard stock solution 1 of four accurately weighed reference compounds (9, 12, 13 and 18) was directly prepared in methanol for TLC. Standard stock solution 2 of eight reference compounds (9, 11, 12, 18, 21–23 and 25) was directly prepared in methanol for HPLC–DAD and HPLC–QTOF/MS. The two kinds of working standard solutions were prepared by diluting the stock solutions with methanol to a series of proper concentrations. An aliquot of 10 ll of the standard solution 1 was applied to the TLC analysis. An aliquot of 5 ll of the standard solution 2 was injected into HPLC for quantitative analysis, and 2 ll of the standard solution 2 was injected into HPLC–QTOF/MS for qualitative analysis.

0.349 ± 0.009 0.199 ± 0.012 0.135 ± 0.008 0.224 ± 0.017 0.154 ± 0.041 0.220 ± 0.033 0.291 ± 0.018 0.125 ± 0.011 0.153 ± 0.009 0.165 ± 0.014 ND ND ND ND ND ND ND ND ND ND

P. Zhao et al. / Food Chemistry 173 (2015) 54–60

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P. Zhao et al. / Food Chemistry 173 (2015) 54–60

2.5. LC–QTOF/MS qualitative analysis The HPLC conditions were consistent with the above HPLC quantitative analysis. All MS experiments were conducted on an Agilent 6530 quadrupole tandem time-of-flight mass spectrometer (Q-TOF–MS) equipped with electro spray ionization (ESI) interface (Agilent Corporation, MA, USA). The QTOF/MS operating parameters were as follows: drying gas (N2) flow rate, 10 L/min; drying gas temperature, 300 °C; nebuliser, 35 psig; sheath gas temperature, 350 °C; sheath gas flow, 10 L/min; capillary, 3500 V; skimmer, 65 V; fragmentor voltage, 150 V. The sample collision energy was set at 20 V. Mass spectra were recorded in positive mode across the range m/z 100–1000 with accurate mass measurement of all mass peaks. The chemical structures of target-isolated compounds were identified by comparison of the retention times (Rt) and the maximum absorbance wavelengths (kmax) with reference compounds and confirmed by high-resolution QTOF–MS/MS analysis. The system was controlled by Mass Hunter software (Agilent Corporation, MA, USA).

2.6. Evaluation of the antioxidant capacity 2.6.1. Determination of total phenolic compounds (TPC) TPC in CWT and CML was determined using a modified Folin– Ciocalteu method (Dewanto, Wu, Adom, & Liu, 2002; Magalhães, Santos, Segundo, Reis, & Lima, 2010). 10 ll of gallic acid standard solution or sample solution was mixed with 160 ll of Folin– Ciocalteu phenol reagent (1:8, v/v). The mixture was allowed to stand for 5 min, and 30 ll of 20% sodium hydroxide solution was added. The absorbance was measured at 765 nm after the microplate was kept in the dark for 60 min. The reagent blank was evaluated by the addition of 10 ll of water instead of standard compound or sample. All experiments were performed in triplicate at room temperature (25 ± 1 °C). All results are expressed as milligram of gallic acid per gram of dry drug (mg GA/g DW).

2.6.2. Determination of total flavonoids content (TFC) The TFC was measured using a colorimetric assay adapted from a previous study (Roux, 1957; Zhang, He, & Hu, 2011). Briefly, 200 ll of 5% aqueous NaNO2 was added to an aliquot (2.6 ml) of each extract and the mixture was vortexed. A reagent blank using 50% aqueous methanol instead of sample was prepared. Samples were allowed to stand for 6 min, then 200 ll of 10% aqueous Al(NO3)3 was added. 2 ml of 1 M NaOH was added 6 min after the addition of aluminium chloride. The solution was mixed and the absorbance was measured after 15 min against the blank at 510 nm. The TFC was calculated with respect to a quercetin standard curve (concentration range: 5–300 lg/ml). Results are expressed in lg of quercetin1 DW of plant material. 2.6.3. DPPH assay The DPPH assay is based on the measurement of the scavenging ability of antioxidants toward the stable radical DPPH (BrandWilliams, Cuvelier, & Berset, 1995). 120 lM of DPPH solution dissolved in 1.9 ml methanol was added to 0.1 ml of each extract and shaken vigorously. Change in absorbance of the sample extract was measured at 515 nm for 30 min. The percentage inhibition of DPPH of the test sample and known solutions of Trolox were calculated by the following formula: % Inhibition = 100  (A0  A)/A0, where A0 was the beginning absorbance at 515 nm, obtained by measuring the same volume of solvent, and A was the final absorbance of the sample extract at 515 nm. Methanol was used as a blank. Results were expressed as lM Trolox equivalent (TE)/g DW.

2.6.4. Ferric-reducing antioxidant power (FRAP) assay The FRAP assay was carried out according to the procedure described in the literature (Benzie & Szeto, 1999). Briefly, the FRAP reagent was prepared according to the Total Antioxidant Capacity Assay Kit with FRAP (Beyotime Institute of Biotechnology). The FRAP reagent was freshly prepared daily and warmed to 37 °C in a water bath before use. 5 ll of the diluted sample was added to 180 ll of the FRAP reagent. The absorbance of the mixture was measured at 593 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek, USA) after 4 min. The standard curve was constructed using FeSO4 solution, and the results were expressed as lM Fe(II)/g dry weight of fruit. Additional dilution was needed if the FRAP value measured was over the linear range of the standard curve. 2.6.5. Trolox equivalent antioxidant capacity (TEAC) assay The TEAC assay was carried out according to the method established in the literature (Re et al., 1999). Total Antioxidant Capacity Assay Kit with ABTS (Beyotime Institute of Biotechnology) was used to prepare the ABTS+ stock solution. 7 mM ABTS and 2.45 mM potassium persulphate in a volume ratio of 1:1, was then incubated in the dark for 16 h at room temperature and used within 2 days. The ABTS+ working solution was prepared by diluting the stock solution with ethanol to an absorbance of 0.70 ± 0.05 at 734 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek, USA). All samples were diluted approximately to provide 20–80% inhibition of the blank absorbance. 5 ll of the diluted samples were mixed with 200 ll ABTS+ working solution. The absorbance of the mixture was measured at 734 nm after 6 min of incubation at room temperature, and the percent of inhibition of absorbance at 734 nm was calculated. Trolox was used as a reference standard, and the results were expressed as lM TE/g dry weight of fruit. 2.7. Statistical analysis All analyses were carried out in triplicates, and the data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed by a Student’s test. Differences were considered to be significant for p < 0.05. Graphpad Prism 5.0 (San Diego, CA, USA) and Microsoft Excel 2010 (Roselle, IL, USA) were used for the statistical and graphical analysis. 3. Results and discussion 3.1. TLC analysis Although CWT and CML are consumed as CF under the same name, they were found here to have significant differences in chemical composition. TLC analysis indicated that the chromatogram of the CWT sample solution exhibited a prominent yellow band corresponding to naringin (9) at RF value of approximately 0.22 (Fig. 1). It also exhibited a narrow and intense blue band corresponding in colour and RF to the band in the chromatogram of the meranzin hydrate (12), whilst the chromatogram of the CML sample solution exhibits two intense blue bands corresponding in colour and RF corresponding to 5,7-dimethoxycoumarin (18) and scoparone (13). 3.2. LC–QTOF/MS analysis HPLC–MS/MS analyses were performed in both positive and negative ion modes. The total ion chromatograms in positive ion mode of the extracts of CF herbs are shown in Fig. 2. Twenty-five major compounds were identified or tentatively characterised by

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P. Zhao et al. / Food Chemistry 173 (2015) 54–60

scoparone 5,7-dimethoxycoumarin

meranzin hydrate naringin

Standard 1

CWT1

CWT2

CWT3

CWT4

CWT5

CML1

CML2

CML3

CML4

Fig. 1. TLC analysis of CWT and CML; CWT1–CWT5 represented 5 CWT samples from different sources, whilst CML1–CML5 represented 5 CML samples from different sources. (naringin, RF = 0.22; meranzin hydrate, RF = 0.34; 5,7-dimethoxycoumarin, RF = 0.74; scoparone, RF = 0.88).

Fig. 2. Positive HPLC–QTOF/MS chromatograph of CWT (A) and CML (B). The peak numbers are in accordance with the compound numbers in Table 2.

comparison with reference substances or literature data. Their retention times and MS data are listed in Table 2. Analysis of the UV absorption and MS spectra led to the identification of two flavone di-C-glycosides (1 and 2), one flavone triglycoside (3), five flavanone-O-diglycosides (4, 5, 9, 11 and 14), three flavone-Odiglycosides (6, 8 and 10), one flavone-C-glycoside (7), ten coumarins (12, 13, 15, 16, 17, 18, 19, 20, 24 and 25), and three limonoids (21, 22 and 23). These results provide helpful chemical information for quality control and clinical application of the two species of CF. 3.2.1. Characterisation of flavonoids Citrus flavonoid-O-glycosides are usually characterised by the linkage of either b-deoxyhexose or b-hexose to the flavonoid skeleton through the C-7 hydroxyl group (Cuyckens, Rozenberg, de Hoffmann, & Claeys, 2001). For example, Compounds 9 and 11 yielded [M+H]+ at m/z 581 and m/z 611 in positive mode and [MH] at m/z 579, m/z 609 in negative mode, respectively. The cleavage of two glycosidic linkages and the successive neutral losses of 146 and 308 amu produced the fragments at m/z 435, 273 for compound 9 and m/z 465, 303 for compound 11, respectively. Compared with the standard compounds, compounds 9 and 11 were unequivocally identified as naringin and hesperidin, respectively. In the present study, the other six flavonoid-O-glycosides (compounds 4, 5, 6, 8, 10 and 14), which showed similar fragmentation patterns, were tentatively identified as eriocitrin, neoeriocitrin, rutin, rhoifolin, diosmin and melitidin in CWT and CML. In the present study, naringin was observed in both CWT and CML samples, eriocitrin, neoeriocitrin, rhoifolin, and melitidin

were only detected in CWT, whilst rutin, hesperidin and diosmin were only detected in CML. Unlike flavonoid-O-glycosides, flavone-C-glycosides showed the main fragments such as ([M+HnH2O]+), ([M+HCH2O2H2O]+). For instance, Compound 1 exhibited quasi-molecular ion ([M+H]+) at m/z 595 in positive mode and m/z 593 ([MH]) in negative mode. Positive ion MS/MS mode focused on secondary fragments at m/z 577 ([M+HH2O]+), 559 ([M+H2H2O]+), 541 ([M+H3H2O]+), 529 ([M+HCH2O2H2O]+), 523 ([M+H4H2O]+), 511 ([M+HCH2O3H2O]+) and 475 ([M+H120]+). Although the authentic standard was not available, the MS spectrum showed fragmentations indicative of cleavage of the saccharide residues and the spectrum was in accordance with that of apigenin-6,8di-C-glucoside (Mencherini et al., 2012; Roowi & Crozier, 2011). Compound 2, 3 and 7 showed the characteristic fragmentation pattern like compound 1, they were tentatively characterised as lucenin-2-40 -methylether, rhoifolin-40 -O-glucoside and scoparin respectively (Barreca, Bellocco, Caristi, Leuzzi, & Gattuso, 2011; Mencherini et al., 2012; Roowi & Crozier, 2011). It was noticeable that apigenin-6,8-di-C-glucoside was present in both CWT and CML, whilst lucenin-2-40 -methylether and scoparin were only detected in CML, and rhoifolin-40 -O-glucoside was detected in CWT. 3.2.2. Characterisation of coumarins In positive ion mode, coumarin compounds could form the base peaks of [M+HCO]+, [M+H2CO]+, [M+HCO2]+ or [M+HCH2O]+ by loss of CO (28 Da) and CO2 (44 Da) groups in their MS/MS

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P. Zhao et al. / Food Chemistry 173 (2015) 54–60

Table 2 Identification of constituents from CWT and CML by HPLC–Q/TOF–MS in positive ion mode.

a

Peak

TR (min)

kmax (nm)

Precursor ion [M+H]+

Formula

Fragment ions (m/z)

Diff. (ppm)

Identification

Crude drug

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

1.8 2.0 2.0 4.3 4.7 5.0 5.3 6.7 6.9 7.0 7.3 9.2 9.3 9.8 10.8 12.5 13.2 13.3 13.48 13.5 13.7 14.2 14.9 16.1 16.6

280 280 280 280 280 280 280 280 280 280 280 330 330 280 330 330 330 330 330 330 210 210 210 330 330

595.1653 625.1767 741.2233 611.1599 597.1815 597.1792 463.1237 579.1731 581.1890 609.1814 611.1986 279.1125 207.0651 725.2304 305.1017 333.1702 261.1124 207.0650 217.0499 261.1150 471.2030 515.2281 455.2043 245.1179 271.0964

C27H30O15 C28H32O16 C33H40O19 C27H30O16 C27H32O15 C27H32O15 C22H22O11 C27H30O14 C27H32O14 C28H32O15 C28H34015 C15H18O5 C11H10O4 C33H40O18 C16H16O6 C19H24O5 C15H16O5 C11H10O4 C12H8O4 C15H16O4 C26H30O8 C28H34O9 C26H30O7 C15H16O3 C16H1404

577,541,457,409,355,307,163 589,541,487,439,355,285,158 595,433,271 303 506,435,399,289,245,195 506,435,399,289,245,195 427,397,343,313,155 217 545,492,383,273 505,463,301 557,489,413,303 261,189,159,131 191,179,163,151 653,509,443,381,273,145 203,175,159,147,131 297,277,255,163,135 243,189,159,131 192,179,151 202,174,146 243,213,189,159,131 425,367,253,161,105 469,369,313,161,111 409,349,315,161 232,203,189,175,145 203,175,147,131

0.3 2.11 0.15 1.24 0.89 0.98 0.25 1.51 1.75 0.28 1.39 0.79 3.74 2.23 0.16 1.37 0.7 1.99 1.63 1.15 0 1.11 1.28 1.39 1.34

Apigenin-6,8-di-C-glucoside Lucenin-2-40 -methylether Rhoifolin-40 -O-glucoside Rutin Eriocitrin Neoeriocitrin Scoparin Rhoifolin Naringina Diosmin Hesperidina Meranzin hydratea Scoparone Melitidin Oxypeucedanin hydrate Marmin Meranzin 5,7-Dimethoxycoumarina Bergapten Isomeranzin Limonina Nomilina Obacunonea Osthole Isoimperatorina

CWT & CML CML CWT CML CWT CWT CML CWT CWT & CML CML CML CWT CML CWT CWT CWT CWT CML CML CWT & CML CWT& CML CWT & CML CWT & CML CWT CWT

Confirmed by standard compounds.

spectra (Siskos, Mazomenos, & Konstantopoulou, 2008). In the present work, a total of ten coumarins were detected and elucidated in CWT and CML. For example, scoparone (6,7-dimethoxcoumarin, compound 13), which had been identified in C. medica var. Sarcodactylis (Chu, Li, Yin, Ye, & Zhang, 2012), yielded the fragments m/z 179 [M+HCO]+, m/z 163 [M+HCO2]+ and m/z 151 [M+H2CO]+. The results demonstrated that there were significant differences in composition of coumarin compounds between the two CF species. A total of seven coumarins were detected in CWT, whilst only four coumarins (scoparone, 5,7-dimethoxcoumarin, bergapten and isomeranzin) were detected in CML.

(21), nomilin (22) and obacunone (23) was at 210 nm. To analyse them at one run, we divided the chromatographic fingerprinting into three segments according to the retention time of target compounds, and set specific detection wavelength for each segment. The DAD wavelength program was set as follows: 0–7.7 min, 283 nm; 7.7–13.4 min, 330 nm; 13.4–20 min, 210 nm. The typical chromatograms of standards and samples are shown in Fig. 3.

3.3. HPLC–DAD analysis

3.3.2. Validation of the developed method The method was validated in terms of linearity, sensitivity, precision, stability and accuracy. The linearity, test range, limit of detection (LOD) and limit of quantification (LOQ) of the eight compounds are shown in Supplementary materials. It was found that the calibration curves for all compounds showed good linearity (R2 > 0.9992) within the test range. The LODs and LOQs were less than 5.6 and 18 ng for DAD, indicating that this method is sufficiently sensitive. The relative standard deviations (RSDs) for intraand inter-day repeatability are also shown in Supplementary materials. It was found that overall intra- and inter-day variations were not more than 1.27% and 1.42%, respectively, suggesting that the developed method was precise. The spike recoveries of eleven components were 93.5–103.1%, demonstrating that this method was also accurate.

3.3.1. Optimisation of HPLC conditions The HPLC conditions, including chromatographic column and mobile phases were optimised to achieve the simultaneous separation of different types of compounds in CWT and CML samples. Finally, the assay was performed on an Agilent Zorbox SB-C18 (4.6 mm  50 mm, 1.8 lM) column and the elution program of 0.1% aqueous formic acid (A) and acetonitrile (B) were used as the mobile phase system. All eight compounds could be eluted with baseline separation in a run time of 20 min. The eight compounds analysed have different UV kmax values, for example, naringin (9) and hesperidin (11) showed distinct maximal UV absorption at 283 nm, meranzin hydrate (12) and 5,7-dimethoxycoumarin (18) had maximal absorptions at 330 nm, and the maximum absorption wavelength of limonin

3.3.3. Sample determination The HPLC–DAD method was subsequently applied for simultaneous quantification of eight marker compounds in CWT and CML samples. The HPLC chromatograms of CWT and CML extracts are shown in Fig. 3 and the assay results are summarised in Table 1. The results indicated that there were remarkable differences in the amounts of the eight compounds in CWT and CML extracts. In CWT, naringin (34.1–68.0 mg/g) was present in the highest amounts, followed by meranzin hydrate (3.8–7.4 mg/g) and nomilin (1.5–4.8 mg/g); whilst nomilin (1.97–4.4 mg/g) was the most dominant constituent in CML, followed by hesperidin (0.75–2.0 mg/g). Hesperidin, 5,7-dimethoxycoumarin and obacunone were not detected in CWT, whereas meranzin hydrate and isoimperatorin were not detected in CML. This finding

3.2.3. Characterisation of limonoids To date, more than 50 individual limonoid aglycones and glucosides have been isolated in Citrus herbs (Li, Zhang, Zhang, Xu, & Liu, 2010; Liu et al., 2012). In the present study, three limonoids, namely limonin (m/z 471 [M+H]+), nomilin (m/z 515 [M+H]+) and obacunone (m/z 455 [M+H]+) were unequivocally characterised in comparison with the authentic standards. It was interesting to note that both CWT and CML were shown to contain the three limonoid compounds by using LC–QTOF/MS analysis.

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No1rm. 400

25

12

DAD1 A, Sig=283,4 Ref=off, TT (STANDARD 2.D)

18

9

21 22

300

A

200

11

23

100 0 No1rm.

6

4

2

8

10

12

14

min

16

DAD1 A, Sig=283,4 Ref=off, TT (CWT.D)

2000

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21 13

25

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15

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12 0 4

2 No1rm.

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175 150

C

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DAD1 A, Sig=283,4 Ref=off, TT (CML.D)

50

125 100

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0 6.5

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Fig. 3. HPLC–DAD chromatograph of Standard stock solutions 2 (A), CWT (B) and CML (C), naringin (9), hesperidin (11), meranzin hydrate (12), 5,7-dimethoxycoumarin (18), limonin (21), nomilin (22), obacunone (23), isoimperatorin (25).

Table 3 Results of the TPC, TFC, and DPPH, ABTS, FRAP free radical-scavenging activity of CWT and CML.

TPC (mg GAE/g) TFC (mg QUE/g) DPPH (TE mM/g) ABTS (TE mM/g) FRAP (FeII mM/g)

CWTa

CMLa

Standard curve

17.59 ± 1.94 17.205 ± 2.19 15.2 ± 1.44 5.09 ± 3.92 1.63 ± 2.09

2.74 ± 1.12 2.41 ± 2.03 1.48 ± 1.82 0.92 ± 2.08 0.38 ± 1.98

Y = 0.0019X  0.0107, R2 = 0.9996 Y = 4.3616X  0.0005, R2 = 0.9998 Y = 0.3394X + 0.0659, R2 = 0.9991 Y = 0.3394X + 0.0659, R2 = 0.9991 Y = 0.3621X  0.0056, R2 = 0.9998

GAE/g: gallic acid equivalent/g; QUE/g: quercetin equivalent/g. a Data are represented as the mean ± SD from three independent experiments (n = 3).

could be elaborated as a means for differentiation of the two species of CF. 3.4. Antioxidant analysis 3.4.1. TPC and TFC The results shown in Table 3 indicated that there were significant differences for TPC and TFC in the two species (p < 0.05). The TPC in CWT was 17.59 ± 1.94 mg GAE/g, compared to 2.74 ± 1.12 mg GAE/g for CML. We also found that CWT had a high TFC (17.205 ± 2.19 mg quercetin equivalent/g). 3.4.2. Determination of antioxidant activity In the present study, the antioxidant activities of CWT and CML extracts were determined by DPPH, ABTS and FRAP assay. As shown in Table 3, the DPPH, ABTS and FRAP values of CWT were 15.2 ± 1.44 lM TE/g, 5.09 ± 3.92 lM TE/g and 1.63 ± 2.09 lM Fe(II)/g, respectively, whilst the DPPH, ABTS and FRAP values of CML was 1.48 ± 1.82 lM TE/g, 0.92 ± 2.08 lM TE/g, 0.38 ± 1.98 lM Fe(II)/g, respectively. It is quite obvious that CWT exhibited higher antioxidant activity than CML. Previous researches have reported the linear positive correlation between bioactive compounds in plant extracts with their antioxidant capacities (Mustafa, Hamid, Mohamed, & Bakar, 2010). Therefore, the TPC,

TFC data and the antioxidant assay results were correlated and compared with each other. The results demonstrated a positive significant relationship (R2 = 0.967, 0.870, 0.960, respectively) between TPC and DPPH, ABTS, FRAP radical scavenging activity. The TFC also showed a positive correlation with DPPH scavenging activity (R2 = 0.977), ABTS (R2 = 0.830) and reducing power FRAP (R2 = 0.980) (shown in Supplementary materials). According to the quantitative and qualitative analysis of the two CF herbs, CWT has a higher level of flavonoids (e.g., naringn and melitidin), whilst CML contains more limonins and coumarins (e.g., nomilin, limonin and 5,7-dimethoxycoumarin). Previous studies demonstrated that flavonoids in CF had higher radical scavenging activity than coumarins and limonins (Kiplimo, Islam, & Koorbanally, 2012; Torres et al., 2006; Yu, Lou, Chiu, & Ho, 2013). Therefore, the higher antioxidant activity of CWT may possibly be attributed to the presence of high level of flavonoids in it. 4. Conclusions In the present study, we analysed the constituents in CWT and CML extracts by TLC, HPLCQTOF/MS and HPLCDAD methods, and compared the antioxidant activity of CWT and CML using three different methods (DPPH, FRAP and ABTS). The TLC and LCQTOF/ MS analysis demonstrated that there were significant differences

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in composition of chemical compounds between the two CF species. A total of 25 compounds including flavonoids, coumarins and limonoids were identified in CWT and CML extracts by comparing their retention time, UV spectra and MS fragmentation behaviour with standard compounds or published data. The HPLC–DAD analysis indicated that naringin (34.1–68.0 mg/g) was present in the highest amounts in CWT, whilst nomilin (1.97–4.4 mg/g) was the most dominant constituent in CML. The antioxidant assay demonstrated that CWT had significantly higher free radical-scavenging activity than CML. The results obtained in the present work might provide very useful information for authentication and quality control of CF. Acknowledgements The authors greatly appreciate financial support from Program for the National Natural Science Foundation of China (81473343), New Century Excellent Talents in University (NECT-13-1034), ‘‘Six Talent Peaks Program’’ of Jiangsu Province of China (2013-YY-001), Fundamental Research Funds for the Central Universities (ZD2014YW0033), Traditional Chinese Medicine Industry Special Scientific Research (201307002), the National New Drug Innovation Major Project of China (2011ZX09307002-02), and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 10.010. References Barreca, D., Bellocco, E., Caristi, C., Leuzzi, U., & Gattuso, G. (2011). Elucidation of the flavonoid and furocoumarin composition and radical-scavenging activity of green and ripe chinotto (Citrus myrtifolia Raf.) fruit tissues, leaves and seeds. Food Chemistry, 129, 1504–1512. Benzie, I. F., & Szeto, Y. T. (1999). Total antioxidant capacity of teas by the ferric reducing/antioxidant power assay. Journal of Agricultural and Food Chemistry, 47, 633–636. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT – Food Science and Technology, 28, 25–30. Chai, X. Y., Li, S. L., & Li, P. (2005). Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSD. Journal of Chromatography A, 1070, 43–48. Chau, C. F., & Wu, S. H. (2006). The development of regulations of Chinese herbal medicines for both medicinal and food uses. Trends in Food Science & Technology, 17, 313–323. Chen, X., Hu, L., Su, X., Kong, L., Ye, M., & Zou, H. (2006). Separation and detection of compounds in Honeysuckle by integration of ion-exchange chromatography fractionation with reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Journal of Pharmaceutical and Biomedical Analysis, 40, 559–570.

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