Food Chemistry xxx (2014) xxx–xxx
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Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities Chanjuan Xu a, Bingxian Yang a, Wei Zhu a, Ximin Li b, Jingkui Tian a, Lin Zhang a,⇑ a b
College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, Zhejiang, China Changshu Qiushi Technology Co. Ltd., Changshu, Jiangsu, China
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Linderae aggregate Polyphenol Flavonoid Quality control Antioxidant assays
a b s t r a c t The leaves of Linderae aggregate (LAL) has been used as a type of tea in China and other Southeast Asian countries. In this study, 11 polyphenols in LAL were clarified for the first time using multiple highperformance liquid chromatographic techniques. An optimal extracting method was developed through the comparison of the amount of quercetin-3-O-a-L-rhamnoside using a uniform design method. From the fingerprint liquid chromatography data, 11 common peaks in the 8 samples collected from April to November were semi-determined. The antioxidant capacities were examined using the 2,2-diphenyl1-picrylhydrazyl free radical scavenging assay and the ferric reducing/antioxidant power assay. All 8 samples contained the same 11 polyphenols in similar ratios. Three samples, S2, S5 and S6 contained higher amount of quercetin-3-O-a-L-rhamnoside and were demonstrated to have stronger antioxidant capacities in both antioxidant assays. These results are critical in optimising harvest time and quality control of LAL. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lindera aggregate (Sims) Kosterm belongs to the Lauraceae family and is used throughout southern China, Japan, and other southeastern Asian countries (Chen, Chou, Yang, Bligh, & Wang, 2012). As a traditional Chinese medicine, the root of L. aggregate is utilised in the treatment of several different symptoms including chest and abdomen pain, inflammation, indigestion, regurgitation, cold hernia and frequent urination (Editorial Committee of the Administration Bureau of Traditional Chinese Medicine, 1999). The leaves of L. aggregate (LAL) is popular in tea drinks due to the protective effects against oxidative stress (Gu et al., 2008). Previous phytochemical investigations showed that the main components of LAL are flavonoid glycosides (Zhang, Sun, Zhao, & Wang, 2001; Zhang, Sun, Chou, & Wang, 2003; Xiao, Cao, Fan, Shen, & Xu, 2011), sesquiterpene lactones as well as other components (Zhang, Sun, Wang, & Chou, 2001). Isoquinoline alkaloids are found in the roots of L. aggregate (Wang et al., 2007), while tannins are in the stems (Zhang, Sun, Wang, Hattori, & Tewtrakul, 2003). Recently, LAL was approved as a new food resource (Ministry of Health of the People’s Republic of China, 2013). However, the key components in LAL have not been well understood and they need to be ⇑ Corresponding author. Tel.: +86 571 87951301. E-mail address: [email protected] (L. Zhang).
further explored before LAL could be widely used as a food resource. Herbal plants have multiple types of components that vary in content; therefore, quality control of these components remains a challenge. In recent years, chromatography methods, such as high-performance liquid chromatography (HPLC) fingerprint, have been used for species’ differentiation and quality control of herbal medicines. This method has been utilised with ginkgo leaf (Beek & Montoro, 2009), Danshen (Zhang, Cui, He, Yu, & Guo, 2005), green tea (Alaerts et al., 2012), ginger (Yudthavorasit, Wongravee, & Leepipatpiboon, 2014), Goji berry fruit (Donno, Beccaro, Mellano, Cerutti, & Bounous, 2014), Rosa (Riffault, Destandau, Pasquier, André, & Elfakir, 2014) and L. aggregata root (Han et al., 2008; Wu et al., 2010). HPLC fingerprinting can be used to detect the complex constituents in herbal extracts both qualitatively and quantitatively, and it is highly suitable for quality control of various herbs. In addition, the chemical profile information of herbal extract can also be identified using multiple detectors such as tandem mass spectrometry (MS/MS) (Guan et al., 2011; Wu et al., 2013). In the Tiantai district of the Zhejiang Province in China, LAL is known as the native ‘‘Tiantai L. aggregate’’ or ‘‘Tai L. aggregate’’ and it is a popular tea drink in that region. Since LAL is a traditional Chinese medicine and popular tea, it’s essential to identify and investigate all the functional ingredients of LAL. Previous studies
http://dx.doi.org/10.1016/j.foodchem.2014.11.042 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
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on LAL have identified 15 flavonoids, 3 sesquiterpene lactones and 3 phenylpropanoid glycosides (Luo, Zhang, Tian, & Yang, 2009; Zhao et al., 2012). To further expand upon the functional components in LAL, this study characterised the ingredients using HPLC fingerprint and MS methods. To explore the optimal harvest time of LAL, quercetin-3-O-a-L-rhamnoside (QR) from LAL was collected over several months and analysed for content. Since flavonoids and other polyphenols are widely used as natural antioxidants to significantly reduce the oxidative damage (Gu et al., 2008; Walle, 2004), the antioxidant capacities of different LAL samples were also measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing/antioxidant power (FRAP) assay. These powerful methods are suited for the identification and quantitation of the complex components found in herbal extracts.
LC/MS). A TC-C18 column (4.6 mm 250 mm, 5 lm i.d., Agilent, USA) was used for separation, and the column temperature was 35 °C. The mobile phase consisted of acetonitrile (A) and water (B, with 0.05% formic acid) with the following gradient program: 0–20 min, 10–23% A; 20–40 min, 23% A; 40–45 min, 23–95% A; 45–55 min, 95% A. The flow rate was set at 0.8 ml/min, and the DAD wavelength was 254 nm, with a sample injection volume of 10 ll. Mass spectra were recorded within 55 min. The negative electronic spray ion (ESI) conditions included a nitrogen nebulizer pressure of 35 psi, a vaporizer temperature of 350 °C at 11 L/min, and a capillary voltage of 4000 V. Selected ion monitoring (SIM) mode was used to record the abundance of the negative ion at m/z 100 to 1200 with a dwell time of 500 ms. Peak identification was performed by comparing the mass spectra and fragmentation ions, with relative retention time to the standards.
2. Experimental 2.1. Reagents and materials Quercetin-3-O-(200 -O-b-D-glucopyranosyl)-a-L-arabinofuranoside (2), quercetin-3-O-(200 -O-b-D-glucopyranosyl)-b-D-xylopyranoside (3), quercetin-3-O-b-D-glucopyranoside (4), quercetin-3-O-(200 -Ob-D-glucopyranosyl)-b-D-rhamnopyranoside (6), quercetin-3-O-aL-arabinofuranoside (7), quercetin-3-O-b-D-xylopyranoside (8), quercetin-3-O-a-L-rhamnoside (9), kaempferol-3-O-(200 -O-b-D-glucopyranosyl)-b-D-rhamnopyranoside (10) and kaempferol-3-O-aL-rhamnopyranoside (11) were prepared and identified in our laboratory as previously reported (Luo et al., 2009; Zhao et al., 2012). The structures are shown in Fig. S1. 1,1-Diphenyl-2picrylhydrazyl (J28Z009) was obtained from Alfa Aesar (USA) and 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ, K1319026) from Aladdin (China). Other reagents (analytical grade) were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). HPLC-grade acetonitrile was purchased from Tedia Company (USA). Ultra pure water was prepared using a Milli-Q50 SP Reagent Water System. 2.2. Sample extraction and optimisation From young leaves of L. aggregate (Sims) Kosterm, 8 seasonal leaf batches (S1–S8) were collected in the Tiantaishan district from April through November 2013 (Table 4). All batches were authenticated by Professor Jingkui Tian in Zhejiang University, where the voucher specimens (No. LA20130108) were deposited. Dried powder of each LAL sample (1 g) was extracted using 12 ml of 95% ethanol under reflux for 2 h and repeated thrice. All of the extract liquors were combined, filtered, concentrated under vacuum, then transferred and diluted to a final volume of 10 ml solution. The resulting solution was utilised for HPLC analysis and antioxidant assay. To optimise the extraction process of LAL, a uniform design method was applied (Liang, Fang, & Xu, 2001; Wu, Zhu, & Xu, 2011) using S1 sample. QR was quantified as a chemical indicator. The process factors that were varied include extracting ethanol concentration (60%, 70%, 80% and 95%), solid to liquid ratio (1:12, 1:14, 1:16, 1:18, 1:20 and 1:22), extraction time (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 h) and extraction frequencies (once, twice and thrice) and were used to optimise the extraction conditions (Table 2). Each test was repeated in triplicate. 2.3. HPLC analysis and total polyphenol content assay 2.3.1. HPLC–DAD–ESI MS/MS analysis The HPLC system was comprised of an Agilent 1200 analytical HPLC equipped with a DAD and a mass detector (6410 Triple Quad
2.3.2. HPLC analysis HPLC analysis was performed using a Shimadzu HPLC with two LC-10AT VP pumps, a photodiode array detector (DAD) and LC solution program. The chromatographic separation was performed on a TC-C18 column (4.6 mm 250 mm, 5 lm i.d., Agilent, USA) with column temperature of 40 °C. The solvent system consisted of a 30 min isocratic elution of 20% acetonitrile with 0.5% formic acid in water. At the end of the isocratic elution the column was flushed with 95% acetonitrile for 10 min to remove strongly retained constituents. The flow rate was 1.0 ml/min with an injection volume of 20 ll. In order to monitor all polyphenols simultaneously, the detection wavelength was set at 254 nm. To determine the contents of QR in the 8 seasonal samples, the stock QR solution (1.993 mg/ml) was applied and diluted if needed. The working calibration curves showed linearity in the range of 0.09965–1.993 mg/ml (r = 0.999) for QR, and the regression curves was Y = 2600X + 3811 (r = 0.999), where Y is the peak area of the analyte and X is the injected quantity (ng) of the QR standard. For fingerprint analysis, the chromatographic peaks were identified by comparing the retention time and UV spectra to the HPLC–DAD–MS analysis results together with those of reference compounds 2–4 and 6–11 which were eluted in parallel under the same conditions. Their relative contents were calculated as QR amount according to their peak area ratio, respectively. 2.3.3. Total polyphenol content assay Total phenolic contents in S1–S8 samples were further determined using the Felin–Ciocalteu method (Małgorzata, Magdalena, Artur, & Dorota, 2014). A 0.1 ml of sample solution was mixed with 2 ml ferrous tartrate solution and 2.9 ml phosphate buffer (pH = 7.5), and water was added to bring the final volume to 10 ml. The absorbance of the mixture was determined at 535 nm versus water blank. Gallic acid was used as a standard, and the linear range was between 0.0998 and 0.5989 mg (r = 0.999). Results were expressed as gallic acid equivalents (mg of gallic acid/g of sample in dried weight). 2.4. Antioxidant assay 2.4.1. DPPH free radical scavenging assay The antioxidant activities of S1–S8 samples were evaluated using a DPPH assay as reported by Sun et al. (2013) with some variations. Each sample extract was diluted from 250 lg/ml to 2500 lg/ml. Sample solutions or blank ethanol solvents (400 ll) were mixed with 0.16 mM DPPH ethanol solution (3 ml) and the action mixtures were kept for 30 min at room temperature. The absorbance of the resulting solution was read at 517 nm by a
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
C. Xu et al. / Food Chemistry xxx (2014) xxx–xxx
spectrophotometer. The capability to scavenge the DPPH radical was calculated by using the following equation:
%Inhibition ¼ ðA0 A1 Þ=A0 100%
ð1Þ
where A1 and A0 was the absorbance of the sample and blank, respectively. 2.4.2. Ferric reducing/antioxidant power assay The FRAP assay was also utilised to measure the total antioxidant capacity of S1–S8 samples as reported by Benzie and Siu-Wai (2014). The FRAP reagent was prepared using 0.3 mol/L acetate buffer (pH = 3.6), 20 mM ferric chloride and 10 mM TPTZ in 40 mM hydrochloric acid. The three solutions were mixed together at a ratio of 25:2.5:2.5 (v/v/v). 3 ml FRAP reagent (freshly prepared) was mixed with 100 ll of test sample. After incubation at room temperature for 30 min, the absorbance of reaction mixture was measured at 593 nm. The FRAP values, expressed in mmol ferrous sulphate equivalents (FSE)/g sample in dry weight, were derived from a standard curve. 2.5. Statistical analysis All measurements were replicated in triplicate. Data were expressed as mean ± standard error of three separate experiments,
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IC50 values were calculated by regression analysis. Evaluation of statistical significance of differences was performed by a oneway analysis of variance (one-way ANOVA). All statistical analyses were performed with EXCEL and SPSS 17.0 software. 3. Results and discussion 3.1. Characterisation of polyphenolic components in LAL Previously, the main chemical components of LAL were identified to involve 15 flavonoids, 3 sesquiterpene lactones as well as other components (Luo et al., 2009; Zhao et al., 2012). To expand upon the previous results new methodologies were used. Seasonal sample S1 was analysed by HPLC–DAD–ESIMS/MS to identify the principle components which are shown in Fig. 1 and Table 1. The UV spectra, mass spectra of pseudo-molecular ions and de-monosaccharide fragmentation ions of S1 were compared to published data and the retention time of S1 samples was compared with reference compounds 2–4 and 6–11. From these comparisons, 11 compounds were identified. Peak 1 was identified as cinnamtannin B1 (Zhang et al., 2003) and peak 5 was identified as quercetin-3-Ob-D-galactopyranoside, an isomer of QR (Zhang et al., 2001). Except 1, all other compounds 2–11 are flavonoid glycosides, with QR (9) as the major component and these are the key ingredients of LAL
Fig. 1. HPLC–PDA–()ESIMS/MS chromatograms of the leaves of Linderae aggregate. For the retention time and respective compound 1–11, see Table 1.
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
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Table 1 Identification of polyphenols in the leaves of Linderae aggregate by HPLC–DAD–() ESIMS/MS analysis. Peak No.
Rt (min)
kmax (nm)
MS [MH]
MS/MS of [MH]
Mw
Identification
1 2 3 4 5 6 7 8 9 10 11
17.03 22.79 24.25 24.65 25.11 26.33 27.78 28.79 29.56 31.69 38.18
278 232, 255, 255, 255, 255, 256, 255, 254, 265, 263,
863.1 595.0 595.0 463.1 463.1 609.0 433.0 433.0 447.1 593.0 431.1
711.1, 572.9 301.0 300.9 301.0 301.0 300.9 301.0 301.0 300.9 413.0, 285.0 285.1, 254.9
864.0 596.0 596.0 464.0 464.0 610.0 434.0 434.0 448.0 594.0 432.0
Cinnamtannin B1 Quercetin-3-O-(200 -O-b-D-glucopyranosyl)-a-L-arabinofuranoside Quercetin-3-O-(200 -O-b-D-glucopyranosyl)-b-D-xylopyranoside Quercetin-3-O-b-D-glucopyranoside Quercetin-3-O-b-D-galactopyranoside Quercetin-3-O-(200 -O-b-D-glucopyranosyl)-b-D-rhamnopyranoside Quercetin-3-O-a-L-arabinofuranoside Quercetin-3-O-b-D-xylopyranoside Quercetin-3-O-a-L-rhamnoside Kaempferol-3-O-(200 -O-b-D-glucopyranosyl)-b-D-rhamnopyranoside Kaempferol-3-O-a-L-rhamnopyranoside
350 352 352 354 349 353 352 348 345 342
Table 2 The content of quercetin-3-O-a-L-rhamnoside in S1 sample of leaves of Linderae aggregate using U12 (124) uniform design test.
a
Level
Ethanol (%)
Solid–liquid ratio (g/ml)
Extraction time (h)
Extraction frequencies
QRa (mg/g)
1 2 3 4 5 6 7 8 9 10 11 12
60 60 60 70 70 70 80 80 80 95 95 95
1:12 1:14 1:16 1:18 1:20 1:22 1:12 1:14 1:16 1:18 1:20 1:22
1.5 2.5 0.5 2 3 1 2.5 0.5 1.5 3 1 2
3 2 1 3 2 1 3 2 1 3 2 1
4.15 ± 0.54 3.56 ± 0.22 0.78 ± 0.17 4.62 ± 0.08 4.09 ± 0.18 1.65 ± 0.10 5.44 ± 0.05 3.60 ± 0.13 3.26 ± 0.40 5.94 ± 0.16 4.67 ± 0.11 4.29 ± 0.16
Data are expressed as mean ± SD values.
observed in Fig. 1. Therefore, further fingerprint analysis of LAL was conducted using these identified constituents as indicators. 3.2. HPLC fingerprint analysis and total polyphenol content assay 3.2.1. Uniform design test for optimal sample extraction The optimal sample extraction was determined using uniform design test. The results obtained from this testing are shown in Fig. 2A, B and Table 2. The regression curve was Y = 0.656X1 0.004X2 + 0.046X3 + 0.097X4 0.322 (r = 0.970), where Y was the QR content (mg/g), X1 was ethanol concentration, X2 was solid–liquid ratio, X3 was extracting time (h) and X4 was extracting frequencies. The ethanol concentration and extraction frequencies were the critical factors in the extraction process. The extracting time also affected extracting efficiency, while the solid–liquid ratio was less important as demonstrated by the SPSS analysis (Table S1). Accordingly, the optimal extracting process was established as follows: LAL was extracted for 2 h with 95% ethanol (1:12, g/ml) under reflux and repeated three times. This condition was repeated and the LAL extract had QR at 5.875 mg/g, which agrees with the QR extracting result of test 10 in Table 2. 3.2.2. Fingerprint analysis of samples of different month The seasonal LAL samples collected over several months were run on HPLC and the overlapped HPLC chromatograms of S1–S8 are shown in Fig. 2C. All samples contain common peaks 1–11 at different ratios, with QR (9) as the most intense peak. A detailed semi-quantitative comparison of these samples was conducted. The relative amounts of all other peaks, 1–8 and 10–11, were calculated by comparing the peak areas with that of QR, respectively. As shown in Table 3, compounds 1, 6 and 9 were major constituents of
LAL, with their amounts (equivalent of QR) generally higher than 3 mg/g. The contents of 1–11 all varied in the samples from different months, with S2 (45.4 mg/g in sum; collected in May) being the highest, followed by S5 (35.0 mg/g in sum; collected in August) and S6 (32.9 mg/g in sum; collected in September). By contrast, all other samples such as S3, S7 and S8 contained a significant less of these constituents (about 19 mg/g in sum, respectively). Apparently, the seasonal collection month affected the contents of flavonoids in LAL dramatically, which is a critical factor in quantity control and optimal collection time selection. 3.2.3. Total polyphenol content analysis The different LAL samples were also tested in the total polyphenol content assay and the results are shown in Table 4. The order of sample for total polyphenol (TP) content was as follows: S2 (49.47 mg/g) > S6 (35.18 mg/g) > S5 (29.72 mg/g) > S4 (24.56 mg/g) > S7 (24.33 mg/g) > S1 (23.13 mg/g) > S8 (21.92 mg/g) > S3 (15.11 mg/g). The rank order of this result is similar to QR amounts, with some slight variations in S5 and S6. The results suggested that flavonoids are a major part of TP, while other polyphenols such as cinnamomin B1 and other tannins also contributing to TP. 3.3. Antioxidant capability analysis of samples obtained from different months 3.3.1. Antioxidant capability analysis with DPPH free radical scavenging assay The free radical scavenging activities of S1–S8 samples were determined by the DPPH assay, using ascorbic acid (IC50 = 30.5 lg/ml) as a positive control. All samples exhibited strong concentration-dependent antioxidant potentials against DPPH free radicals at 250–2500 lg/ml. The IC50 (lg/ml) values are shown in Table 4. The order of the antioxidant capacity against DPPH was as follows: S2 » S5, S6, S7 » S8 > S4 » S1 > S3. S2 showed the strongest free radical scavenging capability (IC50 = 542.4 lg/ml) with the highest QR and TP contents, while S1 and S3 possessed relative lower antioxidant capacities (IC50 > 950 lg/ml). S5, S6 and S7 also showed stronger free radical scavenging capability (IC50 = 641.5 lg/ml) with higher QR and TP contents. Although S4 and S8 showed lower activities than S7, their QR and TP contents did not follow the same ratio. These results indicate that other constituents could also contribute to the total antioxidant capacities of LAL, which needed further exploration. 3.3.2. Antioxidant capability analysis with ferric reducing/antioxidant power assay The total antioxidant capacities of samples S1–S8 were also determined by FRAP assay. FRAP values were expressed in mmol ferrous sulphate equivalent (mmol FSE/g) sample in dry weight. The results are also shown in Table 4. The order of the antioxidant capacity in this assay was: S2 » S5, S6 » S8, S4 > S1, S3, S7. S2
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
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Fig. 2. HPLC chromatograms of (A) Quercetin-3-O-a-L-rhamnoside, (B) representative S1 sample and (C) overlapped S1–S8 samples of the leaves of Linderae aggregate. For respective compounds 1–11, see Table 1.
showed the strongest antioxidant capability (0.76 mmol FSE/g), followed by S5 and S6, while S1, S3 and S7 possessed relative lower antioxidant capacities (60.42 mmol FSE/g). These results were in parallel with those of DPPH order except S7. S7 possessed similar antioxidant capacity and QR/TP contents as that of S1 in this case. In general, both DPPH and FRAP assays suggested higher
antioxidant potentials of LAL accompany with higher amounts of QR and TP. In this study, LAL was shown to have apparent antioxidant capacities, likely due to high amount of flavonoids and other constituents, which is in agreement with a previous report (Gu et al., 2008). Moreover, 11 functional constitutes which contribute to
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
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Table 3 Relative contents of compounds 1–11 in eight samples of Linderae aggregate by HPLC–DAD analysis.a,b
a b
No.
S1
S2
S3
S4
S5
S6
S7
S8
1 2 3 4 5 6 7 8 9 10 11 Sum
3.53 ± 0.02 1.13 ± 0.01 2.60 ± 0.03 0.93 ± 0.01 0.44 ± 0.01 4.29 ± 0.04 0.93 ± 0.02 2.24 ± 0.03 5.87 ± 0.29 0.76 ± 0.01 1.12 ± 0.01 23.84 ± 0.48
5.20 ± 0.01 1.86 ± 0.02 3.95 ± 0.03 2.78 ± 0.02 1.39 ± 0.01 5.00 ± 0.07 1.83 ± 0.01 4.86 ± 0.04 15.58 ± 0.17 0.78 ± 0.01 2.22 ± 0.02 45.45 ± 0.41
3.08 ± 0.02 0.50 ± 0.01 0.99 ± 0.01 0.88 ± 0.01 0.58 ± 0.01 2.27 ± 0.02 0.84 ± 0.01 1.59 ± 0.01 6.86 ± 0.06 0.38 ± 0.01 1.40 ± 0.01 19.37 ± 0.18
3.36 ± 0.03 0.55 ± 0.01 1.11 ± 0.01 1.11 ± 0.01 0.96 ± 0.01 2.73 ± 0.03 0.95 ± 0.01 1.95 ± 0.02 8.63 ± 0.14 0.35 ± 0.01 1.55 ± 0.04 23.25 ± 0.32
3.96 ± 0.04 0.74 ± 0.01 1.49 ± 0.01 1.74 ± 0.02 2.40 ± 0.07 4.17 ± 0.05 1.27 ± 0.02 2.98 ± 0.03 14.04 ± 0.18 0.31 ± 0.01 1.93 ± 0.02 35.03 ± 0.46
3.42 ± 0.01 0.72 ± 0.01 1.45 ± 0.01 1.78 ± 0.02 2.19 ± 0.01 3.98 ± 0.02 1.28 ± 0.01 2.87 ± 0.01 12.98 ± 0.14 0.32 ± 0.01 1.89 ± 0.03 32.88 ± 0.28
3.69 ± 0.08 0.49 ± 0.01 1.43 ± 0.02 0.65 ± 0.01 0.54 ± 0.01 1.95 ± 0.04 0.66 ± 0.02 1.22 ± 0.02 6.45 ± 0.20 0.32 ± 0.01 1.52 ± 0.04 18.92 ± 0.46
3.32 ± 0.02 0.46 ± 0.01 0.99 ± 0.01 0.69 ± 0.01 0.76 ± 0.01 2.09 ± 0.02 0.67 ± 0.01 1.38 ± 0.01 7.23 ± 0.15 0.33 ± 0.01 1.56 ± 0.03 19.48 ± 0.29
Contents of compounds 1–8 and 10–11 were calculated as equivalents of quercetin-3-O-a-L-rhamnoside (mg/g of dried extracts). Data are expressed as mean ± SD values (n = 3).
Table 4 Contents of quercetin-3-O-a-L-rhamnoside and total phenols in 8 batches of the leaves of Linderae aggregate and their antioxidant capacities. No. S1 S2 S3 S4 S5 S6 S7 S8
Batch No. 201304 201305 201306 201307 201308 201309 201310 201311
QRa (mg/g) 5.87 ± 0.29 15.58 ± 0.17 6.86 ± 0.06 8.63 ± 0.14 14.04 ± 0.18 12.98 ± 0.14 6.45 ± 0.20 7.23 ± 0.15
TPa,b (mg/g)
DPPHa,c IC50 (lg/ml) ⁄S3
23.13 ± 0.61 49.47 ± 3.59 15.11 ± 0.26 24.56 ± 1.53 29.72 ± 1.11 35.18 ± 5.34 24.33 ± 0.62 21.92 ± 2.01
954.3 ± 35.7 542.4 ± 12.9 993.9 ± 32.5⁄S1 777.7 ± 32.2⁄S8 637.6 ± 5.5#S6, S7 641.5 ± 1.5#S5, S7 665.7 ± 14.9#S5, S6 735.9 ± 5.8⁄S4
FRAPa,c,d (mmol FSE/g) 0.42 ± 0.06#S3, S7 ⁄S4, S8 0.76 ± 0.03 0.41 ± 0.02 #S1, S7 ⁄S4 0.50 ± 0.01#S8 ⁄S3 0.66 ± 0.04#S6 0.62 ± 0.04#S5 0.39 ± 0.04#S1, S3 0.51 ± 0.04⁄S1
a
Data are expressed as mean ± SD values (n = 3). Results are expressed as gallic acid equivalents (mg of gallic acid/g of dried extracts). c # Differences of the same columns are not significant (P > 0.05); ⁄differences of the same columns are significant (0.01 < P < 0.05); differences between other columns are obvious significant (P < 0.01). d FRAP values are expressed in mmol ferrous sulphate equivalent (FSE)/g sample in dry weight. b
the antioxidant capacities were identified and semi-determined for the first time in this study. Quercetin-3-O-a-rhamnoside (quercitrin) and kaempferol-3-O-a-rhamnoside (afzelin) were also reported to contribute in major to the antioxidant capacities of the leaves of Lindera obtusiloba (Hong, Rhee, Won, Choi, & Lee, 2013). Total alkaloids isolated from the root of L. angustifolia also possessed free radical scavenging activities (Zhao, Zhao, & Wang, 2006), however, whether or not these ingredients existing in LAL remains unknown. Since flavonoids and other polyphenols are the major constituents of LAL with strong antioxidant capacities, the harvesting time points of S2 (collected in May), S5 (collected in August) and S6 (collected in September) are recommended as preferred ones. The contents of polyphenols and other secondary metabolites in plants depend on environmental conditions such as sun light, temperature, water and nutrition as previously reported (Guo et al., 2013). In general, LAL starts growing in May and is lush through August and September in Tiantai district, which may contribute to higher polyphenol amounts in LAL. By contrast, LAL was traditionally believed to be in first class when collected in May but since this is the beginning of the growing season, it would be more productive to harvest between July and August. These results provide critical guidance for monitoring the quantity of LAL and select the optimal harvesting time. 4. Conclusion In this study, the polyphenol profile of LAL was investigated using multiple HPLC fingerprint techniques. For the first time, QR and other 10 polyphenolic compounds in LAL were identified and
semi-determined by LC fingerprint analysis. Using the optimal extracting method developed in this study, 8 seasonal samples (S1–S8) were collected from April to November and evaluated using their fingerprint data. All 8 samples contain 11 polyphenolic compounds at various ratios, while S2, S5 and S6 have the highest amounts of QR and show stronger antioxidant capacities in the DPPH assay. These results have provided fundamental evidence critical for future development of LAL products under direction of their optimised collection time and quality control methods. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was financially supported by the National Science and Technology Major Project of China (No. 2013ZX09103002) and the National Science Foundation of China (No. U1303122). 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. 11.042. References Alaerts, G., Van Erps, J., Pieters, S., Dumarey, M., Nederkassel, A. M., Goodarzi, M., et al. (2012). Similarity analyses of chromatographic fingerprints as tools for
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Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities Chanjuan Xu a, Bingxian Yang a, Wei Zhu a, Ximin Li b, Jingkui Tian a, Lin Zhang a,⇑ a b
College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, Zhejiang, China Changshu Qiushi Technology Co. Ltd., Changshu, Jiangsu, China
a r t i c l e
i n f o
Article history: Available online xxxx Keywords: Linderae aggregate Polyphenol Flavonoid Quality control Antioxidant assays
a b s t r a c t The leaves of Linderae aggregate (LAL) has been used as a type of tea in China and other Southeast Asian countries. In this study, 11 polyphenols in LAL were clarified for the first time using multiple highperformance liquid chromatographic techniques. An optimal extracting method was developed through the comparison of the amount of quercetin-3-O-a-L-rhamnoside using a uniform design method. From the fingerprint liquid chromatography data, 11 common peaks in the 8 samples collected from April to November were semi-determined. The antioxidant capacities were examined using the 2,2-diphenyl1-picrylhydrazyl free radical scavenging assay and the ferric reducing/antioxidant power assay. All 8 samples contained the same 11 polyphenols in similar ratios. Three samples, S2, S5 and S6 contained higher amount of quercetin-3-O-a-L-rhamnoside and were demonstrated to have stronger antioxidant capacities in both antioxidant assays. These results are critical in optimising harvest time and quality control of LAL. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lindera aggregate (Sims) Kosterm belongs to the Lauraceae family and is used throughout southern China, Japan, and other southeastern Asian countries (Chen, Chou, Yang, Bligh, & Wang, 2012). As a traditional Chinese medicine, the root of L. aggregate is utilised in the treatment of several different symptoms including chest and abdomen pain, inflammation, indigestion, regurgitation, cold hernia and frequent urination (Editorial Committee of the Administration Bureau of Traditional Chinese Medicine, 1999). The leaves of L. aggregate (LAL) is popular in tea drinks due to the protective effects against oxidative stress (Gu et al., 2008). Previous phytochemical investigations showed that the main components of LAL are flavonoid glycosides (Zhang, Sun, Zhao, & Wang, 2001; Zhang, Sun, Chou, & Wang, 2003; Xiao, Cao, Fan, Shen, & Xu, 2011), sesquiterpene lactones as well as other components (Zhang, Sun, Wang, & Chou, 2001). Isoquinoline alkaloids are found in the roots of L. aggregate (Wang et al., 2007), while tannins are in the stems (Zhang, Sun, Wang, Hattori, & Tewtrakul, 2003). Recently, LAL was approved as a new food resource (Ministry of Health of the People’s Republic of China, 2013). However, the key components in LAL have not been well understood and they need to be ⇑ Corresponding author. Tel.: +86 571 87951301. E-mail address: [email protected] (L. Zhang).
further explored before LAL could be widely used as a food resource. Herbal plants have multiple types of components that vary in content; therefore, quality control of these components remains a challenge. In recent years, chromatography methods, such as high-performance liquid chromatography (HPLC) fingerprint, have been used for species’ differentiation and quality control of herbal medicines. This method has been utilised with ginkgo leaf (Beek & Montoro, 2009), Danshen (Zhang, Cui, He, Yu, & Guo, 2005), green tea (Alaerts et al., 2012), ginger (Yudthavorasit, Wongravee, & Leepipatpiboon, 2014), Goji berry fruit (Donno, Beccaro, Mellano, Cerutti, & Bounous, 2014), Rosa (Riffault, Destandau, Pasquier, André, & Elfakir, 2014) and L. aggregata root (Han et al., 2008; Wu et al., 2010). HPLC fingerprinting can be used to detect the complex constituents in herbal extracts both qualitatively and quantitatively, and it is highly suitable for quality control of various herbs. In addition, the chemical profile information of herbal extract can also be identified using multiple detectors such as tandem mass spectrometry (MS/MS) (Guan et al., 2011; Wu et al., 2013). In the Tiantai district of the Zhejiang Province in China, LAL is known as the native ‘‘Tiantai L. aggregate’’ or ‘‘Tai L. aggregate’’ and it is a popular tea drink in that region. Since LAL is a traditional Chinese medicine and popular tea, it’s essential to identify and investigate all the functional ingredients of LAL. Previous studies
http://dx.doi.org/10.1016/j.foodchem.2014.11.042 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
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C. Xu et al. / Food Chemistry xxx (2014) xxx–xxx
on LAL have identified 15 flavonoids, 3 sesquiterpene lactones and 3 phenylpropanoid glycosides (Luo, Zhang, Tian, & Yang, 2009; Zhao et al., 2012). To further expand upon the functional components in LAL, this study characterised the ingredients using HPLC fingerprint and MS methods. To explore the optimal harvest time of LAL, quercetin-3-O-a-L-rhamnoside (QR) from LAL was collected over several months and analysed for content. Since flavonoids and other polyphenols are widely used as natural antioxidants to significantly reduce the oxidative damage (Gu et al., 2008; Walle, 2004), the antioxidant capacities of different LAL samples were also measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing/antioxidant power (FRAP) assay. These powerful methods are suited for the identification and quantitation of the complex components found in herbal extracts.
LC/MS). A TC-C18 column (4.6 mm 250 mm, 5 lm i.d., Agilent, USA) was used for separation, and the column temperature was 35 °C. The mobile phase consisted of acetonitrile (A) and water (B, with 0.05% formic acid) with the following gradient program: 0–20 min, 10–23% A; 20–40 min, 23% A; 40–45 min, 23–95% A; 45–55 min, 95% A. The flow rate was set at 0.8 ml/min, and the DAD wavelength was 254 nm, with a sample injection volume of 10 ll. Mass spectra were recorded within 55 min. The negative electronic spray ion (ESI) conditions included a nitrogen nebulizer pressure of 35 psi, a vaporizer temperature of 350 °C at 11 L/min, and a capillary voltage of 4000 V. Selected ion monitoring (SIM) mode was used to record the abundance of the negative ion at m/z 100 to 1200 with a dwell time of 500 ms. Peak identification was performed by comparing the mass spectra and fragmentation ions, with relative retention time to the standards.
2. Experimental 2.1. Reagents and materials Quercetin-3-O-(200 -O-b-D-glucopyranosyl)-a-L-arabinofuranoside (2), quercetin-3-O-(200 -O-b-D-glucopyranosyl)-b-D-xylopyranoside (3), quercetin-3-O-b-D-glucopyranoside (4), quercetin-3-O-(200 -Ob-D-glucopyranosyl)-b-D-rhamnopyranoside (6), quercetin-3-O-aL-arabinofuranoside (7), quercetin-3-O-b-D-xylopyranoside (8), quercetin-3-O-a-L-rhamnoside (9), kaempferol-3-O-(200 -O-b-D-glucopyranosyl)-b-D-rhamnopyranoside (10) and kaempferol-3-O-aL-rhamnopyranoside (11) were prepared and identified in our laboratory as previously reported (Luo et al., 2009; Zhao et al., 2012). The structures are shown in Fig. S1. 1,1-Diphenyl-2picrylhydrazyl (J28Z009) was obtained from Alfa Aesar (USA) and 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ, K1319026) from Aladdin (China). Other reagents (analytical grade) were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). HPLC-grade acetonitrile was purchased from Tedia Company (USA). Ultra pure water was prepared using a Milli-Q50 SP Reagent Water System. 2.2. Sample extraction and optimisation From young leaves of L. aggregate (Sims) Kosterm, 8 seasonal leaf batches (S1–S8) were collected in the Tiantaishan district from April through November 2013 (Table 4). All batches were authenticated by Professor Jingkui Tian in Zhejiang University, where the voucher specimens (No. LA20130108) were deposited. Dried powder of each LAL sample (1 g) was extracted using 12 ml of 95% ethanol under reflux for 2 h and repeated thrice. All of the extract liquors were combined, filtered, concentrated under vacuum, then transferred and diluted to a final volume of 10 ml solution. The resulting solution was utilised for HPLC analysis and antioxidant assay. To optimise the extraction process of LAL, a uniform design method was applied (Liang, Fang, & Xu, 2001; Wu, Zhu, & Xu, 2011) using S1 sample. QR was quantified as a chemical indicator. The process factors that were varied include extracting ethanol concentration (60%, 70%, 80% and 95%), solid to liquid ratio (1:12, 1:14, 1:16, 1:18, 1:20 and 1:22), extraction time (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 h) and extraction frequencies (once, twice and thrice) and were used to optimise the extraction conditions (Table 2). Each test was repeated in triplicate. 2.3. HPLC analysis and total polyphenol content assay 2.3.1. HPLC–DAD–ESI MS/MS analysis The HPLC system was comprised of an Agilent 1200 analytical HPLC equipped with a DAD and a mass detector (6410 Triple Quad
2.3.2. HPLC analysis HPLC analysis was performed using a Shimadzu HPLC with two LC-10AT VP pumps, a photodiode array detector (DAD) and LC solution program. The chromatographic separation was performed on a TC-C18 column (4.6 mm 250 mm, 5 lm i.d., Agilent, USA) with column temperature of 40 °C. The solvent system consisted of a 30 min isocratic elution of 20% acetonitrile with 0.5% formic acid in water. At the end of the isocratic elution the column was flushed with 95% acetonitrile for 10 min to remove strongly retained constituents. The flow rate was 1.0 ml/min with an injection volume of 20 ll. In order to monitor all polyphenols simultaneously, the detection wavelength was set at 254 nm. To determine the contents of QR in the 8 seasonal samples, the stock QR solution (1.993 mg/ml) was applied and diluted if needed. The working calibration curves showed linearity in the range of 0.09965–1.993 mg/ml (r = 0.999) for QR, and the regression curves was Y = 2600X + 3811 (r = 0.999), where Y is the peak area of the analyte and X is the injected quantity (ng) of the QR standard. For fingerprint analysis, the chromatographic peaks were identified by comparing the retention time and UV spectra to the HPLC–DAD–MS analysis results together with those of reference compounds 2–4 and 6–11 which were eluted in parallel under the same conditions. Their relative contents were calculated as QR amount according to their peak area ratio, respectively. 2.3.3. Total polyphenol content assay Total phenolic contents in S1–S8 samples were further determined using the Felin–Ciocalteu method (Małgorzata, Magdalena, Artur, & Dorota, 2014). A 0.1 ml of sample solution was mixed with 2 ml ferrous tartrate solution and 2.9 ml phosphate buffer (pH = 7.5), and water was added to bring the final volume to 10 ml. The absorbance of the mixture was determined at 535 nm versus water blank. Gallic acid was used as a standard, and the linear range was between 0.0998 and 0.5989 mg (r = 0.999). Results were expressed as gallic acid equivalents (mg of gallic acid/g of sample in dried weight). 2.4. Antioxidant assay 2.4.1. DPPH free radical scavenging assay The antioxidant activities of S1–S8 samples were evaluated using a DPPH assay as reported by Sun et al. (2013) with some variations. Each sample extract was diluted from 250 lg/ml to 2500 lg/ml. Sample solutions or blank ethanol solvents (400 ll) were mixed with 0.16 mM DPPH ethanol solution (3 ml) and the action mixtures were kept for 30 min at room temperature. The absorbance of the resulting solution was read at 517 nm by a
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
C. Xu et al. / Food Chemistry xxx (2014) xxx–xxx
spectrophotometer. The capability to scavenge the DPPH radical was calculated by using the following equation:
%Inhibition ¼ ðA0 A1 Þ=A0 100%
ð1Þ
where A1 and A0 was the absorbance of the sample and blank, respectively. 2.4.2. Ferric reducing/antioxidant power assay The FRAP assay was also utilised to measure the total antioxidant capacity of S1–S8 samples as reported by Benzie and Siu-Wai (2014). The FRAP reagent was prepared using 0.3 mol/L acetate buffer (pH = 3.6), 20 mM ferric chloride and 10 mM TPTZ in 40 mM hydrochloric acid. The three solutions were mixed together at a ratio of 25:2.5:2.5 (v/v/v). 3 ml FRAP reagent (freshly prepared) was mixed with 100 ll of test sample. After incubation at room temperature for 30 min, the absorbance of reaction mixture was measured at 593 nm. The FRAP values, expressed in mmol ferrous sulphate equivalents (FSE)/g sample in dry weight, were derived from a standard curve. 2.5. Statistical analysis All measurements were replicated in triplicate. Data were expressed as mean ± standard error of three separate experiments,
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IC50 values were calculated by regression analysis. Evaluation of statistical significance of differences was performed by a oneway analysis of variance (one-way ANOVA). All statistical analyses were performed with EXCEL and SPSS 17.0 software. 3. Results and discussion 3.1. Characterisation of polyphenolic components in LAL Previously, the main chemical components of LAL were identified to involve 15 flavonoids, 3 sesquiterpene lactones as well as other components (Luo et al., 2009; Zhao et al., 2012). To expand upon the previous results new methodologies were used. Seasonal sample S1 was analysed by HPLC–DAD–ESIMS/MS to identify the principle components which are shown in Fig. 1 and Table 1. The UV spectra, mass spectra of pseudo-molecular ions and de-monosaccharide fragmentation ions of S1 were compared to published data and the retention time of S1 samples was compared with reference compounds 2–4 and 6–11. From these comparisons, 11 compounds were identified. Peak 1 was identified as cinnamtannin B1 (Zhang et al., 2003) and peak 5 was identified as quercetin-3-Ob-D-galactopyranoside, an isomer of QR (Zhang et al., 2001). Except 1, all other compounds 2–11 are flavonoid glycosides, with QR (9) as the major component and these are the key ingredients of LAL
Fig. 1. HPLC–PDA–()ESIMS/MS chromatograms of the leaves of Linderae aggregate. For the retention time and respective compound 1–11, see Table 1.
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
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C. Xu et al. / Food Chemistry xxx (2014) xxx–xxx
Table 1 Identification of polyphenols in the leaves of Linderae aggregate by HPLC–DAD–() ESIMS/MS analysis. Peak No.
Rt (min)
kmax (nm)
MS [MH]
MS/MS of [MH]
Mw
Identification
1 2 3 4 5 6 7 8 9 10 11
17.03 22.79 24.25 24.65 25.11 26.33 27.78 28.79 29.56 31.69 38.18
278 232, 255, 255, 255, 255, 256, 255, 254, 265, 263,
863.1 595.0 595.0 463.1 463.1 609.0 433.0 433.0 447.1 593.0 431.1
711.1, 572.9 301.0 300.9 301.0 301.0 300.9 301.0 301.0 300.9 413.0, 285.0 285.1, 254.9
864.0 596.0 596.0 464.0 464.0 610.0 434.0 434.0 448.0 594.0 432.0
Cinnamtannin B1 Quercetin-3-O-(200 -O-b-D-glucopyranosyl)-a-L-arabinofuranoside Quercetin-3-O-(200 -O-b-D-glucopyranosyl)-b-D-xylopyranoside Quercetin-3-O-b-D-glucopyranoside Quercetin-3-O-b-D-galactopyranoside Quercetin-3-O-(200 -O-b-D-glucopyranosyl)-b-D-rhamnopyranoside Quercetin-3-O-a-L-arabinofuranoside Quercetin-3-O-b-D-xylopyranoside Quercetin-3-O-a-L-rhamnoside Kaempferol-3-O-(200 -O-b-D-glucopyranosyl)-b-D-rhamnopyranoside Kaempferol-3-O-a-L-rhamnopyranoside
350 352 352 354 349 353 352 348 345 342
Table 2 The content of quercetin-3-O-a-L-rhamnoside in S1 sample of leaves of Linderae aggregate using U12 (124) uniform design test.
a
Level
Ethanol (%)
Solid–liquid ratio (g/ml)
Extraction time (h)
Extraction frequencies
QRa (mg/g)
1 2 3 4 5 6 7 8 9 10 11 12
60 60 60 70 70 70 80 80 80 95 95 95
1:12 1:14 1:16 1:18 1:20 1:22 1:12 1:14 1:16 1:18 1:20 1:22
1.5 2.5 0.5 2 3 1 2.5 0.5 1.5 3 1 2
3 2 1 3 2 1 3 2 1 3 2 1
4.15 ± 0.54 3.56 ± 0.22 0.78 ± 0.17 4.62 ± 0.08 4.09 ± 0.18 1.65 ± 0.10 5.44 ± 0.05 3.60 ± 0.13 3.26 ± 0.40 5.94 ± 0.16 4.67 ± 0.11 4.29 ± 0.16
Data are expressed as mean ± SD values.
observed in Fig. 1. Therefore, further fingerprint analysis of LAL was conducted using these identified constituents as indicators. 3.2. HPLC fingerprint analysis and total polyphenol content assay 3.2.1. Uniform design test for optimal sample extraction The optimal sample extraction was determined using uniform design test. The results obtained from this testing are shown in Fig. 2A, B and Table 2. The regression curve was Y = 0.656X1 0.004X2 + 0.046X3 + 0.097X4 0.322 (r = 0.970), where Y was the QR content (mg/g), X1 was ethanol concentration, X2 was solid–liquid ratio, X3 was extracting time (h) and X4 was extracting frequencies. The ethanol concentration and extraction frequencies were the critical factors in the extraction process. The extracting time also affected extracting efficiency, while the solid–liquid ratio was less important as demonstrated by the SPSS analysis (Table S1). Accordingly, the optimal extracting process was established as follows: LAL was extracted for 2 h with 95% ethanol (1:12, g/ml) under reflux and repeated three times. This condition was repeated and the LAL extract had QR at 5.875 mg/g, which agrees with the QR extracting result of test 10 in Table 2. 3.2.2. Fingerprint analysis of samples of different month The seasonal LAL samples collected over several months were run on HPLC and the overlapped HPLC chromatograms of S1–S8 are shown in Fig. 2C. All samples contain common peaks 1–11 at different ratios, with QR (9) as the most intense peak. A detailed semi-quantitative comparison of these samples was conducted. The relative amounts of all other peaks, 1–8 and 10–11, were calculated by comparing the peak areas with that of QR, respectively. As shown in Table 3, compounds 1, 6 and 9 were major constituents of
LAL, with their amounts (equivalent of QR) generally higher than 3 mg/g. The contents of 1–11 all varied in the samples from different months, with S2 (45.4 mg/g in sum; collected in May) being the highest, followed by S5 (35.0 mg/g in sum; collected in August) and S6 (32.9 mg/g in sum; collected in September). By contrast, all other samples such as S3, S7 and S8 contained a significant less of these constituents (about 19 mg/g in sum, respectively). Apparently, the seasonal collection month affected the contents of flavonoids in LAL dramatically, which is a critical factor in quantity control and optimal collection time selection. 3.2.3. Total polyphenol content analysis The different LAL samples were also tested in the total polyphenol content assay and the results are shown in Table 4. The order of sample for total polyphenol (TP) content was as follows: S2 (49.47 mg/g) > S6 (35.18 mg/g) > S5 (29.72 mg/g) > S4 (24.56 mg/g) > S7 (24.33 mg/g) > S1 (23.13 mg/g) > S8 (21.92 mg/g) > S3 (15.11 mg/g). The rank order of this result is similar to QR amounts, with some slight variations in S5 and S6. The results suggested that flavonoids are a major part of TP, while other polyphenols such as cinnamomin B1 and other tannins also contributing to TP. 3.3. Antioxidant capability analysis of samples obtained from different months 3.3.1. Antioxidant capability analysis with DPPH free radical scavenging assay The free radical scavenging activities of S1–S8 samples were determined by the DPPH assay, using ascorbic acid (IC50 = 30.5 lg/ml) as a positive control. All samples exhibited strong concentration-dependent antioxidant potentials against DPPH free radicals at 250–2500 lg/ml. The IC50 (lg/ml) values are shown in Table 4. The order of the antioxidant capacity against DPPH was as follows: S2 » S5, S6, S7 » S8 > S4 » S1 > S3. S2 showed the strongest free radical scavenging capability (IC50 = 542.4 lg/ml) with the highest QR and TP contents, while S1 and S3 possessed relative lower antioxidant capacities (IC50 > 950 lg/ml). S5, S6 and S7 also showed stronger free radical scavenging capability (IC50 = 641.5 lg/ml) with higher QR and TP contents. Although S4 and S8 showed lower activities than S7, their QR and TP contents did not follow the same ratio. These results indicate that other constituents could also contribute to the total antioxidant capacities of LAL, which needed further exploration. 3.3.2. Antioxidant capability analysis with ferric reducing/antioxidant power assay The total antioxidant capacities of samples S1–S8 were also determined by FRAP assay. FRAP values were expressed in mmol ferrous sulphate equivalent (mmol FSE/g) sample in dry weight. The results are also shown in Table 4. The order of the antioxidant capacity in this assay was: S2 » S5, S6 » S8, S4 > S1, S3, S7. S2
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
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Fig. 2. HPLC chromatograms of (A) Quercetin-3-O-a-L-rhamnoside, (B) representative S1 sample and (C) overlapped S1–S8 samples of the leaves of Linderae aggregate. For respective compounds 1–11, see Table 1.
showed the strongest antioxidant capability (0.76 mmol FSE/g), followed by S5 and S6, while S1, S3 and S7 possessed relative lower antioxidant capacities (60.42 mmol FSE/g). These results were in parallel with those of DPPH order except S7. S7 possessed similar antioxidant capacity and QR/TP contents as that of S1 in this case. In general, both DPPH and FRAP assays suggested higher
antioxidant potentials of LAL accompany with higher amounts of QR and TP. In this study, LAL was shown to have apparent antioxidant capacities, likely due to high amount of flavonoids and other constituents, which is in agreement with a previous report (Gu et al., 2008). Moreover, 11 functional constitutes which contribute to
Please cite this article in press as: Xu, C., et al. Characterisation of polyphenol constituents of Linderae aggregate leaves using HPLC fingerprint analysis and their antioxidant activities. Food Chemistry (2014), http://dx.doi.org/10.1016/j.foodchem.2014.11.042
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C. Xu et al. / Food Chemistry xxx (2014) xxx–xxx
Table 3 Relative contents of compounds 1–11 in eight samples of Linderae aggregate by HPLC–DAD analysis.a,b
a b
No.
S1
S2
S3
S4
S5
S6
S7
S8
1 2 3 4 5 6 7 8 9 10 11 Sum
3.53 ± 0.02 1.13 ± 0.01 2.60 ± 0.03 0.93 ± 0.01 0.44 ± 0.01 4.29 ± 0.04 0.93 ± 0.02 2.24 ± 0.03 5.87 ± 0.29 0.76 ± 0.01 1.12 ± 0.01 23.84 ± 0.48
5.20 ± 0.01 1.86 ± 0.02 3.95 ± 0.03 2.78 ± 0.02 1.39 ± 0.01 5.00 ± 0.07 1.83 ± 0.01 4.86 ± 0.04 15.58 ± 0.17 0.78 ± 0.01 2.22 ± 0.02 45.45 ± 0.41
3.08 ± 0.02 0.50 ± 0.01 0.99 ± 0.01 0.88 ± 0.01 0.58 ± 0.01 2.27 ± 0.02 0.84 ± 0.01 1.59 ± 0.01 6.86 ± 0.06 0.38 ± 0.01 1.40 ± 0.01 19.37 ± 0.18
3.36 ± 0.03 0.55 ± 0.01 1.11 ± 0.01 1.11 ± 0.01 0.96 ± 0.01 2.73 ± 0.03 0.95 ± 0.01 1.95 ± 0.02 8.63 ± 0.14 0.35 ± 0.01 1.55 ± 0.04 23.25 ± 0.32
3.96 ± 0.04 0.74 ± 0.01 1.49 ± 0.01 1.74 ± 0.02 2.40 ± 0.07 4.17 ± 0.05 1.27 ± 0.02 2.98 ± 0.03 14.04 ± 0.18 0.31 ± 0.01 1.93 ± 0.02 35.03 ± 0.46
3.42 ± 0.01 0.72 ± 0.01 1.45 ± 0.01 1.78 ± 0.02 2.19 ± 0.01 3.98 ± 0.02 1.28 ± 0.01 2.87 ± 0.01 12.98 ± 0.14 0.32 ± 0.01 1.89 ± 0.03 32.88 ± 0.28
3.69 ± 0.08 0.49 ± 0.01 1.43 ± 0.02 0.65 ± 0.01 0.54 ± 0.01 1.95 ± 0.04 0.66 ± 0.02 1.22 ± 0.02 6.45 ± 0.20 0.32 ± 0.01 1.52 ± 0.04 18.92 ± 0.46
3.32 ± 0.02 0.46 ± 0.01 0.99 ± 0.01 0.69 ± 0.01 0.76 ± 0.01 2.09 ± 0.02 0.67 ± 0.01 1.38 ± 0.01 7.23 ± 0.15 0.33 ± 0.01 1.56 ± 0.03 19.48 ± 0.29
Contents of compounds 1–8 and 10–11 were calculated as equivalents of quercetin-3-O-a-L-rhamnoside (mg/g of dried extracts). Data are expressed as mean ± SD values (n = 3).
Table 4 Contents of quercetin-3-O-a-L-rhamnoside and total phenols in 8 batches of the leaves of Linderae aggregate and their antioxidant capacities. No. S1 S2 S3 S4 S5 S6 S7 S8
Batch No. 201304 201305 201306 201307 201308 201309 201310 201311
QRa (mg/g) 5.87 ± 0.29 15.58 ± 0.17 6.86 ± 0.06 8.63 ± 0.14 14.04 ± 0.18 12.98 ± 0.14 6.45 ± 0.20 7.23 ± 0.15
TPa,b (mg/g)
DPPHa,c IC50 (lg/ml) ⁄S3
23.13 ± 0.61 49.47 ± 3.59 15.11 ± 0.26 24.56 ± 1.53 29.72 ± 1.11 35.18 ± 5.34 24.33 ± 0.62 21.92 ± 2.01
954.3 ± 35.7 542.4 ± 12.9 993.9 ± 32.5⁄S1 777.7 ± 32.2⁄S8 637.6 ± 5.5#S6, S7 641.5 ± 1.5#S5, S7 665.7 ± 14.9#S5, S6 735.9 ± 5.8⁄S4
FRAPa,c,d (mmol FSE/g) 0.42 ± 0.06#S3, S7 ⁄S4, S8 0.76 ± 0.03 0.41 ± 0.02 #S1, S7 ⁄S4 0.50 ± 0.01#S8 ⁄S3 0.66 ± 0.04#S6 0.62 ± 0.04#S5 0.39 ± 0.04#S1, S3 0.51 ± 0.04⁄S1
a
Data are expressed as mean ± SD values (n = 3). Results are expressed as gallic acid equivalents (mg of gallic acid/g of dried extracts). c # Differences of the same columns are not significant (P > 0.05); ⁄differences of the same columns are significant (0.01 < P < 0.05); differences between other columns are obvious significant (P < 0.01). d FRAP values are expressed in mmol ferrous sulphate equivalent (FSE)/g sample in dry weight. b
the antioxidant capacities were identified and semi-determined for the first time in this study. Quercetin-3-O-a-rhamnoside (quercitrin) and kaempferol-3-O-a-rhamnoside (afzelin) were also reported to contribute in major to the antioxidant capacities of the leaves of Lindera obtusiloba (Hong, Rhee, Won, Choi, & Lee, 2013). Total alkaloids isolated from the root of L. angustifolia also possessed free radical scavenging activities (Zhao, Zhao, & Wang, 2006), however, whether or not these ingredients existing in LAL remains unknown. Since flavonoids and other polyphenols are the major constituents of LAL with strong antioxidant capacities, the harvesting time points of S2 (collected in May), S5 (collected in August) and S6 (collected in September) are recommended as preferred ones. The contents of polyphenols and other secondary metabolites in plants depend on environmental conditions such as sun light, temperature, water and nutrition as previously reported (Guo et al., 2013). In general, LAL starts growing in May and is lush through August and September in Tiantai district, which may contribute to higher polyphenol amounts in LAL. By contrast, LAL was traditionally believed to be in first class when collected in May but since this is the beginning of the growing season, it would be more productive to harvest between July and August. These results provide critical guidance for monitoring the quantity of LAL and select the optimal harvesting time. 4. Conclusion In this study, the polyphenol profile of LAL was investigated using multiple HPLC fingerprint techniques. For the first time, QR and other 10 polyphenolic compounds in LAL were identified and
semi-determined by LC fingerprint analysis. Using the optimal extracting method developed in this study, 8 seasonal samples (S1–S8) were collected from April to November and evaluated using their fingerprint data. All 8 samples contain 11 polyphenolic compounds at various ratios, while S2, S5 and S6 have the highest amounts of QR and show stronger antioxidant capacities in the DPPH assay. These results have provided fundamental evidence critical for future development of LAL products under direction of their optimised collection time and quality control methods. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was financially supported by the National Science and Technology Major Project of China (No. 2013ZX09103002) and the National Science Foundation of China (No. U1303122). 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. 11.042. References Alaerts, G., Van Erps, J., Pieters, S., Dumarey, M., Nederkassel, A. M., Goodarzi, M., et al. (2012). Similarity analyses of chromatographic fingerprints as tools for
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