Quality evaluation and chemometric discrimination of Zanthoxylum bungeanum Maxim leaves based on flavonoids profiles, bioactivity and HPLC-fingerprint in a common garden experiment

Industrial Crops & Products 134 (2019) 225–233

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Quality evaluation and chemometric discrimination of Zanthoxylum bungeanum Maxim leaves based on flavonoids profiles, bioactivity and HPLC-fingerprint in a common garden experiment ⁎

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Xiaoqi Chen1, , Wei Wang1, Cheng Wang, Zijia Liu, Qi Sun, Dongmei Wang College of Forestry, Northwest A & F University, Yangling, Shaanxi, 712100, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Zanthoxylum bungeanum leaves (ZBL) Flavonoids profiles Bioactivity HPLC fingerprint Chemometric discrimination Quality evaluation

Zanthoxylum bungeanum leaves (ZBL) have rich flavonoids with a variety of bioactivities, which has great values of development and utilization. In this paper, the quality of thirteen varieties of ZBL collected from a common garden was evaluated based on flavonoids compositions, bioactivity and reversed-phase high performance liquid chromatography (RP-HPLC) fingerprint coupled with chemometrics analysis. The values revealed that You Huajiao (S13) exhibited the highest contents of flavonoids compounds and strongest bioactivity, followed by Hancheng Dangcun stingless (S1). Among different varieties HPLC-fingerprint, eight common characteristic peaks were selected and the similarity lay within the range of 0.502-0.999. Meanwhile, thirteen varieties were divided into four groups (G1-G4) and two discriminant functions with the discriminant rate of 100% were constructed. Additionally, quercitrin and afzelin were found to be key compounds of quality evaluation. In conclusion, G4 (You Huajiao, S13) was selected as the best variety, followed by G1 (S1, S3, S5, S6, S7, S10, S12) and G2 (S4, S8, S9, S11), respectively, and G3 (Fugu late-maturing, S2) was at the last level. Consequently, this study screened for fine varieties of ZBL and provided a basis for the development and utilization of high-quality resources of Z. bungeanum.

1. Introduction Zanthoxylum bungeanum Maxim belonged to the Zanthoxylum genus of the family Rutaceae is a deciduous shrub and native to East Asian countries. In China, Z. bungeanum has a long history of nearly three thousand years of cultivation and utilization. Its planting area and output are both the largest in the world. (Rahman et al., 2002; Zhang et al., 2017a). The pericarp and leaf of Z. bungeanum which often used as peppery spices in Chinese cooking are due to its special fragrance and distinctive tingling taste (Yang, 2008). More scholars have devoted to specializing in Z. bungeanum leaves (ZBL) with its high contents of polyphenols and flavonoids with a variety of physiological activities having been discovered recently. In previous studies, nine flavonoids and three phenolic acids from ZBL were identified by HPLC-MS/MS (Yang et al., 2013). Ma and coworkers found that ZBL extracts exhibited good ability to against hyperlipidemia and atherosclerosis (Ma et al., 2015). ZBL extracts also could increase endogenous antioxidant enzyme activities and displayed a protective effect on anti-lipid oxidation in

salted fish (Li et al., 2015). In addition, there was reported that hyperoside from ZBL exerted the antiproliferative effect (Zhang et al., 2017b), antihyperglycemic and hepatocyte-protective effects (Zhang et al., 2017c). In our previous work, nine flavonoids were isolated from the ZBL of Fengxian Dahongpao for the first time, and quercitrin, hyperoside, rutin and afzelin were dominant flavonoid compounds, which played a major role in the antioxidant and antimicrobial activities (Zhang et al., 2014a, b; Zhang et al., 2015). Furthermore, four main flavonoids were successfully purified by an aqueous two-phase system (He et al., 2016). On this basis, an oral liquid of ZBL which had high phytochemical contents and good antioxidant activities was prepared, and its shelf life was more than 12 months after high temperature steam treatment (Zhang et al., 2017d). These results strongly suggested that ZBL has great potential for the development and utilization of functional products as ingredients. Although it is very rich in Z. bungeanum germplasms resources in China, there are still some deficiencies in the cultivation and management. On the one hand, under the influence of cross breeding, natural

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Corresponding author. E-mail addresses: [email protected] (X. Chen), [email protected] (W. Wang), [email protected] (C. Wang), [email protected] (Z. Liu), [email protected] (Q. Sun), [email protected] (D. Wang). 1 These two authors contributed equally to this work. https://doi.org/10.1016/j.indcrop.2019.04.017 Received 3 November 2018; Received in revised form 5 April 2019; Accepted 7 April 2019 0926-6690/ © 2019 Published by Elsevier B.V.

Table 1 Sample information. 226

555 472 472 613 555 555 555 486.3 491.7 555 400 555 491.7 13.5 9.1 9.1 11.4 13.5 13.5 13.5 11.0 11.0 13.5 14.6 13.5 11.0 515.88 821.82 821.82 1243.09 515.88 515.88 515.88 1775.45 1344.74 515.88 1344.74 515.88 1344.74 Hancheng Dangcun stingless Fugu late-maturing Fugu early-maturing Fengxian Dahongpao Gelao stingless Hancheng Dahongpao Hancheng stingless Maoxian Dahongpao Qin’an Yihao Hancheng Shizitou Wudu Dahongpao Xinong stingless You Huajiao S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13

2.1. Materials and reagents The thirteen varieties of ZBL were collected in September 2016, from a common garden (the Research Centre for Engineering and Technology of Zanthoxylum, State Forestry Administration, in Fengxian County, Shaanxi Province, China, latitude 33°59′61″N, longitude 106°39′28″E, at an altitude of 1027 m), and samples information was shown in Table 1. All samples of thirteen varieties of Z. bungeanum without wormhole and bacteria were air-dried in the dark at home temperature. The powder obtained after crushed in a laboratory grinder was stored avoiding light in -20℃. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-

E110.35 °N35.37° E111.07 °N39.03° E111.07 °N39.03° E106.66 °N33.97° E110.35 °N35.37° E110.35 °N35.37° E110.35 °N35.37° E103.49 °N31.45° E104.96 °N33.46° E110.35 °N35.37° E104.96 °N33.46° E110.35 °N35.37° E104.96 °N33.46°

Varieties Sample number

2. Materials and methods

Hancheng, Shaanxi Fugu, Shaanxi Fugu, Shaanxi Fengxian, Shaanxi Hancheng, Shaanxi Hancheng, Shaanxi Hancheng, Shaanxi Maoxian, Sichuan Tianshui, Gansu Hancheng, Shaanxi Wudu, Gansu Hancheng, Shaanxi Tianshui, Gansu

Location

Coordinates

Altitude(m)

Mean annual temperature (℃)

Annual precipitation (mm)

Annual sunshine hours (h)

variation and other factors, many Z. bungeanum varieties are very similar in morphology, bringing about the difficulty in distinguishing them by appearance (Li et al., 2016). On the other hand, due to the lack of authoritative and unified naming rules, the phenomenon of homonyms and synonym is existing widely (Feng et al., 2015). All these restrict the deep utilization of high quality ZBL resources seriously. Besides, considering the huge potential of ZBL in functional products, it is necessary to evaluate the quality of different varieties of ZBL and to screen out the fine quality germplasm resource. Nowadays, the quality evaluation and control of natural products and pharmaceuticals are essential, which is closely related to a variety of active ingredients and directly affects the products effect (Zhu et al., 2013). HPLC fingerprinting techniques have been wildly applied for quality assessment of functional products and traditional Chinese medicines. It has been introduced and accepted by World Health Organization (WHO) and US Food and Drug Administration (FDA) (Zhong et al., 2015). HPLC fingerprinting is usually analyzed in combination with chemometric methods, such as similarity analysis (SA), hierarchical cluster analysis (HCA), principal component analysis (PCA) and discriminant analysis (DA), which can simplify a large number of complex and multidimensional original data to understand and interpret the variable information easily. Moreover, classification information about known samples and the prediction model about unknown samples also can be provided. At present, HPLC fingerprinting combined with chemometrics has been successfully applied to food and crop analysis, such as Goji berry (Donno et al., 2015), Pyrrosiae Folium (Cui et al., 2016), beech bark (Hofmann et al., 2017), basil (Teofilović et al., 2017) and so on. Common garden experiment is a study that analyses the behavior of different species or varieties of plants collected from different areas under the same environmental conditions (Moloney et al., 2009). Because all living organisms are grown under the same conditions, the influence of natural environment and geographical differences on secondary metabolites is eliminated, and the effect of genes on organisms is highlighted. (Nord-Larsen and Pretzsch, 2017). Therefore, it has become an important means to evaluate quality of plant germplasm resources. (Liang et al., 2016). Unfortunately, to the best of our knowledge, there have been no previous reports on HPLC fingerprints combined with chemometric analysis for the quality evaluation of different varieties of ZBL. This study is aimed to determine the flavonoids profiles (total flavonoid and five flavonoids compounds) and bioactivities (antioxidant activity and antibacterial activity) of thirteen varieties of ZBL collected from a common garden, set up an effective analysis method of classification and quality evaluation of ZBL by HPLC fingerprinting techniques combined with SA, HCA, PCA and DA, then to select a good variety and provide a basis of the development and utilization of high-quality resources of Z. bungeanum, and provided valuable reference for further study and application of ZBL.

2436.0 2894.9 2894.9 1840.0 2436.0 2436.0 2436.0 1549.4 2100.0 2436.0 1911.7 2436.0 2100.0

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method as described previously (Brand-Williams et al., 1995; Wang et al., 2015). Trolox and 80% ethanol were used as the positive and reference blank control, respectively, and all samples were tested in triplicate. A lower IC50 value represents the stronger DPPH radical scavenging activity. The method of decolourisation of free radical ABTS·+ was performed as expressed previously (Re et al., 1999; Wang et al., 2013). The results were expressed as micromoles of Trolox equivalent per gram. All determinations were carried out in triplicate. Ferric reducing antioxidant power (FRAP) activity was determined using the protocol of Benzie & Strain with some modifications (Benzie and Strain, 1996; Wang et al., 2013). The results of FRAP activity were expressed in terms of micromoles Trolox equivalent per gram dry extract weight (mmol equiv. Trolox/g). All experiments were performed in triplicate.

carboxylic acid (Trolox) were purchased from Sigma-Aldrich (St. Louis, Miss., U.S.A.). Five chemical standards were obtained by method 2.2. Tetrachloro benzoquinone was brought from Aladdin Reagent Shanghai Co., Ltd. (Shanghai, China). HPLC-grade methanol and acetonitrile were obtained from TEDIA Chemical Co., Ltd. (Fairfield, Ohio, USA). Deionized water (18 MΩ cm) was used to prepare aqueous solutions. 2.2. Preparation of chemical standards In our previous work, the powdered ZBL was extracted with 95% ethanol at room temperature, and the total filtrate were concentrated by rotary evaporation in vacuo. The ethanol extracts were further loaded onto silica gel chromatography columns and Sephadex LH20 columns to yield nine compounds. All the isolated compounds were characterized and identified by spectroscopic methods, five of these compounds were identified as rutin, hyperoside, trifolin,quercitrin,afzelin respectively through comparison with published data (Zhang et al., 2014a, b; Zhang et al., 2015).

2.7. Antibacterial activity 2.7.1. Microorganisms The microorganisms, Escherichia coli (ATCC25922), Klebsiella. pneumoniae (ATCC46117), Pseudomonas aeruginosa (ATCC27853), Salmonella enteritidis (ATCC14028), Listeria monocytogenes (ATCC19115), and Candida albicans (ATCC10231) were used for the studies (from microbial preservation center of Guangdong Institute of Microbiology, Guangzhou, China.). All strains were cultured at 37 °C on Mueller-Hinton medium.

2.3. Preparation of the extracts The powdered ZBL and 400 mL 60% ethanol were actually added into the flask before reflux extraction. The mixture was extracted twice at 45℃ for 90 min and collecting filtrate by suction filtration. The total filtrate was concentrated under the conditions of 45 °C by a rotary evaporator to obtain the ethanol extracts, which was stored at −20 °C until further analysis (Wu et al., 2018).

2.7.2. Disc-diffusion method Antimicrobial activity was determined via the paper disc agar diffusion method (Özer et al., 2007; Meng et al., 2016). The bacteriostatic agent was prepared by dissolving 20 mg/mL crude extracts in dimethyl sulfoxide (DMSO). Tetracycline, and chloramphenicol (10 μg/mL) were used as positive controls and DMSO solution as the negative control. Sterilized filter paper discs (6 mm) were soaked in samples for 2 h. In the ultra-clean bench, sterilized filter paper discs were pasted on the solidified petri dish surface after coated 100 μL bacterial suspension evenly. The diameter of the inhibition zone (DIZ) was measured every 12 h into the incubator (37℃). Each sample was assessed in triplicate. DIZ were ranked as follows: > 20 mm (extreme sensitivity); 15–20 mm (high sensitivity); 10–14 mm (moderate sensitivity); 7–9 mm (low sensitivity); < 7 mm (insensitivity). DIZ is proportional to the antibacterial activity of extracts.

2.4. Determination of total flavonoid content (TFC) The TFC was determined based on the SBC assay using sodium borohydride/tetrachloro benzoquinone as described previously (He et al., 2008; Liu et al., 2017). In this research, different concentrations of quercetin (0.3–10 mmol/L) were used for constructing a calibration curve (y = 0.0651x + 0.0037, R2 = 0.9949). Sample solutions were diluted to a concentration of 10 mg/mL. Then the absorbance of samples was measured at 490 nm using spectrophotometer. Data was reported as mean ± SD for three replicates. The results were expressed as millimole quercetin equivalents per 100 g (mmol equiv. QUE/100 g) of dry weight. 2.5. HPLC instrument and chromatographic conditions

2.7.3. Minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) The values of MIC and MBC were determined according to the agar dilution method, with minor modifications (Wang et al., 2013; Mocan et al., 2014). Samples dissolved in DMSO solution by double dilution method were added to Mueller Hinton broth to obtain final concentrations of 40, 20, 10, 5, 2.5 and 1.25 mg/mL. Tetracycline and ampicillin were used as positive control, with final concentrations of 10, 5, 2.5, 1.25, 0.625 and 0.3125 g/mL. 2% DMSO solution was used as negative control. MIC was the lowest concentration that observed no bactericidal growth visually after 24 h while MBC was after 48 h. Tests were performed in triplicate.

The HPLC analysis was conducted with an Agilent Series 1260 liquid chromatograph equipped with a quaternary gradient pump system and variable-wavelength detector system connected to a reversed-phase (RP) SB-C 18 column (5 μm, 4.6 × 250 mm, Agilent, USA). Data collection was performed using ChemStation software (Agilent, USA). All ZBL crude extracts were dissolved in HPLC-grade methanol, filtered through 0.22 μm membrane filters and analyzed by RP-HPLC at ambient temperature. The chromatographic separation was using deionized water (solvent A) and acetonitrile (solvent B) as mobile phase at a flow rate of 0.8 mL/min. The gradient elution program was configured as follows: 0–30 min, 15–35% B; 30–35 min, 35–65% B; 35–55 min, 65%–100% B; keep B at 100% in the following 10 min. The injection volume of each sample and standard solutions was 20 μL, detected at a wavelength of 254 nm. All experiments were performed in triplicate. In our previous study, the precision, repeatability and recovery rate of the HPLC procedure was validated, and all the values of relative standard deviation (RSD) were < 3%, which suggested that the methods of fingerprinting analysis were acceptable and precise (Zhang et al., 2014a).

2.8. Statistical analysis For evaluating the similarity between different varieties of ZBL, conduct the similarity analysis (SA) of different chromatograms. These chromatograms obtained by developed methods were analyzed using the software of Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004 A) (SES software). Hierarchical cluster analysis (HCA), principal component analysis (PCA) and discriminant analysis (DA) were performed using SPSS software (SPSS for Windows 20.0, SPSS Inc., USA).

2.6. Determination of antioxidant activity The DPPH radical scavenging capacity was measured using the 227

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Table 2 Contents of total flavonoids and five flavonoids compounds. Samples

Total flavonoids content (mmol equiv. QUE/100 g)

Content (mg/g) Rutin

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13

b

165.21 ± 2.45 109.37 ± 2.50g 125.80 ± 1.23e 119.89 ± 0.43f 101.55 ± 0.62h 106.86 ± 4.32g 131.62 ± 1.49d 92.97 ± 4.44j 126.46 ± 0.18e 96.85 ± 0.43hi 137.92 ± 1.05c 98.61 ± 1.66j 191.16 ± 1.71a

ND ND ND 9.125 ± 0.151c ND 6.775 ± 0.177f ND 8.016 ± 0.410d 9.560 ± 0.085b ND 7.050 ± 0.145f ND 11.025 ± 0.268a

Hyperoside

Trifolin i

14.531 ± 0.037 7.090 ± 0.094m 11.948 ± 0.231k 21.721 ± 0.021a 15.485 ± 0.107g 11.410 ± 0.104l 16.563 ± 0.022e 17.601 ± 0.085d 20.428 ± 0.215c 15.087 ± 0.082h 16.204 ± 0.119f 13.754 ± 0.065j 20.750 ± 0.115b

0.864 ND 0.565 1.671 0.552 0.712 0.972 1.489 1.618 1.006 0.988 0.486 1.306

Quercitrin

± 0.024 ± ± ± ± ± ± ± ± ± ± ±

f

0.029h 0.046a 0.017h 0.028g 0.036e 0.027c 0.054b 0.001e 0.038e 0.002i 0.024d

Afzelin f

12.965 ± 0.199 22.723 ± 0.163a 18.016 ± 0.217b 12.430 ± 0.206g 14.765 ± 0.136e 17.944 ± 0.141b 15.917 ± 0.040d 11.565 ± 0.061h 11.661 ± 0.157h 14.806 ± 0.064e 9.401 ± 0.055i 16.586 ± 0.008c 5.398 ± 0.016j

1.178 1.561 1.694 1.942 1.806 2.493 1.526 2.015 1.916 1.900 1.726 1.601 0.393

± ± ± ± ± ± ± ± ± ± ± ± ±

0.020h 0.021fg 0.047e 0.064c 0.043d 0.027a 0.009g 0.026b 0.026c 0.004c 0.057e 0.024f 0.020i

Note: Values are mean ± SD (n = 3). Means with different letters within a column are significantly different (p < 0.05). ND means not detected.

All measurements were carried out in triplicate and expressed as the mean ± standard deviation (SD). The significant difference was calculated by SPSS one-way ANOVA followed by Duncan’s test, values < 0.05 were considered to be significant.

3. Results and discussion 3.1. Total flavonoids contents (TFC) The results of TFC were presented in Table 2, significant variations (p < 0.05) among all of the ZBL varieties were observed, the range of TFC was from 92.97 ± 4.44 (Maoxian Dahongpao, S8) to 191.16 ± 1.71 (You Huajiao, S13) mmol equiv. QUE/100 g. It was obvious that You Huajiao (S13) (191.16 ± 1.71 mmol equiv. QUE/100 g) exhibited the highest TFC, followed by Hancheng Dangcun stingless (S1) (165.21 ± 2.45 mmol equiv. QUE/100 g). On the contrary, the TFC of Maoxian Dahongpao (S8) and Xinong stingless (S12) were relatively low. Moreover, the highest values of TFC in all varieties investigated were twice more than the minimum.

Fig. 1. HPLC chromatograms of thirteen varieties of Z. bungeanum leaves. Five compounds were identified by their retention times (min): rutin (11.55, peak 9), hyperoside (12.81, peak 10), trifolin (14.94, peak 12), quercitrin (16.24, peak 13), afzelin (19.84, peak 14). Eight common peaks: peak 1–5, 10, 13 and 14.

3.2. RP-HPLC analysis of five flavonoids compounds

Table 3 Antioxidant activities of different varieties Z. bungeanum leaves.

The contents of five kinds of flavonoids compounds in different varieties of ZBL were quantitative analyzed by RP-HPLC. Based on comparison of their retention times with the standard chemicals and our previous study (Zhang et al., 2014a), peaks 9, 10, and 12–14 were identified as rutin, hyperoside, trifolin, quercitrin, and afzelin, respectively (Fig. 1). According to the Table 2, hypericin, quercetin and afzelin were detected in all varieties, but only six samples containing all five flavonoids compounds, which were Fengxian Dahongpao (S4), Hancheng Dahongpao (S6), Maoxian Dahongpao (S8), Qin’an Yihao (S9), Wudu Dahongpao (S11) and You Huajiao (S13), respectively. However, only hyperoside, quercitrin and afzelin were found in Fugu late-maturing (S2). In Table 2, it could be found that hyperoside and quercitrin were the predominant flavonoids compounds in ZBL, which was consistent with our previous research (Zhang, et al., 2015). Nevertheless, the content of trifolin was usually lower than that of other flavonoids compounds, and its content in S4 was the highest with only 1.671 ± 0.046 mg/g. The contents of the same compounds containing in different varieties were varying obviously. For example, the content of afzelin was 2.493 ± 0.027 mg/g in S6, while in S13 was 0.393 ± 0.020 mg/g, which the maximum value was more than six times greater than the minimum. These results suggested that there was observable diversity of the chemical compositions of different varieties of ZBL.

Samples

DPPH IC50 (μg/ml)

ABTS (μmol Trolox/g)

FRAP (μmol Trolox/g)

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 Trolox

29.40 ± 0.03c 39.86 ± 0.57g 31.94 ± 0.32d 47.86 ± 0.40h 30.49 ± 0.66c 33.40 ± 0.28e 35.31 ± 0.30e 53.07 ± 0.50i 37.29 ± 0.14f 32.63 ± 0.78de 55.29 ± 0.62j 36.51 ± 0.28g 20.79 ± 0.35b 4.37 ± 0.03a

1461.58 ± 7.31b 1006.19 ± 23.25h 1247.89 ± 35.53e 983.39 ± 20.89hi 1336.87 ± 31.51d 1390.90 ± 34.40c 1352.10 ± 10.16d 961.82 ± 5.72i 1150.56 ± 29.25g 1386.36 ± 22.16c 992.85 ± 11.32ij 1267.57 ± 14.15f 1957.24 ± 25.60a –

587.32 414.02 630.02 353.89 497.96 471.21 492.77 350.21 457.65 512.37 382.39 516.05 855.95 –

± ± ± ± ± ± ± ± ± ± ± ± ±

1.55c 2.04h 3.65b 1.99j 10.53e 7.59f 8.63e 4.13j 1.18g 7.76d 6.93i 0.72d 0.84a

Note: DPPH IC50 values are the effective concentrations at which DPPH radicals were scavenged by 50%. ABTS values and FRAP values show ABTS•+ radical cation scavenging assay results and Ferric reducing power assay results respectively, and they are both expressed as micromoles of trolox equivalent per g. Values are mean ± SD (n = 3). Means with different letters within a column are significantly different (p < 0.05).

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activity of Fugu late-maturing (S2) was weaker. What’s more, the results of antibacterial activity were in accordance with the TFC, indicating that antibacterial activity was closely the contents of active substances. In our previous studies, we found that ZBL extracts exhibited good sensitivity against B. cinerea, P. oryzae, G. cingulata and V. pyrina (Zhang et al., 2014a). These results have revealed that ZBL can be harnessed as a potential source of antimicrobial agents for human health.

3.3. Antioxidant activity For evaluation of antioxidant activity of ZBL extracts, DPPH radical scavenging activity, ABTS radical cation scavenging activity and FRAP assay were determined among thirteen varieties (Table 3). Definite antioxidant activity appeared in all varieties and demonstrated in the study. You Huajiao (S13) exhibited the strongest antioxidant activity with the lowest DDPH IC50 value (20.79 ± 0.35 μg/mL), ABTS radical cation scavenging capacity (1957.24 ± 25.60 μmol Trolox/g) and Ferric reducing power (855.95 ± 0.84 μmol Trolox/g) were also the highest. While the antioxidant activity of Maoxian Dahongpao (S8) and Wudu Dahongpao (S11) was lower than others. Compared with the results of TFC, the varieties with abundant flavonoids compounds were also performed better antioxidant activity. The S13 showed the strongest antioxidant activity, which may be related to the high contents of rutin, and hyperoside.

3.5. Establishment of HPLC fingerprinting Under the developed HPLC condition, the HPLC fingerprint of thirteen varieties of ZBL collected from a common garden was established, and a simulative median chromatogram was generated by the SES software. Then, eight common characteristic peaks (peaks 1–5, 10, 13 and 14) were also obtained. The retention time (RT) and peak area (PA) of these peaks were shown in Table S1. According to the RSD of RT and PA, the RT of all common peaks were highly consistent (RSD < 0.496), which means this HPLC method was stable and reliable. Meanwhile, the RSD value of PA is greater than 12.048%, which indicated that the difference in content of flavonoids compounds seems to be larger among different varieties of ZBL. Finally, these eight common peaks were further identified and classified by chemometrics.

3.4. Antibacterial activity The antibacterial activity of thirteen varieties of ZBL against six kinds of pathogenic bacteria was studied using the paper disc diffusion method and the agar dilution method, which were expressed as DIZ and MIC/MBC values, respectively. Two antibiotics (tetracycline and penbritin) were used as positive controls and 2% DMSO solution as the negative control. As shown in Table 4, all extracts expressed antibacterial activity for six tested strains with the DIZ values ranged from 6.92 ± 0.38 mm to 14.67 ± 0.76 mm. The DIZ values of negative control were 6 mm without antibacterial ability. The largest DIZ value was obtained for P. aeruginosa, followed by S. enteritidis and L. monocytogenes, whereas for K. pneumoniae was the lowest. It was obvious that ethanol extracts of ZBL exhibited a certain inhibitory effect on Gram positive bacteria, Gram negative bacteria and fungi. Among all test varieties, You Huajiao (S13) exhibited the best antibacterial activity, with the best inhibition zone diameters for P. aeruginosa (14.67 ± 0.76 mm), followed by S. enteritidis (13.17 ± 0.76 mm), C. albicans (12.92 ± 0.35 mm) and L. monocytogenes (12.88 ± 0.53 mm). MIC and MBC method was carried out for further evaluation of the antibacterial activity. In Table 5, the values of MIC and MBC for tested microorganisms ranged from 5 to 40 mg/mL. You Huajiao (S13) also showed the best antibacterial activity against P. aeruginosa, S. enteritidis, and L. monocytogenes, with the values of MIC were all 5 mg/mL, and MBC values were 10 mg/mL, 20 mg/mL and 20 mg/mL, respectively. Consistent with the results of DIZ, the ZBL varieties of You Huajiao (S13) showed the strongest antibacterial activity but the antibacterial

3.6. Chemometric analysis 3.6.1. Similarity analysis (SA) The results of SA were obtained by the SES software, which calculated according to the correlation coefficient of raw data. A mean chromatogram of all ZBL varieties was also produced and named reference fingerprint (R). As shown in Table 6, the similarity values between different varieties were ranged from 0.502 to 0.999. Meanwhile, the correlation coefficients of all of the ZBL to the R were all above 0.9 except S13, which had distinctive chemical compositions, and the similarity value was 0.775. By comparison, the similarity between S13 and other varieties was lower, especially the similarity between S13 and S2 with the lowest value of 0.502. However, the similarity between Hancheng Dangcun stingless (S1) and Hancheng stingless (S7) was the closest, which similarity value was 0.999. The results showed that the similarity between diverse samples was reasonable and associated with phytochemical constituents. 3.6.2. Hierarchical cluster analysis (HCA) HCA is an unsupervised multivariate analysis method that provides visual representation information about raw data (Wang et al., 2016).

Table 4 Inhibition zone diameter of different varieties Z. bungeanum leaves extracts against bacteria. Samples

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 Tetracycline Penbritin

Inhibition zone diameter (mm) E. coli

K. pneumoniae

P. aeruginosa

S. enteritidis

L. monocytogenes

C. albicans

7.42 ± 0.38h 8.75 ± 0.66g 9.75 ± 0.35d 9.83 ± 1.76d 8.25 ± 0.35g 10.00 ± 0.71cd 9.25 ± 0.35f 8.63 ± 0.88g 8.00 ± 0.00g 10.17 ± 0.58c 9.42 ± 0.88def 9.75 ± 1.06d 10.25 ± 1.39c 14.50 ± 0.71b 21.5 ± 0.5a

7.92 ± 0.14h 9.08 ± 0.80de 11.17 ± 0.38c 8.88 ± 0.38efg 9.08 ± 0.52de 7.83 ± 0.14h 7.75 ± 0.66h 9.58 ± 0.76d 8.42 ± 0.38efg 8.67 ± 0.63efg 7.50 ± 0.43i 8.13 ± 0.53fgh 10.42 ± 0.38c 14.67 ± 1.04b 22.37 ± 1.41a

8.08 ± 0.76h 8.67 ± 0.29g 7.92 ± 0.29h 9.00 ± 0.25fg 10.58 ± 0.29e 9.33 ± 0.14fg 11.17 ± 0.29de 11.00 ± 0.66e 10.67 ± 0.14e 9.83 ± 1.04f 11.67 ± 0.38d 8.33 ± 0.14h 14.67 ± 0.76c 14.67 ± 1.89b 25.92 ± 1.18a

8.92 ± 0.52e 8.08 ± 0.63f 8.83 ± 0.63e 9.75 ± 0.43d 10.42 ± 0.38cd 8.42 ± 0.38ef 7.92 ± 0.38f 8.17 ± 0.38f 7.92 ± 0.14f 8.33 ± 0.29f 11.00 ± 0.75c 8.50 ± 0.66e 13.17 ± 0.76b 17.67 ± 1.89a 17.25 ± 0.71a

7.63 ± 0.88g 7.92 ± 0.52g 6.92 ± 0.38h 10.33 ± 0.52de 9.67 ± 0.63ef 10.42 ± 0.52de 9.58 ± 0.41ef 7.83 ± 0.88g 9.50 ± 0.43ef 11.08 ± 1.18d 8.25 ± 0.43g 9.00 ± 1.09fg 12.88 ± 0.53c 14.42 ± 0.63b 21.75 ± 2.84a

7.58 ± 0.14i 8.67 ± 0.18g 9.08 ± 0.29f 9.28 ± 0.52f 10.75 ± 1.32d 9.33 ± 0.29ef 9.50 ± 0.25ef 9.58 ± 1.13e 9.83 ± 0.13de 10.17 ± 0.33de 8.33 ± 0.29h 9.08 ± 1.04f 12.92 ± 0.35c 15.81 ± 0.73b 25.92 ± 0.52a

Note: Values are the mean of three replicates ± SD (n = 3). Means with different letters within a column are significantly different (p < 0.05). 229

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Table 5 MIC and MBC values of different varieties Z. bungeanum leaves extracts against bacteria. Strains Samples (mg/mL)

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13

Positive control (μg/mL)

Tetracycline Penbritin

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

E. coli

K. pneumoniae

P. aeruginosa

S. enteritidis

L. monocytogenes

C. albicans

10 40 10 40 10 40 10 20 10 40 5 20 10 40 20 40 20 40 10 20 5 20 10 40 10 20 0.625 2.5 1.25 1.25

20 > 40 10 20 10 > 40 20 > 40 10 20 20 40 40 > 40 10 40 20 > 40 20 40 20 > 40 20 > 40 10 20 1.25 2.5 2.5 5

10 40 10 40 20 40 20 40 5 20 20 40 5 20 5 20 5 20 10 20 5 20 20 40 5 10 1.25 2.5 2.5 10

10 40 20 40 10 20 10 40 5 20 20 40 20 40 20 40 40 20 20 40 5 20 20 40 5 20 0.625 1.25 5 10

10 20 20 40 20 40 5 20 10 10 10 20 10 20 10 40 10 40 5 10 10 40 10 40 5 20 0.625 1.25 1.25 1.25

20 40 20 40 10 20 5 20 10 20 10 20 10 20 10 20 10 20 10 20 20 > 40 20 40 10 40 1.25 2.5 0.625 1.25

Note: MIC values express minimum inhibitory concentration. MBC values express minimal bactericidal concentration.

compounds, but they showed a lower antioxidant activity, which caused by their similar chemical compositions. Then, all of these varieties from Hancheng, Shaanxi (S1, S5, S6, S7 and S10) were belonged to the G1, which most varieties of had higher TFC with better biological activity than G2. Next, because of their unique chemical constitutions, S2 was clustered into G3 and S13 was assigned to G4, respectively. For S2, it only contained hyperoside, quercitrin and afzelin, and the antibacterial activity was poor, but the content of quercitrin with 22.723 ± 0.163 mg/g was the highest. By contrast, S13 not only had the highest TFC, and rutin content, higher hyperoside and trifolin content but also exhibited the best the antioxidant activity and antibacterial activity in all the ZBL varieties, which led to S13 was clustered into G4 and the similarity with other sample chromatograms was the lowest.

The peak area data of the eight common characteristic peaks formed a data matrix of 8 × 13. After that, the data matrix was inputted into IBM SPSS Statistics software and used the average linkage between groups and the squared Euclidean distance method for HCA. In Fig. 2a, it was clear that the thirteen varieties were classified into four quality groups (G1-G4) when squared Euclidean distance was 7.5. Firstly, G2 consists of Fengxian Dahongpao (S4), Maoxian Dahongpao (S8), Qin’an Yihao (S9) and Wudu Dahongpao (S11), while G3 and G4 were only composed of Fugu late-maturing (S2) and You Huajiao (S13), respectively. The remaining seven varieties, Hancheng Dangcun stingless (S1), Fugu early-maturing (S3), Gelao stingless (S5), Hancheng Dahongpao (S6), Hancheng stingless (S7), Hancheng Shizitou (S10) and Xinong stingless (S12) formed the G1. Furthermore, in combination with Table 6 and Fig. 2a, the consequences of HCA were found to be in conformity with the results of SA. It is clear that the similarity of the chromatograms of the varieties in the group was higher than that between the groups. Secondly, four varieties from G2 contained all five flavonoids

3.6.3. Principal component analysis (PCA) PCA is a typical multivariate data analysis method that is commonly

Table 6 Similarity calculation results of HPLC fingerprints against simulative mean chromatogram. Samples

S1

S2

S3

S4

S5

S6

S7

S8

S9

S10

S11

S12

S13

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 R

1 0.907 0.981 0.943 0.989 0.963 0.999 0.956 0.942 0.998 0.945 0.986 0.777 0.998

1 0.963 0.749 0.930 0.951 0.903 0.821 0.761 0.920 0.765 0.954 0.502 0.914

1 0.881 0.977 0.987 0.979 0.912 0.880 0.987 0.896 0.997 0.691 0.984

1 0.920 0.890 0.946 0.982 0.995 0.937 0.995 0.897 0.894 0.941

1 0.956 0.988 0.951 0.927 0.992 0.915 0.982 0.715 0.989

1 0.963 0.925 0.889 0.971 0.911 0.986 0.713 0.970

1 0.957 0.944 0.998 0.949 0.986 0.784 0.999

1 0.991 0.952 0.978 0.926 0.840 0.954

1 0.935 0.986 0.896 0.871 0.938

1 0.941 0.992 0.764 0.999

1 0.909 0.899 0.946

1 0.720 0.991

1 0.775

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Fig. 2. Dendrograms of HCA for thirteen varieties of Z. bungeanum leaves (a), 2-D scores plot of the PCA (b), 3-D loadings plot of the PCA (c) and discrimination analysis plot (d).

used in the research of HPLC fingerprinting. It could explain the correlation between a large number of variables with a smaller number of variables (principal components) that do not lose much information, thus reducing the dimensions of the original data, and it has a good ability to generalize variables (Yudthavorasit et al., 2014; Sabir et al., 2017). In this study, the same 8 × 13 data matrix as HCA was implemented for PCA. Based on the eigenvalues > 1, there were first three principal components (PC1, PC2, and PC3) were selected to represent the variable information, which contained 49.59%, 23.65% and 13.63%, respectively. Three principal components could jointly account for 86.87% of the total variance, which contained most information on raw data. Hence, to provide an intuitive classification, PC1 and PC2 were employed to form a 2-D score plot of PCA. As shown in Fig. 2b, it was noticeable that the thirteen varieties of ZBL could be classified into four groups. PC1 could discriminate G3 (S2) and G4 (S13) from other groups while G1 and G2 were differentiated by PC2. Moreover, it can be noted that the profiles of ZBL from G4 were close to G2, whereas the relationship between G3 and G1 was closer. In Fig. 2c, the PCA 3-D loadings plot showed that different degrees of influence of eight common characteristic peaks on the quality evaluation of ZBL. The results revealed that peaks 1, 2, 3 and 5 had the greatest contribution to PC1, while peaks 10 and 13 had an effect on PC2, and peaks 4 and 14

mainly embodied the situation of PC3. In addition, hyperoside (peak 10), quercitrin (peak 13) and afzelin (peak 14) were key compounds and had significant influence on the quality evaluation of different varieties due to these loadings of characteristic peaks were far away from other loadings. Because PC1 could contribute more variables than PC2, it was inferred that the varieties of the first quadrant of 2-D score plot owned a high quality. Therefore, You Huajiao (S13) was rated as the best variety, but the quality of Fugu late-maturing (S2) was the last, which was in agreement with the results of flavonoids compositions and biological activity. 3.6.4. Discriminant analysis (DA) Unlike HCA and PCA, DA is a supervised pattern recognition that provides a prediction model to classify the members of the group. According to the data matrix same as HCA and PCA, one or more discriminant functions can be generated. Although these functions were obtained from the data of the known samples, they apply equally to identify the unknown samples (Liu et al., 2016). There were two kinds of discriminant functions were obtained by SPSS software. Canonical discrimination functions: Function 1 = -0.066X5 + 0.004X13 + 0.013X14 + 1.998 Function 2 = 0.048X5 + 0.005X13 - 0.045X14 - 8.449 231

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through DA with a 100% discrimination ratio, which could discriminate and classify the unknown membership. Moreover, according to the results of PCA and DA, quercitrin and afzelin were considered as crucial compounds for quality assessment and control. Combing the above analysis, G4 (You Huajiao, S13) was stood out from the others, followed by G1 (S1, S3, S5, S6, S7, S10, S12) and G2 (S4, S8, S9, S11), respectively, but G3 (Fugu late-maturing, S2) was the last. In order to establish a full-scale quality evaluation system for ZBL, in the following research, we should increase the sampling range, collect more varieties of Z. bungeanum and evaluate their quality. Then, molecular biology method should be brought together to evaluate the quality at the gene level. Conclusively, this study not only provided a basis of the development and utilization of high-quality resources of Z. bungeanum, but also provided a comprehensive information and valuable reference for the utilization of ZBL as pharmaceutical products and functional food ingredients.

Fisher’s discrimination functions: G1=0.720X5 + 0.046X13 - 0.232X14 - 75.873 G2=0.787X5+ 0.020X13 - 0.138X14 - 61.525 G3=0.776X5+ 0.074X13 - 0.382X14 - 126.928 G4=1.689X5+ 0.011X13 - 0.535X14 - 190.363 Where X5, X13 and X14 denoted the peak area of peak 5, peak 13 (quercitrin) and peak 14 (afzelin) respectively, G1, G2, G3 and G4 represented the unknown sample from Group 1, 2, 3 and 4 respectively. For canonical discrimination functions, Function 1 and Function 2 contained 73.0% and 26.9% of the total variance respectively, with a total of 99.9% variable information, demonstrating a favourable result. As shown in Fig. 2d, Function 1 and Function 2 constituted the abscissa and the ordinate in the discriminant scores plot respectively, which all of the known samples were properly classified and the results were consistent with HCA and PCA. The peak area of peaks 5, 13 and 14 were substituted into both functions respectively, which the corresponding points in the discriminant score plot was obtained, depending on the distance between each point and the centroid of the four groups, it can distinguish which group the samples should belong to. As for Fisher’s discrimination functions, the standard of discrimination is as follows: the three independent variables were substituted into four equations and compared the value of G1, G2, G3 and G4, then the unknown samples were assigned to a group on the basis of the highest function value. It was found that all of samples were correctly classified. Besides, cross-validation also proved that the discriminant accuracy of Fisher’s discrimination functions for all ZBL samples was 100%, which suggested DA provided a high resolution and satisfactory performance prediction model for identifying unknown samples. In this study, there were significant variations in phytochemical among different varieties of ZBL. This diversity may make a significant difference in the efficacy of functional foods and nutritional additives. By common garden experiments, the effects of environmental factors, farming methods and harvest time were eliminated, only genetic factors played a major role in influencing the chemical constituents of different varieties. The genotypes of different varieties in the same group may also serve as similar. Furthermore, four different chemometric analysis methods, SA, HCA, PCA and DA, were deeply used for analyzing the HPLC fingerprinting of thirteen varieties of ZBL, and these results were all consistent. Based on the results of PCA and DA, we concluded that quercitrin (peak 13) and afzelin (peak 14) were chosen as critical chemical markers for quality control and assessment of ZBL. To sum up, the chemometric analysis provided visualized, effective and reliable information for identifying and discriminating different varieties of ZBL.

Conflict of interest All the authors declare that they have no conflict of interests. Acknowledgements This work was supported by the program from The National Natural Science Foundation of China (No. 31872706). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.04.017. References Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 239, 70–76. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci. Technol. 28, 25–30. https://doi.org/ 10.1016/S0023-6438(95)80008-5. Cui, L.L., Zhang, Y.Y., Shao, W., Gao, D.M., 2016. Analysis of the HPLC fingerprint and QAMS from Pyrrosia species. Ind. Crop. Prod. 85, 29–37. Feng, S.J., Yang, T.X., Liu, Z.S., Chen, L., Hou, N., Wang, Y., Wei, A.Z., 2015. Genetic diversity and relationships of wild and cultivated Zanthoxylum, germplasms based on sequence-related amplified polymorphism (SRAP) markers. Genet. Resour. Crop. Ev. 62, 1193–1204. He, X., Liu, D., Liu, R.H., 2008. Sodium borohydride/chloranil-based assay for quantifying total flavonoids. J. Agric. Food Chem. 56, 9337–9344. He, F.Y., Li, D.W., Wang, D.M., Deng, M., 2016. Extraction and purification of quercitrin, hyperoside, rutin, and afzelin from Zanthoxylum bungeanum Maxim leaves using an aqueous two-phase system. J. Food Sci. 81, C1593–C1602. Hofmann, T., Tálos-Nebehaj, E., Albert, L., Németh, L., 2017. Antioxidant efficiency of Beech (Fagus sylvatica L.) bark polyphenols assessed by chemometric methods. Ind. Crop. Prod. 108, 26–35. Li, J.K., Hui, T., Wang, F.L., Li, S., Cui, B.W., Cui, Y.Q., Peng, Z.Q., 2015. Chinese red pepper (Zanthoxylum bungeanum Maxim.) leaf extract as natural antioxidants in salted silver carp (Hypophthalmichthys molitrix) in dorsal and ventral muscles during processing. Food Control 56, 9–17. Li, L.X., Yang, T.X., Wei, A.Z., Feng, S.J., Chen, L., Hou, N., 2016. Genetic diversity and population structure analysis of Zanthoxylum germplasms by SRAP. Acta Agriculturae Boreali-sinica. 31, 122–128. https://doi.org/10.7668/hbnxb.2016.05.018. (In Chinese). Liang, Q., Zhang, Y.J., Chen, J.J., Huang, H.W., Wang, Y., 2016. Quality variation of ten geographic populations of Epimedium sagittatum, as evaluated in common garden practice. Genet. Resour. Crop. Ev. 63, 733–743. Liu, W., Wang, D.M., Liu, J.J., Li, D.W., Yin, D.X., 2016. Quality evaluation of Potentilla fruticosa L. By high performance liquid chromatography fingerprinting associated with chemometric methods. PLoS One 11, e0149197. Liu, Z.H., Wang, D.M., Li, D.W., Zhang, S., 2017. Quality evaluation of Juniperus rigida Sieb. Et Zucc. Based on phenolic profiles, bioactivity, and HPLC fingerprint combined with chemometrics. Front. Pharmacol. 8, 198. https://doi.org/10.3389/fphar.2017. 00198. Ma, L.M., Li, K., Wei, D.D., Xiao, H.Y., Niu, H., Huang, W., 2015. High anti-oxidative and lipid-lowering activities of flavonoid glycosides-rich extract from the leaves of Zanthoxylum bungeanum in multi-system. J. Food Nutr. Res. 3, 62–68. Meng, X.X., Li, D.W., Dan, Z., Wang, D.M., Liu, Q.X., Fan, S.F., 2016. Chemical composition, antibacterial activity and related mechanism of the essential oil from the

4. Conclusions In this study, the quality of thirteen varieties of ZBL collected from a common garden was evaluated. Flavonoids profiles (TFC, and five flavonoids compounds), antioxidant activities and antibacterial activities were determined. The results showed that the flavonoids profiles of the thirteen ZBL varieties were significant variations. You Huajiao (S13) exhibited the highest of TFC and the strongest bioactivity, followed by Hancheng Dangcun stingless (S1). In order to further reflect the phytochemical compositions and assess quality of ZBL, HPLC fingerprint was established and chemometric studies (SA, HCA, PCA and DA) were performed. Eight common characteristic peaks were selected and the similarity values varied from 0.502 to 0.999 between different varieties. Thirteen varieties of ZBL were classified into four groups (G1-G4) by HCA. Using PCA, three principal components were obtained, which could account for 86.87% of the total variance. Canonical discrimination function and Fisher’s discrimination function were constructed 232

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