Climate Effects on Flavonoid Content of Zanthoxylum bungeanum Leaves in Different Development Stages

Abstract

To investigate associations between meteorological parameters and flavonoid content of Zanthoxylum bungeanum leaves (ZBL) in different development stages, three cultivars of Meifengjaio, Shizitou, Qin'an Yihao were selected as experimental material. The flavonoid contents of ZBL were evaluated based on HPLC-MS technology (High Performance Liquid Chromatography - mass spectrograph). Results showed that the total flavonoid content of three ZBLs had significant difference, presented as Shizitou > Qin'an Yihao > Meifengjiao. However, its variation tendency remained nearly constant, with maximums between May and June, and lowest in August. Seven key flavonoids contents (quercitrin, hyperoside, afzelin, quercetin-3-O-glucoside, epicatechin, catechin, rutin.) were identified. The Pearson correlation analysis of 36 meteorological parameters and 7 flavonoids contents indicated that 16 meteorological significantly affected the flavonoid content in ZBL, of which temperature, water vapor pressure and other parameters were significantly negatively correlated with flavonoid content, and wind speed was significantly positive. Quercetin-3-O-glucoside, afzelin, epicatechin and catechin were significantly affected by meteorological parameters, while rutin, quercitrin and ghyperoside were less. Finally, we selected the total flavonoid content in Shizitou as representative to fit with 16 key meteorological parameters respectively, and obtained the specific relationship expression, which revealed the functional relationship between climate and total flavonoid content. This study provided a theoretical basis for the rational harvesting of ZBL and promoted the further development and utilization of it.

Introduction

Zanthoxylum bungeanum (Rutaceae), a deciduous small tree, is widely distributed in Asian countries (Zhang et al., 2017). China as the native of Z. bungeanum, its planting area and output are both the largest in the world (Chen et al., 2019). As one of the most important economic species, Z. bungeanum has been widely recognized for its medicinal and edible value (Shi et al., 2019; Yu et al., 2020). The pericarps, branches, roots, leaves of it are all medicine, which has been widely used to treat dyspepsia, eczema, toothache, and so on (Fan et al., 2019). In addition, it contains several types of compounds, including alkaloids, amides, volatile oil, lignans, flavones, fatty acids, sterols, terpenes, coumarins and hydrocarbons (Ji et al., 2019; Liu et al., 2014).

The mature leaves of Z. bungeanum were used as condiments in traditional Chinese cuisine, and the young leaves have been consumed as foodstuffs (Zhang et al., 2014a). In recent years, researchers have focused more on ZBL. Zhang and her group isolated and characterized nine flavonoids from the leaves of Z. bungeanum. Furthermore, they found quercitrin and hyperoside play crucial roles in the antioxidant activity (Zhang et al., 2015; Zhang et al., 2014b). Zhang and Wang demonstrated that hyperosides from ZBL exerted significant hepatocyte-protective and anti-hyperglycemic effects in mice with diabetes (Zhang et al., 2018). Li and coworkers found the extract of its leaves displayed a protective effect against lipid oxidation of salted fish (Li et al., 2015). Yang's research showed that there are lots of flavonoids in the extract of ZBL which have strong radical scavenging activities (Yang et al., 2013). Therefore, it has attracted researchers to pay more attention on flavonoids of ZBL.

Flavonoids can be roughly divided into six categories (flavanone, flavone, flavonol, flavanol, flavanonol, anthocyanin). And flavonoids have rich biological activities, such as anti-bacterial (Mahunu et al., 2018), anti-inflammatory (Li et al., 2020; Spagnuolo et al., 2018), anticancer (Ma et al., 2019), antidiabetic (Liu et al., 2018) and antioxidant activities (Su et al., 2018; Zhang et al., 2017). According to previous studies, the flavonoids content in the skins and seeds of grapes were strongly influenced by the environment. The total anthocyanin, flavonol and flavan-3-ol contents in winter berry skins were significantly higher than those in summer berry skins (Oliveira et al., 2019; Zhu et al., 2017). Furthermore, Katerina Biniari's research showed that the total phenolics (in grape skins and seeds), were closely linked to air temperature and wind speed (Biniari et al., 2020). Du and his group demonstrated that the accumulation of phenolic compounds was favored under summer conditions, while flavonoids accumulated under lower temperature (winter climate) conditions (Du et al., 2019).

However, they only roughly measured the impact of temperature changes on the content of flavonoids in each quarter, and few people further analyzed the relationship between them. Almost nobody combined climate parameters with ZBL. In our study, seven key flavonoids were detected in three varieties of ZBL, and 36 climate parameters were selected to analyze their relationship with flavonoids content. Our result reveals the correlation between climate and flavonoids and will help its further utilization.

Material and Methods

Meteorological Data    As shown in Appendix 1 and Appendix 2, we collected the meteorological data from May to September 2017–2018, which contained 36 meteorological parameters (average air pressure, mean maximum air pressure, mean minimum air pressure, extreme maximum air pressure, extreme minimum air pressure, average temperature, mean maximum temperature, mean minimum temperature, extreme maximum temperature, extreme minimum temperature, mean vapor pressure, maximum vapor pressure, minimum vapor pressure, mean relative humidity, minimum relative humidity, total precipitation, maximum daily precipitation, mean ground temperature, mean maximum ground temperature, mean minimum ground temperature, extreme maximum ground temperature, extreme minimum ground temperature, ground temperature at 5 cm, ground temperature at 10 cm, ground temperature at 15 cm, ground temperature at 20 cm, ground temperature at 40 cm, ground temperature at 0.8 m, ground temperature at 1.6 m, ground temperature at 3.2 m, average wind speed, maximum wind speed, extreme wind speed, total sunshine hours, percentage of sunshine, actual sunshine hours). All meteorological data were gathered from Yangling Meteorological Bureau of Xianyang City, Shaanxi Province.

Plant Materials    We selected three cultivars of ZBL (Meifengjiao, Shizitou, Qin'an Yihao) as experimental material. The samples (9 years olds) were selected for uniformity in shape, size and color without signs of mechanical damage or disease, and collected at the same time of each month (May to September in 2017 and 2018). The leaves of the three cultivars are shown in Figure 1.

Fig. 1.

Leaves of three cultivated varieties.

They were collected at the same height from a common garden (located in Northwest A and F University, Yangling District, Shaanxi Province, China) with three biological replicates. All samples of three varieties of Z. bungeanum were air-dried at home temperature in the dark. In the laboratory, the leaves were crushed with a grinder and turned into powder and stored at −20 °C.

Extraction and Determination of Flavonoids    In order to get better experimental results, we chose 80% methanol as the extraction solvent instead of ethanol using in traditional methods (Zhang et al., 2017). Air dried and powdered ZBL (1 g) were subjected to ultrasound-assisted extraction with 30 mL of 80% methanol at 40 °C for 40 min, and the extracts were collected and combined after filtration (Bhatt et al., 2017).

The flavonoid contents of ZBL were evaluated based on HPLC-MS (High Performance Liquid Chromatography - mass spectrometry) technology.

The chromatographic conditions used were as follows: The column was a Waters XSelect HSS T3 (2.1 × 150 mm, 2.5 µm). Distilled-water-0.1% formic acid (solvent A) and methanol (solvent B) were used as the mobile phase. The injection volume was 5 µL. Gradient elution was performed as follows: 30–50% B (0–15 min); 50–70% B (15–40 min); 70%–100% B (40–45 min); 100% B (45–60 min) and the flowrate is 0.3 mL/min with a temperature of 40 °C (Yang et al., 2013).

The mass spectrum condition: Using nitrogen as atomizing gas. All analysis was detected in the positive ionization mode using a spray voltage of 5 500 V, and the heated probe temperature and cone temperature were both kept at 550 °C (Schlatt et al., 2020).

Statistical Analysis    We used SPSS 20.0 software (SPSS for Windows, Base System User's Guide, SPSS Inc., Chicago, IL, USA) to do correlation analysis, using MATLAB R2012a software (MATLAB for Windows, Base System User's Guide, MathWorks. Inc., Natick, Massachusetts, USA) to draw thermodynamic diagram, bar and line chart.

Results

The composition and content of flavonoids    The HPLC-MS analysis identified 7 flavonoids in every Z. bungeanum varieties. They were afzelin, epicatechin, hyperoside, quercitrin, rutin, quercetin-3-O-glucoside, and catechin. Among all of them, quercitrin had the highest content, followed by hyperoside (Table 1), which means those two compositions were the dominant flavonoids compounds of ZBL. Our results were similar to Wu's, in addition, their research found those two compositions also play major roles in the antioxidant activity (Wu et al., 2018).

Table 1. The content of flavonoid components

Samples	Contents (mean ± SD, mg·g−1) (n=3)
	Afzelin	Epicatechin	Hyperoside	Quercitrin	Rutin	Quercetin-3-O-glucoside	catechin
M-2017-5	3.426±0.107	2.390±0.049	6.258±0.087	13.124±0.240	0.356±0.005	3.284±0.069	2.388±0.068
M-2017-6	2.307±0.068	3.554±0.096	6.904±0.099	12.782±0.205	0.440±0.014	1.855±0.053	1.509±0.061
M-2017-7	1.881±0.096	0.996±0.047	4.030±0.078	12.558±0.274	0.451±0.006	1.490±0.041	0.845±0.007
M-2017-8	1.790±0.042	1.594±0.086	4.894±0.130	12.032±0.405	0.394±0.004	1.628±0.045	0.639±0.018
M-2017-9	1.147±0.089	1.093±0.076	3.953±0.120	10.598±0.303	0.231±0.014	1.979±0.063	0.873±0.025
Q-2017-5	8.336±0.372	5.081±0.068	8.283±0.260	22.280±0.310	0.122±0.003	7.206±0.030	1.850±0.012
Q-2017-6	5.577±0.018	4.085±0.051	8.738±0.202	14.786±0.086	0.100±0.006	5.146±0.202	1.397±0.039
Q-2017-7	5.866±0.183	2.174±0.047	6.408±0.139	21.501±0.908	0.127±0.004	3.380±0.145	0.340±0.013
Q-2017-8	2.412±0.066	0.993±0.069	5.254±0.179	9.354±0.550	0.632±0.018	2.301±0.119	0.783±0.019
Q-2017-9	3.686±0.085	1.787±0.053	9.558±0.374	12.082±0.569	0.692±0.009	3.932±0.082	0.676±0.027
S-2017-5	7.986±0.127	7.604±0.177	6.609±0.106	21.760±0.608	0.249±0.010	6.646±0.098	6.494±0.076
S-2017-6	3.647±0.120	5.366±0.350	7.682±0.150	16.756±0.568	0.649±0.007	3.720±0.102	2.492±0.069
S-2017-7	3.310±0.113	4.554±0.172	8.330±0.085	17.640±0.377	0.555±0.006	4.201±0.158	3.260±0.113
S-2017-8	2.882±0.076	2.342±0.080	6.946±0.180	12.760±0.115	0.457±0.013	3.840±0.090	1.605±0.022
S-2017-9	2.465±0.068	3.518±0.083	6.182±0.119	17.576±0.270	0.445±0.005	4.464±0.049	5.957±0.106
M-2018-5	3.213±0.202	2.999±0.083	6.267±0.082	10.019±0.227	0.770±0.008	3.700±0.052	0.998±0.036
M-2018-6	2.231±0.147	2.525±0.168	5.554±0.048	14.305±0.378	0.273±0.008	1.778±0.096	1.105±0.068
M-2018-7	1.834±0.087	0.969±0.021	3.802±0.096	10.489±0.288	0.203±0.005	1.223±0,090	0.976±0.496
M-2018-8	2.022±0.040	1.268±0.085	3.188±0.071	12.782±0.238	0.235±0.007	1.267±0.051	0.818±0.071
M-2018-9	1.286±0.100	3.276±0.059	4.416±0.198	6.156±0.117	0.401±0.009	2.038±0.060	2.896±0.107
Q-2018-5	9.242±0.286	6.128±0.100	7.670±0.162	21.040±0.546	0.119±0.092	7.583±0.280	2.274±0.038
Q-2018-6	3.649±0.078	1.294±0.101	2.337±0.056	14.181±0.545	0.831±0.004	2.758±0.074	0.500±0.011
Q-2018-7	4.432±0.136	3.061±0.112	7.619±0.253	17.510±0.274	0.805±0.008	3.708±0.049	0.171±0.014
Q-2018-8	3.912±0.115	1.286±0.133	4.000±0.139	7.582±0.206	0.109±0.007	2.282±0.075	0.744±0.021
Q-2018-9	5.596±0.155	3.394±0.086	7.172±0.195	15.802±0.409	0.118±0.014	4.230±0.231	0.612±0.018
S-2018-5	6.182±0.110	8.358±0.188	7.920±0.069	18.354±0.414	0.128±0.015	7.114±0.120	6.258±0.093
S-2018-6	2.628±0.104	5.677±0.156	6.742±0.070	15.394±0.681	0.423±0.006	3.048±0.098	4.524±0.167
S-2018-7	1.873±0.112	5.344±0.322	5.758±0.016	17.950±0.402	0.541±0.013	3.646±0.188	4.678±0.163
S-2018-8	1.784±0.078	4.474±0.095	5.058±0.095	9.324±0.158	0.436±0.008	3.254±0.180	2.832±0.238
S-2018-9	2.500±0.083	4.692±0.231	5.067±0.086	18.038±0.107	0.614±0.007	4.762±0.217	5.382±0.083

The trend of total flavonoids content in 2017 and 2018 was basically similar (Figure 2). Shizitou had the highest content, Qin'an Yihao is the second, while Meifengjiao is the least. Combined with previous research (Sun et al., 2019), we speculated that this difference might be caused by different genes among varieties.

Fig. 2.

Histogram shows the changes of flavonoids content of each component from May to September in 2017 and 2018. M: Meifengjiao; Q: Qin'an Yihao; S: Shizitou.

In addition, the total flavonoids content of Shizitou and Qin'an Yihao had similar wave trends, highest in May and lowest in August. However, Meifengjiao was different from them, as its highest content of flavonoids was between May and June, while lowest in September. This might be due to Meifengjiao ripening later than Shizitou and Qin'an Yihao.

Correlation analysis of flavonoids and climate parameters    Based on the correlation analysis of SPSS, the heatmap of 7 flavonoids in each variety with climate change were obtained by using Origin drawing (Figure 3). Study showed the relationships between flavonoids and climate were similar in three varieties of Zanthoxylum bungeanum. Rutin, quercitrin and hyperoside were less affected by climate, while quercetin-3-O-glucoside, afzelin, epicatechin and catechin were highly correlated with some meteorological parameters.

Fig. 3.

Heat map of the correlation between flavonoids and meteorological parameters. Blue stands for negative correlation; Red stands for positive correlation.

In Katerina Biniari's research, they speculated some key meteorological parameters correlates positively or negatively with other climate parameters, thus resulting in the correlation of flavonoids to those climatic parameters (Biniari et al., 2020). Our study found those key meteorological parameters containing vapor pressure, ground temperature and wind speed.

According to Figure 3, quercetin-3-O-glucoside, afzelin, epicatechin and catechin had a significant negative correlation with vapor pressure (mean vapor pressure, maximum vapor pressure, minimum vapor pressure), ground temperature (ground temperature at 40 cm, ground temperature at 0.8 m, ground temperature at 1.6 m, ground temperature at 3.2 m) and other meteorological parameters (mean minimum temperature, extreme minimum temperature, mean ground temperature, extreme minimum ground temperature), the correlation coefficient more than 0.45, even high to 0.86. And they had a positive correlation with wind speed (average wind speed, maximum wind speed, extreme wind speed), the correlation is about 0.5.

It appears that the mean and extreme minimum temperature were more likely to affect the flavonoid's content than the mean or extreme maximum temperature, while deep ground temperature was easier to affect the content of flavonoids than shallow ground temperature.

Meteorological effect the content of total flavonoids    Through the above correlation analysis results, 16 meteorological parameters with high correlation were selected for further analysis. And we chose Shizitou, the highest total flavonoids content variety, to match with 16 meteorological parameters, the results were exhibited in Figure 4.

Fig. 4.

The fitting results of 16 meteorological parameters and total flavonoids content in Shizitou. Green stands for negative correlation. Red stands for positive correlation.

The green fitting curve represents that the total content of flavonoids was negatively correlated with it, while the red fitting curve showed the positive correlation. The abscissa represented the total content of flavonoids, and the ordinate represents the value of each meteorological factor.

According to Figure 4, the wind speed was in direct proportion to the total flavonoids content, while the air temperature and vapor pressure were in inverse proportion to the total flavonoids content. What's more, the change trend of total flavone content with weather was the same as that of each flavonoid component with weather.

Our results were partially similar to what Yu Xiaofeng and his team explored in mulberry leaves that lower temperature had a significant positive effect on the accumulation of total flavonoids (Yu et al., 2016). In addition, previously reported the flavonoids were negatively correlated with the precipitation, and positively correlated with the sunshine hours. This also agreed with our research results (Sun et al., 1997).

The Principal Component Analysis    The Principal Component Analysis was further used to demonstrate the correlation between the climatic parameters and total flavonoids content (Biniari et al., 2020).

The PCA 95% confidence ellipse and its loading plot are presented in Figure 5. It appears that air temperature correlates positively with vapor pressure and ground temperature, thus resulting in the correlation of the flavonoids content to those climatic parameters. In addition, we speculated that higher wind speed can reduce the temperature and humidity of leaf surface, which indirectly affects the ZBL flavonoids content.

Fig. 5.

Evaluation of the links between the prevailing climatic parameters and their effect on the flavonoids content.

Discussion

Some researches focused on the influence of meteorological factors on the accumulation of secondary metabolites in plants. Olha Mykhailenko and his team found that sunshine duration had a significant positive effect on the accumulation of phenolic compounds in Iris species (Mykhailenko et al., 2020). In Yang's study, they speculated that the accumulation of phenolic compounds was a complicated biosynthetic process and different environmental conditions could regulate the expression of the relative genes and proteins, which resulted in the same mulberry cultivars with different phenolic compositions. And Sven W. Meckelmann got the same conclusion in his work (Meckelmann et al., 2014; Yang et al., 2017). Our research concentrated on ZBL which had been widely used as medicine, due to its rich flavonoids (He et al., 2016). We found that places with low ground temperature, low vapor pressure and high wind speed were helpful to the accumulation of flavonoids in ZBL. Our conclusions were consistent with other researchers' studies on mulberry and onions. Their results generally showed that temperature, rainfall and humidity as significant parameters, was negatively correlated with the accumulation of flavonoids (Rodrigues et al., 2011; Yang et al., 2019).

Conclusions

In summary, our study was designed to extract different flavonoids components from ZBL, and investigated their relationship with meteorological parameters. Results found that the suitable picking time of ZBL was on June. And places with low ground temperature, low vapor pressure and high wind speed might be helpful to the accumulation of flavonoids in its leaves. Among seven key flavonoids, quercetin-3-O-glucoside, afzelin, epicatechin and catechin were highly correlated with meteorological, while the others were less affected by climate, which facilitated targeted harvesting as needed. In addition, the fitting results of total flavonoids could be used to estimate the content in different climatic conditions. This paper provides was helpful for the further development and utilization of Zanthoxylum bungeanum leaves.

Acknowledgements    This article was supported by the project “the demonstration and promotion of efficient cultivation and management techniques of Zanthoxylum bungeanum in Hanyuan Weibei (No. [2017]18).”

Appendices

Table 2. Abbreviation of meteorological parameters

Symbol	Full Title	Symbol	Full Title
AP	Atmospheric Pressure	RH_1	Mean Relative Humidity
AT	Air Temperature	RH_2	Minimum Relative Humidity
VP	Vapor Pressure	P_1	Total Precipitation
RH	Relative Humidity	P_2	Maximum Daily Precipitation
P	Precipitation	GT_1	Mean Ground Temperature
GT	Ground Temperature	GT_2	Mean Maximum Ground Temperature
SG	Shallow Geothermal	GT_3	Mean Minimum Ground Temperature
DG	Deep Geothermal	GT_4	Extreme Maximum Ground Temperature
WS	Wind Speed	GT_5	Extreme Minimum Ground Temperature
SD	Sunshine Duration	SG_1	Ground Temperature at 5cm
AP_1	Average Air Pressure	SG_2	Ground Temperature at 10cm
AP_2	Mean Maximum Air Pressure	SG_3	Ground Temperature at 15cm
AP_3	Mean Minimum Air Pressure	SG_4	Ground Temperature at 20cm
AP_4	Extreme Maximum Air Pressure	SG_5	Ground Temperature at 40cm
AP_5	Extreme Minimum Air Pressure	DG_1	Ground Temperature at 0.8m
AT_1	Average Temperature	DG_2	Ground Temperature at 1.6m
AT_2	Mean Maximum Temperature	DG_3	Ground Temperature at 3.2m
AT_3	Mean Minimum Temperature	WS_1	Average Wind Speed
AT_4	Extreme Maximum Temperature	WS_2	Maximum Wind Speed
AT_5	Extreme Minimum Temperature	WS_3	Extreme Wind Speed
VP_1	Mean Vapor Pressure	SD_1	Total Sunshine Hours
VP_2	Maximum Vapor Pressure	SD_2	Percentage of Sunshine
VP_3	Minimum Vapor Pressure	SD_3	Actual Sunshine Hours

Table 3. Monthly meteorological data

months		May-17	Jun-17	Jul-17	Aug-17	Sep-17	May-18	Jun-18	Jul-18	Aug-18	Sep-18
AP	AP_1	9521	9469	9442	9480	9532	9505	9461	9441	9465	556
	AP_2	9548	9490	9463	9501	9551	9533	9481	9457	9484	9577
	AP_3	9483	9443	9413	9454	9511	9466	9433	9418	9437	9530
	AP_4	9654	9539	9527	9597	9608	9652	9569	9494	9537	9641
	AP_5	9402	9388	9379	9386	9473	9342	9375	9386	9405	9449
AT	AT_1	199	244	290	247	199	198	246	266	271	189
	AT_2	274	304	358	298	247	257	307	313	329	243
	AT_3	135	187	227	206	165	145	189	230	226	153
	AT_4	349	352	419	372	301	337	373	375	360	320
	AT_5	67	148	185	147	113	87	119	199	175	87
VP	VP_1	134	179	222	224	193	149	193	264	260	169
	VP_2	217	234	305	297	233	237	305	340	313	270
	VP_3	41	77	120	143	114	63	58	186	189	94
RH	RH_1	60	62	58	75	84	67	64	78	74	79
	RH_2	10	14	13	24	25	15	12	35	39	26
P	P_1	597	1330	485	797	1053	427	764	1362	1645	642
	P_2	220	621	221	317	343	128	252	433	845	250
GT	GT_1	242	295	354	286	219	241	292	313	331	220
	GT_2	459	488	573	449	340	430	486	485	537	359
	GT_3	124	182	221	205	161	134	178	229	224	153
	GT_4	628	639	688	702	499	602	619	650	651	588
	GT_5	56	142	170	157	116	68	112	200	174	75
SG	SG_1	228	274	329	276	218	225	272	295	308	219
	SG_2	225	270	325	276	218	221	266	290	304	221
	SG_3	222	266	320	277	220	218	262	286	302	225
	SG_4	218	262	316	277	221	215	258	283	299	228
	SG_5	177	221	264	259	220	192	227	262	279	234
DG	DG_1	163	203	242	252	221	177	209	244	265	239
	DG_2	144	175	206	226	216	153	179	208	234	233
	DG_3	133	145	160	176	185	134	147	162	178	191
WS	WS_1	19	17	17	17	13	17	17	15	13	14
	WS_2	108	84	73	66	75	118	77	55	93	71
	WS_3	172	163	156	121	130	203	137	113	156	112
SD	SD_1	2339	2466	3143	1724	888	1684	1483	1453	2586	1297
	SD_2	54	57	72	42	24	39	34	33	62	35
	SD_3	1263	1405	2262	724	213	656	504	479	1603	453

References

•  Bhatt,  V.,  Sharma,  S.,  Kumar,  N.,  Sharma,  U., and  Singh,  B. (2017). Simultaneous quantification and identification of flavonoids, lignans, coumarin and amides in leaves of Zanthoxylum armatum using UPLC-DAD-ESI-QTOF-MS/MS. J. Pharm. Biomed. Anal., 132, 46-55.
•  Biniari,  K.,  Xenaki,  M.,  Daskalakis,  I.,  Rusjan,  D.,  Bouza,  D., and  Stavrakaki,  M. (2020). Polyphenolic compounds and antioxidants of skin and berry grapes of Greek Vitis vinifera cultivars in relation to climate conditions. Food Chem., 307, 125-518.
•  Chen,  X.,  Wang,  W.,  Wang,  C.,  Liu,  Z.,  Sun,  Q., and  Wang,  D. (2019). 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 and Products, 134, 225-233.
•  de Oliveira,  J. B.,  Egipto,  R.,  Laureano,  O.,  de Castro,  R.,  Pereira,  G. E., and  Ricardo-da-Silva,  J. M. (2019). Climate effects on physicochemical composition of Syrah grapes at low and high altitude sites from tropical grown regions of Brazil. Food Res. Int., 121, 870-879.
•  Du,  B.,  Kruse,  J.,  Winkler,  J. B.,  Alfarray,  S.,  Schnitzler,  J. P.,  Ache,  P.,  Hedrich,  R., and  Rennenberg,  H. (2019). Climate and development modulate the metabolome and antioxidative system of date palm leaves. J. Exp. Bot., 70, 5959-5969.
•  Fan,  L.,  Huang,  Y.,  Zhao,  R.,  Fan,  W.,  Zhang,  M.,  Zhang,  H.,  Yan,  X.,  Li,  L., and  Wu,  C. (2019). Geographical-origin discrimination and volatile oil quantitative analysis of Zanthoxylum bungeanum Maxim. with a portable near-infrared spectrometer. Analytical Methods, 11, 5301-5310.
•  He,  F.,  Li,  D.,  Wang,  D., and  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, 1593-1602.
•  Ji,  Y.,  Li,  S. M., and  Ho,  C. T. (2019). Chemical composition, sensory properties and application of Sichuan pepper (Zanthoxylum genus). Food Science and Human Wellness, 8, 115-125.
•  Li,  J.,  Hui,  T.,  Wang,  F.,  Li,  S.,  Cui,  B.,  Cui,  Y., and  Peng,  Z. (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,  J.,  Liu,  Q.,  Wang,  J.,  Liu,  Q., and  Peng,  Z. (2020). Effect of red pepper (Zanthoxylum bungeanum Maxim.) leaf extract on volatile flavor compounds of salted silver carp. Food Sci. Nutr., 8, 1355-1364.
•  Liu,  Y.-Y.,  Shen,  X.,  Zhang,  Y.,  Cao,  W.,  Wang,  K.,  Xu,  S.-Z., and  Wang,  S.-W. (2014). Analysis of chemical constituents of volatiles and petroleum ether extract from the branch of Zanthoxylum bungeanum by GC-MS. Asian Journal of Chemistry, 26, 3863-3867.
•  Liu,  Y.,  Cao,  Y.,  Fang,  S.,  Wang,  T.,  Yin,  Z.,  Shang,  X.,  Yang,  W., and  Fu,  X. (2018). Antidiabetic effect of Cyclocarya paliurus leaves depends on the contents of antihyperglycemic flavonoids and antihyperlipidemic triterpenoids. Molecules, 23, 1042-1059.
•  Ma,  Y.,  Li,  X.,  Hou,  L.-X., and  Wei,  A.-Z. (2019). Extraction solvent affects the antioxidant, antimicrobial, cholinesterase and HepG2 human hepatocellular carcinoma cell inhibitory activities of Zanthoxylum bungeanum pericarps and the major chemical components. Industrial Crops and Products, 142, 1872-1900.
•  Mahunu,  G. K.,  Zhang,  H.,  Apaliya,  M. T.,  Yang,  Q.,  Zhang,  X., and  Zhao,  L. (2018). Bamboo leaf flavonoid enhances the control effect of Pichia caribbica against Penicillium expansum growth and patulin accumulation in apples. Postharvest Biology and Technology, 141, 1-7.
•  Meckelmann,  S. W.,  Riegel,  D. W.,  van Zonneveld,  M.,  Ríos,  L.,  Peña,  K.,  Mueller-Seitz,  E., and  Petz,  M. (2014). Capsaicinoids, flavonoids, tocopherols, antioxidant capacity and color attributes in 23 native Peruvian chili peppers (Capsicum spp.) grown in three different locations. European Food Research and Technology, 240, 273-283.
•  Mykhailenko,  O.,  Gudzinskas,  Z.,  Kovalyov,  V.,  Desenko,  V.,  Ivanauskas,  L.,  Bezruk,  I., and  Georgiyants,  V. (2020). Effect of ecological factors on the accumulation of phenolic compounds in Iris species from Latvia, Lithuania and Ukraine. Phytochem. Anal., 2020, 1-19.
•  Rodrigues,  A. S.,  Pérez-Gregorio,  M. R.,  García-Falcón,  M. S.,  Simal-Gándara,  J., and  Almeida,  D. P. F. (2011). Effect of meteorological conditions on antioxidant flavonoids in Portuguese cultivars of white and red onions. Food Chemistry, 124, 303-308.
•  Schlatt,  L.,  Costa,  A. C. C.,  Barz,  V.,  Ciarimboli,  G., and  Karst,  U. (2020). Fast simultaneous quantification of gabapentin and cetirizine in cell lysates by means of HPLC-MS/MS. J. Pharm. Biomed. Anal., 184, 113-172.
•  Shi,  J.,  Fei,  X.,  Hu,  Y.,  Liu,  Y., and  Wei,  A. (2019). Identification of key genes in the synthesis pathway of volatile terpenoids in fruit of Zanthoxylum bungeanum Maxim. Forests, 10, 328-344.
•  Spagnuolo,  C.,  Moccia,  S., and  Russo,  G. L. (2018). Anti-inflammatory effects of flavonoids in neurodegenerative disorders. Eur. J. Med. Chem., 153, 105-115.
•  Su,  D.,  Li,  N.,  Chen,  M.,  Yuan,  Y.,  He,  S.,  Wang,  Y.,  Wu,  Q.,  Li,  L.,  Yang,  H., and  Zeng,  Q. (2018). Effects ofin vitrodigestion on the composition of flavonoids and antioxidant activities of the lotus leaf at different growth stages. International Journal of Food Science and Technology, 53, 1631-1639.
•  Sun,  J. and  Ding,  a. (1997). Effects of geographical environment on isoflavones accumulation in soybean seeds. Soybean Science, 16, 298-302.
•  Sun,  L.,  Yu,  D.,  Wu,  Z.,  Wang,  C.,  Yu,  L.,  Wei,  A., and  Wang,  D. (2019). Comparative transcriptome analysis and expression of genes reveal the biosynthesis and accumulation patterns of key flavonoids in different varieties of Zanthoxylum bungeanum leaves. J. Agric. Food Chem., 67, 13258-13268.
•  Wu,  Z.,  Wang,  W.,  He,  F.,  Li,  D., and  Wang,  D. (2018). Simultaneous enrichment and separation of four flavonoids from Zanthoxylum bungeanum leaves by ultrasound-assisted extraction and macroporous resins with evaluation of antioxidant activities. J. Food Sci., 83, 2109-2118.
•  Yang,  J.,  Zhang,  X.,  Jin,  Q.,  Gu,  L.,  Liu,  X.,  Li,  J., and  Ni,  Y. (2017). Effect of meteorological parameters and regions on accumulation pattern of phenolic compounds in different mulberry cultivars grown in China. Nat. Prod. Res., 31, 1091-1096.
•  Yang,  L. C.,  Li,  R.,  Tan,  J., and  Jiang,  Z. T. (2013). Polyphenolics composition of the leaves of Zanthoxylum bungeanum Maxim. grown in Hebei, China, and their radical scavenging activities. J. Agric. Food Chem., 61, 1772-1778.
•  Yang,  W.,  Ma,  X.,  Laaksonen,  O.,  He,  W.,  Kallio,  H., and  Yang,  B. (2019). Effects of latitude and weather conditions on proanthocyanidins in blackcurrant (Ribes nigrum) of finnish commercial cultivars. J. Agric. Food Chem., 67, 14038-14047.
•  Yu,  L.,  Wu,  W.,  Pan,  Y.,  Wang,  W.,  Sun,  L.,  Liu,  Y.,  Wang,  D., and  Li,  D. (2020). Quality evaluation of different varieties of Zanthoxylum bungeanum Maxim. peels based on phenolic profiles, bioactivity, and HPLC fingerprint. J. Food Sci., 85, 1090-1097.
•  Yu,  X.,  Li,  Y.,  Zhang,  W.,  Wng,  D.,  Wei,  y., and  Ou,  Y. (2016). Mulberry leaves harvested before and after frost: correlation between phenylalanine ammonia lyase activity and total flavonoid content as well as air temperature. Food Science, 37, 21-25.
•  Zhang,  M.,  Wang,  J.,  Zhu,  L.,  Li,  T.,  Jiang,  W.,  Zhou,  J.,  Peng,  W., and  Wu,  C. (2017). Zanthoxylum bungeanum Maxim. (Rutaceae): A systematic review of its traditional uses, botany, phytochemistry, pharmacology, pharmacokinetics, and toxicology. Int. J. Mol. Sci., 18, 2172-2200.
•  Zhang,  Y.,  He,  F.,  Li,  D., and  Wang,  D. (2017). Effects of high temperature steam treatment on microbial and phytochemical contents, antioxidant activities, chemical stability, and shelf life of oral liquid prepared from the leaves of Zanthoxylum bungeanum Maxim. Journal of Food Processing and Preservation, 41, 1-7.
•  Zhang,  Y.,  Luo,  Z., and  Wang,  D. (2015). Efficient quantification of the phenolic profiles of Zanthoxylum bungeanum leaves and correlation between chromatographic fingerprint and antioxidant activity. Nat. Prod. Res., 29, 2024-2029.
•  Zhang,  Y.,  Wang,  M.,  Dong,  H.,  Yu,  X., and  Zhang,  J. (2018). Anti-hypoglycemic and hepatocyte-protective effects of hyperoside from Zanthoxylum bungeanum leaves in mice with high-carbohydrate/high-fat diet and alloxan-induced diabetes. Int. J. Mol. Med., 41, 77-86.
•  Zhang,  Y. J.,  Luo,  Z. W.,  Wang,  D. M.,  He,  F. Y., and  Li,  D. W. (2014a). Phytochemical profiles and antioxidant and antimicrobial activities of the leaves of Zanthoxylum bungeanum. Scientific World Journal, 2014, 1-13.
•  Zhang,  Y. J.,  Wang,  D. M.,  Yang,  L. N.,  Zhou,  D., and  Zhang,  J. F. (2014b). Purification and characterization of flavonoids from the leaves of Zanthoxylum bungeanum and correlation between their structure and antioxidant activity. Plos One, 9, 1-11.
•  Zhu,  L.,  Huang,  Y.,  Zhang,  Y.,  Xu,  C.,  Lu,  J., and  Wang,  Y. (2017). The growing season impacts the accumulation and composition of flavonoids in grape skins in two-crop-a-year viticulture. J. Food Sci. Technol., 54, 2861-2870.