Geographic Variation in the Chemical Composition and Antioxidant Properties of Phenolic Compounds from Cyclocarya paliurus (Batal) Iljinskaja Leaves

Cyclocarya paliurus has been widely used as an ingredient in functional foods in China. However, the antioxidant properties of phenolic compounds and the effect of the plant origin remain unclear. The present study evaluated the geographical variation of this plant in term of its phenolic composition and antioxidant activities based on leaf materials collected from five regions. high-performance liquid chromatography (HPLC) analysis showed that there are three major components, quercetin-3-O-glucuronide, kaempferol-3-O-glucuronide, and kaempferol-3-O-rhamnoside, and their contents varied significantly among sampling locations. The investigated phenolic compounds showed substantial antioxidant activities, both in vitro and in vivo, with the highest capacity observed from Wufeng and Jinzhongshan. Correlation analysis revealed that quercetin and kaempferol glycosides might be responsible for the antioxidant activities. Our results indicate the importance of geographic origin, with sunny hours and temperature as the main drivers affecting the accumulation of C. paliurus phenolics and their antioxidant properties.


Introduction
It is well known that excess reactive oxygen species (ROS) can lead to so-called oxidative stress in the human body, which is a major cause of aging and disease [1]. Treatments with synthetic antioxidants often have toxic or side effects [2]. Hence, attention in recent years has focused on screening natural antioxidants from plants, as they can help maintain the biological balance between oxidation and antioxidation [1]. Polyphenols, a large category of secondary plant metabolites, have been widely studied for their biological functions in combating chronic disease due to their high antioxidant activities [3][4][5]. In this context, fruits, vegetables, and plants that are rich in polyphenols such as Ginkgo biloba, Crataegus pinnatifida, and Cyclocarya paliurus, are receiving increasing attention from pharmacological researchers and nutritionists [6][7][8].
Cyclocarya paliurus (Batal) Iljinskaja is a multifunction tree species that belongs to the Juglandaceae family and is mainly distributed in the subtropical highlands of China [9]. Leaves of this plant have been used in China as functional foods or a nutraceutical tea for the treatment of hyperhidrosis, hypertension,

Phytochemical Composition
In order to identify the phenolic compounds responsible for the antioxidant activities, the phytochemical composition of C. paliurus extracts was clarified by using both quantitative and qualitative analyses through high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). The data of retention time, and MS and MS 2 fragment ions are summarized in Table 1. The chemical structures of the 10 components are shown in Figure 1. We found that extracts from different locations were rich in flavonoids (Table 2), which yielded from 2.30 mg/g to 6.88 mg/g. The phenolic yields among the five locations studied were significantly different. The highest content of total phenolics (7.89 ± 0.02 mg/g dry weight (DW)) was found in the extract from WF, while the highest content of total flavonoids (6.87 ± 0.01 mg/g, DW) was observed in the extract from JZS. The five locations were ranked in the following order: WF > JZS > MW > MC > SN. Table 1. The data of retention time, and MS and MS 2 fragment ions of 10 phenolics from leaves of C. paliurus by developed high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). was observed in the extract from JZS. The five locations were ranked in the following order: WF > JZS > MW > MC > SN.      The quantities of individual phytochemicals were also given in Table 2. Three phenolic acids and seven flavonoids were identified. Meanwhile, quercetin-3-O-glucuronide (0.78-2.38 mg/g DW), kaempferol-3-O-glucuronide (0.51-1.88 mg/g DW), and kaempferol-3-O-rhamnoside (0.68-2.30 mg/g DW) were found to be the predominant components. These results were comparable to those of a previous study [13]. Furthermore, the canonical correspondence analysis (CCA) test in our study showed that the higher yields of phenolic compounds from JZS, WF, and MW were associated with increasing mean annual temperatures and sunshine hours ( Figure 2). A previous study also indicated that genetic diversity was low among these C. paliurus populations based on the simple-sequence repeat (SSR) and inter-SSR (ISSR) tests [24]. Thus, compared with environmental factors, the genotype may have a slight impact on phytochemical accumulation, which is inconsistent with the results of Sosa et al. [25]. Overall, higher radiance and drier climate in these locations might lead to increased use of secondary metabolites, such as phenolic acids and flavonoids, by plants to protect cells from oxidative damages caused by ROS [22]. The quantities of individual phytochemicals were also given in Table 2. Three phenolic acids and seven flavonoids were identified. Meanwhile, quercetin-3-O-glucuronide (0.78-2.38 mg/g DW), kaempferol-3-O-glucuronide (0.51-1.88 mg/g DW), and kaempferol-3-O-rhamnoside (0.68-2.30 mg/g DW) were found to be the predominant components. These results were comparable to those of a previous study [13]. Furthermore, the canonical correspondence analysis (CCA) test in our study showed that the higher yields of phenolic compounds from JZS, WF, and MW were associated with increasing mean annual temperatures and sunshine hours ( Figure 2). A previous study also indicated that genetic diversity was low among these C. paliurus populations based on the simple-sequence repeat (SSR) and inter-SSR (ISSR) tests [24]. Thus, compared with environmental factors, the genotype may have a slight impact on phytochemical accumulation, which is inconsistent with the results of Sosa et al. [25]. Overall, higher radiance and drier climate in these locations might lead to increased use of secondary metabolites, such as phenolic acids and flavonoids, by plants to protect cells from oxidative damages caused by ROS [22].

Variation in Antioxidant Activity
The DPPH and FRAP methods are commonly applied to determine the antioxidant activities of phenolic compounds by measuring the capacity of plant extracts to donate hydrogen of DPPH or

Variation in Antioxidant Activity
The DPPH and FRAP methods are commonly applied to determine the antioxidant activities of phenolic compounds by measuring the capacity of plant extracts to donate hydrogen of DPPH or reduce ferric iron to ferrous in vitro [26]. The antioxidant activity from DPPH and FRAP tests is presented in Figure 3. The investigated C. paliurus phenolic compounds showed substantial antioxidant activities, although the two tests yielded different values of antioxidant activities. IC 50 values of DPPH and EC 50 values of FRAP ranged from 6.1 to 68.8 µg/mL, and from 0.29 to 1.18 mg/mL, respectively. The highest antioxidant of DPPH and FRAP tests was observed from the WF location, while the lowest antioxidant activity was observed from the SN region. Compared with other species, the antioxidant activity of C. paliurus phenolics is much higher than that of Thymus daenensis (IC 50 of DPPH: 273.4 µg/mL), Folium eucommiae (IC 50 of DPPH: 100.0 µg/mL), Copaifera langsdorffii (IC 50 of DPPH: 510.0 µg/mL), Ginkgo biloba (IC 50 of DPPH: 40.7 µg/mL), and comparable with that of Fagopyrum tataricum (IC 50 of DPPH: 8.4 µg/mL), Anadenanthera peregrina (IC 50 of DPPH: 11.6 µg/mL), and Algerian Mentha (IC 50 of DPPH: 16.2 µg/mL) [6,23,[27][28][29][30].  Malonaldehyde (MDA) is an important index that represents the degree of lipid peroxidation in tissue [31]. Compared to normal mice, MDA levels in STZ-induced diabetic mice were significantly increased (Figure 4), which was in consistence with a previous report [29]. Treatment with C. paliurus phenolics from JZS, MC, WF, and MW significantly decreased MDA levels, which revealed their protective effects in diabetes treatment by reducing oxidative stress (Figure 4). It is well known that the activities of antioxidant enzymes, such as SOD and GSH-px, are very low in diabetic animals as compared to those of normal animals [32]. Our study showed that treatment with C. paliurus extracts markedly restored SOD and GSH-px activities in diabetic mice. Results also revealed that variation of the antioxidant activities of C. paliurus phenolics from different locations in vivo was significant (Figure 4), with the best effect observed from the JZS treated group. Malonaldehyde (MDA) is an important index that represents the degree of lipid peroxidation in tissue [31]. Compared to normal mice, MDA levels in STZ-induced diabetic mice were significantly increased (Figure 4), which was in consistence with a previous report [29]. Treatment with C. paliurus phenolics from JZS, MC, WF, and MW significantly decreased MDA levels, which revealed their protective effects in diabetes treatment by reducing oxidative stress (Figure 4). It is well known that the activities of antioxidant enzymes, such as SOD and GSH-px, are very low in diabetic animals as compared to those of normal animals [32]. Our study showed that treatment with C. paliurus extracts markedly restored SOD and GSH-px activities in diabetic mice. Results also revealed that variation of the antioxidant activities of C. paliurus phenolics from different locations in vivo was significant (Figure 4), with the best effect observed from the JZS treated group.
as compared to those of normal animals [32]. Our study showed that treatment with C. paliurus extracts markedly restored SOD and GSH-px activities in diabetic mice. Results also revealed that variation of the antioxidant activities of C. paliurus phenolics from different locations in vivo was significant (Figure 4), with the best effect observed from the JZS treated group.

Correlation of Phenolic Compounds and Antioxidant Activity
Correlation between the obtained antioxidant activities and studied phenolic contents is shown in Table 3. Positive correlations were presented between the contents of total phenolic or total flavonoid compounds and antioxidant ability. Total phenolics were negatively correlated with IC50 of DPPH (r = -0.991, p < 0.01) and MDA (r = -0.878, p < 0.05). Similarly, total flavonoids were also negatively correlated with IC50 of DPPH (r = -0.950, p < 0.05) and MDA (r = -0.903, p < 0.05). These negative relationships revealed the antioxidant effects of total phenolics and total flavonoids of this plant, which can be used as an alternative additive in foods and pharmaceuticals [4].

Correlation of Phenolic Compounds and Antioxidant Activity
Correlation between the obtained antioxidant activities and studied phenolic contents is shown in Table 3. Positive correlations were presented between the contents of total phenolic or total flavonoid compounds and antioxidant ability. Total phenolics were negatively correlated with IC 50 of DPPH (r = −0.991, p < 0.01) and MDA (r = −0.878, p < 0.05). Similarly, total flavonoids were also negatively Molecules 2018, 23, 2440 7 of 12 correlated with IC 50 of DPPH (r = −0.950, p < 0.05) and MDA (r = −0.903, p < 0.05). These negative relationships revealed the antioxidant effects of total phenolics and total flavonoids of this plant, which can be used as an alternative additive in foods and pharmaceuticals [4].  (Table 3). Additionally, strong correlation was observed between the antioxidant tests in vivo (SOD, GSH-px, and MDA values) and the contents of kaempferol-3-O-glucoside (F5) and kaempferol-3-O-rhamnoside (F7). These results suggest that the antioxidant effects of C. paliurus might be mainly dependent on these individual phenolics. Previous studies have also indicated that quercetin or kaempferol glycosides from plants exhibited various activities such as antitumor, antioxidant, immunoregulatory, and antihyperglycemic effects [4,[33][34][35]. However, in order to determine the individual compounds responsible for antioxidant activities, isolation and purification should in the future be done using chromatographic techniques.  Table 4. Information on sample collecting and pretreatment was the same as our previous study [36]. Briefly, leaf sampling was carried out in September 2014. At each location, 6-30 sample trees (dominant or codominant trees) were chosen based on stem form, tree age, and population size. Leaves (about 400 g per tree from the middle crown) of each location were mixed and dried to constant weight in the lab, and then ground into fine powder for the preparation of C. paliurus extracts.

Preparation of C. paliurus Extracts
Extraction of C. paliurus samples was carried out as described previously, with slight modifications, in August 2015 [4]. The leaf powder (about 75 g) from each sample was extracted with 80% ethanol (1000 mL) and incubated at 90 • C for 1 hour. Afterward, the mixture was shaken at 25 • C for 15 min. Centrifugation for the mixture was done at 8000 g for 10 min, and the supernatants were then evaporated at 40 • C to yield an 80% ethanol extract (JZS: 36.5 g, 48.7% yield; MC: 37.6 g, 50.1% yield; WF: 36.8 g, 49.1% yield; MW: 36.1 g, 48.1% yield; SN: 36.4 g, 48.5% yield). All extracts were stored in the lab at 4 • C before antioxidant tests and chemical analysis.

HPLC-Q-TOF-MS Confirmation
Liquid chromatography (LC)-mass spectrometry (MS) analysis was carried out to confirm the peak identities. The identification was performed on an Agilent 6520 Q-TOF mass spectrometer system equipped with a diode array detector (DAD) and electrospray interface (ESI) (Agilent Technologies, Santa Clara, CA, USA). The MS system was operated in negative ionization modes with the mass scan range set at m/z 100-1200. The mass spectral parameters were a gas temperature of 300 • C; gas flow of 10 L/min; nebulizer pressure of 30 psi; capillary voltage of 4000 V; cone voltage of 100 V; and collision voltage: 60 V. The chromatographic conditions were same as those described above. Agilent Mass Hunter version B.04.00 software was used for data acquisition and processing. Peaks were identified on the basis of comparison of retention times and MS spectra with standards.

Determination of Phenolic Compounds by HPLC
Phenolic profiles of C. paliurus from different locations were measured using an HPLC system according to a previous method, with slight modifications, in September 2015 (Waters, Milford, MA, USA) [13]. Separation for C. paliurus phenolics was carried out on an X-Bridge C18 column by a stepwise elution with acetonitrile containing 0.01% formic acid (solution A) and water containing 0.01% formic acid (solution B). The gradient elution included 0-13 min, 8% A; 13-28 min, 19% A; and 28-40 min, 21% A. The flow rate was kept at 1.0 mL/min and the injection volume was 10 µL. Meanwhile, column temperature was kept at 45 • C and the wavelength for detection was 205 nm. Contents of individual compounds were quantified from their external standards. The representative HPLC chromatogram of 80% ethanol extract of the C.

DPPH Radical-Scavenging Activity
The DPPH radical-scavenging activities of C. paliurus phenolics were carried out as described by a previous study, with some modifications, in September 2015 [37]. C. paliurus extracts were dissolved in methanol. Samples of various concentrations (0.1-2.0 mg/mL, 2 mL) were added to the DPPH methanol solution (0.5 mM, 2 mL). Afterward, incubation of the mixture was done at 25 • C for 30 min in a water bath. Sample absorbance was then measured at 517 nm against a blank. The radical-scavenging activities of C. paliurus phenolics were calculated from the equation: radical scavenging activity (%) = [(A 0 − A 1 )/A 0 ] × 100, where A 0 was the absorbance of the DPPH solution without the sample and A 1 was the absorbance of the tested samples. The assay was carried out in triplicate. The extract concentration at 50% radical-scavenging activity (IC 50 ) was then calculated from the graph of scavenging activities against six gradient concentrations.

FRAP
The reducing power of C. paliurus phenolics was determined according to the Fe 3+ reduction method with modifications in September 2015 [38]. Different ethanolic concentrations (1 mL, 0.2-2.0 mg/mL) of the C. paliurus extracts were mixed with 2 mL phosphate buffer (0.2 M, pH 6.6) and 2 mL potassium ferricyanide (K 3 Fe(CN) 6 , 1%). Afterward, incubation of the mixture was done at 50 • C for 20 min in a water bath. Trichloroacetic acid (4 mL, 10%) was then added and the mixture was centrifuged at 8000 rpm for 5 min. Finally, the supernatants of the solution (2 mL) were mixed with distilled water (2 mL) and ferric chloride (FeCl 3 ; 0.4 mL, 1%), and sample absorbance was measured at 700 nm. The extract concentration at 0.5 absorbance (EC 50 ) was calculated by plotting absorbance at 700 nm against the six tested gradient concentrations.

Antioxidant Activity In Vivo
3.6.1. Animals and Experimental Design C57BL/6 diabetic mice (male, 17.9-18.1 g, certificate number: SCXK (HU) 2013-0018) were used for the in vivo antioxidant analysis of C. paliurus phenolics at the same time. Animal resources, management, and the development of diabetes were the same as our previous study, following the guidelines and policy set forth by the Chinese Experimental Animals Administration Legislation (Ethical Protocol #SL-008-01) [4]. Diabetic mice were then treated with C. paliurus extract from each of the five locations (JZS, MC, WF, MW, and SN), respectively (8 g/kg body weight once a day, n = 6). Diabetic mice treated with distilled water (10 mL/kg body weight) or Metformin hydrochloride tablets (250 mg/kg body weight) were defined as negative (DC) and positive (PC) control groups (n = 6), respectively [39]. Meanwhile, normal mice treated with distilled water (10 mL//kg body weight) was defined as the normal control (NC) group (n = 6).

Biochemical Assay
Animals in each group of 2.5.1 were administered by gavage with a basal control diet once a day for 28 days. Blood samples were then collected from the abdominal aortic at the end of the trial, and levels of SOD, GSH-px, and MDA content were then measured using commercial kits.

Data Analysis
Statistical analysis was performed using Duncan multiple range tests (SPSS, Chicago, IL, USA, p < 0.05). The impact of geographical and environmental factors on phenolic variability was measured using CCA (multivariate statistical package (MVSP) V5.2). To assess the variability of antioxidant activities of phenolics among sampling locations, we used PCA based on matrix-linking values of the antioxidant index, both in vitro and in vivo, to different locations.

Conclusions
Our results confirmed the hypothesis that geographical location significantly affects the phenolic composition and antioxidant activities of C. paliurus extracts, and the magnitude of antioxidant activities mainly depends on some key phenolic components in the extracts. The highest antioxidant activity in vitro was observed from the Wufeng location, and the lowest activity was observed from the Suining region. Results also demonstrated that variation in the antioxidant activities of C. paliurus phenolics in vivo was significant among different locations, and the best effect was observed from the JZS location. Correlation analysis revealed that quercetin-3-O-glucuronide and quercetin-3-O-rhamnoside, and kaempferol-3-O-glucoside and kaempferol-3-O-rhamnoside might be responsible for the antioxidant capacity both in vitro and in vivo, respectively. These results suggested the importance of growing environment selection for better use of this plant in pharmaceutical and food industries. To determine the specific compounds responsible for antioxidant activities, isolation, purification, and bioactivity analysis of C. paliurus phenolics should be carried out in the future.
Supplementary Materials: The following are available online. Figure S1: the representative HPLC chromatogram of 80% ethanol extract of the C. paliurus leaves collected from (a) JZS and (b) mixed standards.
Author Contributions: S.F. and X.F. conceived and designed the experiments; X.S. collected the leaf samples; Y.L. performed the experiments, analyzed the data, and wrote the manuscript; P.C. and M.Z. helped perform the experiment; S.F. and T.W. revised the manuscript.
Funding: This research received no external funding.