Resource evaluation and novel germplasm excavation of wild Chinese prickly ash in Qinling mountains

Wild Chinese prickly ash with elevated antioxidants is a valuable genetic resource for Zanthoxylum bungeanum Maxim improvement. There are rich wild germplasm resources in the Qinling Mountains. In a study with wild germplasm resources from different altitudes and six cultivated varieties, the phenolic and flavonoid compounds were analyzed by high performance liquid chromatography (HPLC). The chromatograms of them were basically the same, although their chemical composition content was greatly different. The thirty samples were divided into three categories through the hierarchical clustering analysis. And catechin, hyperoside and quercitrin were considered to be key compound for the quality evaluation, by contrast, the wild samples with an altitude of 2300±50 m (IV group) had the highest content of key compounds, and showed stronger antioxidant activity and antibacterial ability, indicating that these wild samples could be used as an excellent breeding resource. This is the first time to evaluate the quality of wild Chinese prickly ash in different altitude areas of Qinling Mountains. These excellent wild germplasm resources provided substantial potential accessions for use directly in Chinese prickly ash breeding programs.

Qinling mountains are the abundant sources for wild Chinese prickly ashes, which provides an excellent source to promote the cultivation. A survey of the literature and additional work indicated that wild Chinese prickly ashes had be found at the altitudes of 1300-2500 m above sea level, which grew mainly along streams, in low forest or in stone heap. Unfortunately, the wild Chinese prickly ash resources are abundant and distributed widely in Qinling mountains, but there lacks a fine cultivar and strain, and their system breeding work has not been started. The studies of identification, preservation, and quality evaluation for wild Chinese prickly ash resources are of special importance.
Traditionally, only a few markers or bioactive components were used to assess the authenticity and quality of the medicinal germplasm resources. 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 [11][12][13]. However, the traditional methods used for quality control of herbal medicines are inefficient, and a more efficient and accurate method is needed [14,15]. HPLC fingerprinting combined with chemometrics is a comprehensive and quantifiable identification method, and has been successfully applied to food and crop analysis [16,17]. It is, therefore, desirable to determine a reliable and accurate methodology to differentiate the samples collected from this area.
In this study, we aimed to adopt HPLC fingerprint technology combined with multivariate statistical methods to establish an effective set of CPP classification and quality evaluation of wild Chinese prickly ash samples collected at different altitudes in Qinling mountains, and understand how wild Chinese prickly ash differ from cultivated. The primary objective of this study was to characterize the flavonoids and phenolic compounds in wild clones and cultivated varieties, and screen out the excellent germplasm resources containing special physiological active ingredients, which provided valuable reference for the further research and improvement program Chinese prickly ash.  Table 1). With the prerequisite of protecting the local germplasm resources and ecological environment, representative plant samples were collected in replicates of three at each site, with a distance of more than 50 m between any two plants. All samples were collected in July to August 2020.

Materials and methods
Those wild samples were authenticated by Professor Zhenhai Wu (College of Life Sciences, Northwest Agriculture and Forestry University), and all voucher specimens were deposited at the College of Sciences, Northwest Agriculture and Forestry University, Yangling, China. The peels with no signs of mechanical damage or disease were dried in an oven at 45 °C until they reached a constant weight.

Sample preparations
The dried peels were pulverized and sieved through a no. 60 mesh (< 0.250 mm). Each sample was accurately weighed at 1.0 g and extracted with 30 mL of 80% methanol at 50 ℃ for 40 min by ultrasonication and then centrifuged at 15000 rpm for 10 min. The supernatant was filtered through a 0.22μm membrane before injection.

Determination of total flavonoid content and total phenolic content
The total phenolic content of sample was detected by photometric method using Folin-Ciocalteu reagent assay [18]. Gallic acid standard solution (0 to 5 mg/L) was used for constructing the calibration curve (Y=0.0939X-0.0055, R 2 =0.9991). Total flavonoid content was determined using NaNO2-Al (NO3)3 coloration assay [19]. The total flavonoid content was calculated by rutinum standard curve (Y=0.0184X+0.001, R 2 =0.9995) of different concentrations (0.0-20.0 mg/L). The results of total phenolic content and total flavonoid content were calculated as gallic acid equivalents per 100 grams of dry extract and rutinum equivalents per 100 grams of dried extracts, respectively.

HPLC analysis of eight compounds
The quantitative analysis of eight effective components in Chinese prickly ash peels was carried out by HPLC. Chromatographic separation was achieved on a Xterra MS C18 column (5µm, 4.6 × 250 mm, Agilent Technologies Inc., Santa Clara, CA, USA). The gradient elution system is consisted of (A)  Table S1. All the relative standard deviation (RSD) values were less than 2.34%, which indicated that this HPLC method was stable and reliable.

Determination of antioxidant activity.
The DPPH radical scavenging capacities were measured using the methods described previously [20]. The Trolox and 80% methanol were used as positive control and control blank. interpreted as a concentration of the sample at a DPPH radical scavenging rate of 50%, and was inversely proportional to the DPPH radical scavenging ability.
Ferric reducing antioxidant power (FRAP) activity was determined using the protocol of Benzie & Strain with some modifications [21]. The results of FRAP activity were expressed in terms of micromoles Trolox equivalent per gram dry extract weight (mmol equiv. Trolox/g). A higher FRAP value represents a stronger antioxidant activity.
The ABTS inhibitory ability was evaluated using the method of previous study [22]. One milligram per milliliter sample was reacted with ABTS•+ solution at 37 °C for 10 min. After that, the absorbance was measured at 734 nm. Trolox standard solution (0 to 600 µmol/L) was used for constructing a standard curve. The ABTS inhibitory ability was expressed as micromoles of Trolox equiv. per grams.

Determination of antibacterial activity
This study selected Candida Albicans (C.Albicans) as the representative of the fungi, Staphylococcus aureus (S. aureus) as the bacteria representative to determine the antibacterial activity .
Antibacterial activity was tested using filter paper diffusion method. All the CPP extracts were diluted to 0.5, 0.8, 1.1, 1.4 and 1.7 mg/L with 80% methanol, using 80% methanol solution as blank control, and then soaked in a circular white filter for 24 h with a size of 5 mm×5 mm.
The C. Albicans was cultured on rose-bengal medium, and the S. aureus was cultured on Buffer protein hydrophobic (BP) medium at 37 °C. In the ultra-clean bench, 0.5 mL of 10 6 cfu/mL-10 7 cfu/mL of bacterial fluid was absorbed to coat each plate, and then sterilized filter paper discs soaked in crude extracts were placed on the solidified petri dish surface. All strains were tested with 5 replicates and cultured in Biochemical incubator for 24 h at 37 ℃. The diameter of the inhibition zone (DIZ) was measured using a cursor caliper using the cross-patch method, and the inhibition rate was calculated based on the diameter of the inhibition zone. Finally, the minimum inhibitory concentration (MIC50) was calculated according to the inhibition rate and the corresponding crude extracts concentration.

Statistical analysis
Chemometric analyses, such as hierarchical cluster analysis, principal component analysis, were performed to systematically analyze the difference of the flavonoids and phenolic compounds between wild and cultivated varieties peels. Hierarchical cluster analysis and principal component analysis were generated using the Origin software for statistical and computing (Origin Pro 2020b, Origin Lab, USA).
The significant difference was calculated by SPSS one-way ANOVA followed by Duncan's test, values <0.05 were considered to be significant (SPSS 24.0 for Windows, SPSS Inc., Chicago, IL, USA).

Total flavonoids contents (TFC) and total phenolic contents (TPC)
As shown in

Quantification of the bioactive metabolites of Chinese prickly ash
The reliable and replicable HPLC method was used to simultaneously determine eight flavonoid and phenolic components in 30 populations of Chinese prickly ash peels ( Fig.2(C)). The compounds were extracted and analyzed in triplicate (summarized in Table 2). Significant differences (P < 0.05) were observed in the contents of the eight active ingredients of Chinese prickly ash peels. The contents of hyperoside, quercitrin and catechin were significantly higher than that of other substances, and they were the main compounds of CPP at different altitudes, which was consistent with our previous research results. The  content was even higher than that of cultivars, which indicated that the group IV might be the excellent wild germplasm resource.

HCA and PCA analysis
Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were performed, so as to analyze the correlation between multiple compounds of Chinese prickly ash peels. The HCA was used to sort samples into groups by applying the inter-group connection, which used the pearson correlation as the measurement standard. The Z-score method was used to standardize the related variables to obtain the clustering diagram. The results of the HCA were shown in Fig. 3(A). It was clear that the thirteen varieties were classified into three clusters (C Ⅰ-C Ⅲ). Firstly, the C Ⅰ consists of group I, group II and group III. Both of them were wild samples and the TFC and TPC were lower than the remaining samples. Samples S25 ('Hancheng Dahongpao'), S26 ('Hancheng Shizitou') and S30 ('Xiaohongguan') were grouped together in C Ⅲ . Both of them came from Shaanxi Province and the content of YLU, YKP, YQI and YCA were higher than that of all other samples. Samples S19, S20, S21, S22, S23, S24, S27 ('Fengxian Dahongpao'), S28 ('Qin'an Yihao') and S29 ('Wudu Dahongpao') were clustered into a larger group (C Ⅱ) and the total flavonoid contents and total phenolic contents in these samples was higher. Besides, the content of YHY, YQI, YC and YCA were close. According to these results, the clustering results were closely related to the concentration of secondary metabolites and the quality of Chinese prickly ashes.
In the PCA, three principal components were constructed, the first three main components (PC1, PC2, and PC3) was 63.790%, 14.016%, 12.281% respectively, and the cumulative contributory ratio was 90.087%, indicating that the PC1, PC2, and PC3 could reflect the most information on raw data. As the first two principal components represented 77.806% of the total variance, two-dimensional score plots were generated (Fig.3(B)), in which each sample is represented by a marker. In the 2D score plot, the tested samples were separated as relatively independent clusters on PC1, PC2 and PC3, which were approximately in accordance with the HCA.  (Fig. 3 (C)). Therefore, YHY, YC and YQI 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 the contribution rate of PC1 was higher than that of PC2 and PC3, it was inferred that the varieties in the first quadrant of the 2D scoring map had high quality. Therefore, group IV, S25 ('Hancheng Dahongpao'), S26 ('Hancheng Shizitou') and S30 ('Xiaohongguan') were rated as the best variety, but the quality of group II and group III were the last, which was in agreement with the results of flavonoids compositions. It meant that these samples of group IV were the excellent wild germplasm resource and were thus screened for further analysis.

Dimensionality reduction analysis
In order to simplify the analysis and reduce the identification difficulty, three key chemical compounds with higher loading values such as YHY, YQI, and YC were selected for dimensionality reduction analysis. The PCA results after dimension reduction are shown in Fig. 3 (D). Compared with the common pattern with eight compounds, the analysis results were consistent. It meant that reducing characteristic peaks from eight to three was still satisfactory for analytical effects. That was to say, YHY, YQI, and YC could be optimized as markers of different locations and all of them might be suitable for evaluating the quality of Chinese prickly ash.  PCA on 3D map after dimensionality reduction.

Antioxidant activity and antibacterial activity
In order to further evaluate the quality of wild germplasm resources, we measured the antioxidant capacity and antibacterial ability, and compared with the six cultivated varieties.

Antioxidant activity
The antioxidant activity of Chinese prickly ashes peels was evaluated by integrating the results of these three methods (DPPH, ABTS, FRAP). According to μmol Trolox/g). Among them, the antioxidant activity of CPP at the altitude of 1700±50m was lower than that of other varieties. However, the samples (S19, S20, S21, S22, S23, S24) exhibited stronger antioxidant activity than the remaining wild samples, and exceeded cultivated varieties S28 ('Qin'an Yihao') and S29 ('Wudu Dahongpao'). In addition, the samples with strong antioxidant activity may be related to the high contents of the key compounds (YHY, YC and YQI). 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).

Antibacterial activity
The antibacterial activity of 30 crude extracts of CPP against C. Albicans and S. aureus was studied by paper disc diffusion method. The size of the antibacterial ring was measured by the cursor caliper using the cross-method, and the antibacterial rate was calculated according to the diameter of the anti-  activity. The antioxidant activity and antibacterial activity were positively related to the TFC, indicating that antibacterial activity was closely related to the content of active substances. These results suggested that Chinese prickly ashes of group IV can be used as the potential germplasm resources for breeding.

Discussion
Crop wild relatives are an invaluable reservoir of productivity enhancement related characters having resilience to climate change and farming system, and are source of novel traits [23]. The accurate evaluation of crop wild relatives is a prerequisite for identifying interested target traits, and then they are infiltrated into the background of cultivated varieties to improve genetic gains [24][25][26]. Any new varieties are modified, machined, and improved on the basis of the original plant resources through the methods of selection, hybridization, backcrossing, and mutation [27,28]. The precise evaluation of crop wild relatives is pre-requisite to identify target traits of interest followed by their introgression into the background of cultivated varieties for enhancing genetic gains [9,29]. with hierarchical cluster analysis, and chose excellent sources from these wild resources [32]. A wide range of variation in wild Chinese prickly ash accessions was observed against the target traits flavonoids, antioxidant activity and antibacterial activity suggesting diverse genetic makeup and geographical origins of wild Chinese prickly ashes collections.
The wild germplasm resources in high altitude areas are known to be resistant against major biotic and abiotic stresses [33]. Those promising resources belonging to different taxa and ecological niches may be useful accessions to enhance genetic gains of cultivated varieties. Collection of new germplasm in stress-prone areas will augment the sources of new genes for abiotic stress tolerance in developing stress-tolerant varieties [27,34]. Screening germplasm resources rich in flavonoids with strong resistance can effectively improve the medicinal value of Chinese prickly ash pericarp and provide high-quality resources for the cultivation of Chinese prickly ash varieties.
The flavonoids and phenolic compounds are the important bioactive component of Chinese prickly ash pericarps, and its content determines its medicinal value and quality [35,36]. From the results, it can be seen that the content of flavonoids of CPP varied greatly with the change of altitude. The TFC of group Ⅳ (2300±50 m) was significantly higher than those at other altitudes. Through the comparison of wild samples with the cultivated varieties, it was found that the content of quercetin and catechin of wild samples at the altitude of 2300±50m was close to that of cultivated varieties, and the content of In our study, these wild samples at the altitude of 2300±50m can be used in breeding programs to produce cultivars with high antioxidant properties. The germplasm resources rich in flavonoids and phenolic compounds are particularly valuable, and in the next step, the flavonoids content traits should be mapped to find QTLs closely linked to the flavone content traits, providing a theoretical basis for fine mapping and cloning in the future, which has far-reaching significance for the quality breeding and improvement of Chinese prickly ash.

Conclusions
In the present study, the HPLC fingerprint analysis method for flavonoids and phenolic compounds of Chinese prickly ash was established, and 24 batches of Chinese prickly ash from different altitude in