Acer Buergerianum as a New Resource for the Extraction of Corilagin: Determination of Corilaginin Different Cultivars of A. Buergerianum by UPLC

: Corilagin is a promising anticancer drug candidate. Acer nikoense and A. ammonium are both known to contain corilagin. To explore whether corilagin occurs in congeneric species and to fully develop the medicinal value of maple, the corilagin content in different varieties of maple was determined by Ultrahigh-Performance Liquid Chromatography (UPLC). Acer buergerianum leaves contained corilagin and the corilagin content in leaves of different varieties at the deciduous stage varied from 1.2371 to 6.8887 mg/g, with an average of 4.3791 mg/g. Among the cultivars, ‘Xingwang’ and ‘Qianlihong' showed relatively high corilagin contents and the content in ‘Qilujin’ was the lowest. The range of corilagin contents at the new leaf stage was 8.7452~11.2441 mg/g. Many parts of A. buergerianum contained corilagin, with the highest content in the leaves; based on this considerable corilagin concentration, this plant can be developed into raw material for corilagin extraction. There are large differences among varieties. To increase the output value and efficiency, it is necessary to further screen high-content varieties for industrial production; harvesting can be performed in spring and autumn, with autumn as the main season and spring as a supplemental season.


Introduction
Corilagin,-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-d-glucose, a water-soluble tannin, has a variety of pharmacological activities, including antitumor, antioxidant, antiatherosclerotic, antifibrinolytic, blood pressure-lowering, antiviral, antibacterial andantiinflammatory activities (Yeo et al., 2015, Li et al., 2018aHuang et al., 2013;Wu et al., 2021), especially antitumor activity. Studies have shown that this compound has a significant inhibitory effect on the cell growth of cancers such as liver cancer, cholangiocarcinoma, ovarian cancer, cervical cancer, and gastric cancer (Ming et al., 2013;Gu et al., 2016;Attar et al., 2017). In addition, corilagin has low toxicity to normal human cells and tissues. Therefore, this tannin has become a promising anticancer drug candidate. In addition, it has been reported in the latest research that corilagin can be used as a small molecule to interfere with the interaction between the new coronavirus (SARS-CoV-2) and host cells (Hanson et al., 2020). At present, corilagin is mainly extracted from plants. Schmidt and Lademann (1951) were the first to isolate corilagin from a plant, Caesalpinia coriaria, and identified it as tannin. To date, corilagin has been identified in 15 families and 50 species of plants (Li et al, 2018b),mainly in the families Euphorbiaceae (19 species), Geraniaceae (10 species), and Combretaceae (7 species), including, Phyllanthus emblica (Liu et al., 2020), Phyllanthus urinaria (Fan, 2016), Mallotus japonicus (Tabata et al., 2010), 206 Erodium stephanianum, Erodium cicutarium (Fecka and Cisowski., 2015), Geranium carolinianum, Geranium sibiricum, Geranium thunbergii, Geranium wilfordii (Bastian et al., 2018), Terminalia catappa (Kinoshita et al., 2007), Terminalia bellirica and Terminalia chebula (Avula et al., 2013). Longan and rambutan in the Sapindusgenus (Rakariyatham et al., 2020) and Acer nikoense and Acer amoenum in the Acer genus have also been identified as containing corilagin (Okabe et al., 2001;Honma et al., 2010). Since the only known synthetic pathway of gallic acid, an important precursor for the synthesis of corilagin, is the shikimic acid pathway (Werner et al., 1997), the entire biosynthesis pathway of corilagin is not clear and artificial synthesis cannot be carried out at present. Therefore, it is of great significance to continue to expand the exploration of raw materials of corilagin. In particular, the abovementioned families and genera have been reported to contain corilagin.
Acer buergerianum is a deciduous tree of the Sapindaceae family (Acer); it is native to China and distributed in the middle and lower reaches of the Yangtze River. It is often used as an autumn leaf tree in garden applications and as sidewalk trees, garden trees, and hedges (Xu et al., 2017). In recent years, its medicinal and food value has been explored. Studies have shown that its seeds can be pressed into oil. The seed oil content can reach 30.56%, with a nervonic acid content of approximately 6% and it contains 21.85% protein and 36.07% vitamin E. The mature leaves are very richin volatile components. In particular, many of these chemical components have aromatic or pharmacological effects and have applicationpotential in the fields of flavors and fragrances or medical treatment (Li et al., 2018c), but they have not been fully developed for use. The leaves of A. nikoense and A. amoenum are known to contain corilagin (Okabe et al., 2001;Honma et al., 2010). Whether A. buergerianum contains corilagin has not been reported. With the increasing demand for corilagin, it is necessary to identify high corilagin-content plants. Thus, it is of great significance to explore whether there are high corilagin-content Acer species. Furthermore, in recent years, as the'slow sweep of fallen leaves' has become popular, urban management has begun to pay attention to the landscape of fallen leaves, which adds natural beauty to the city in autumn. However, fallen leaves should not only be used for viewing but also be developed concerning their economic value to turn fallen leaves into "treasures". As an important garden landscape tree species, A. buergerianum has concentrated autumn leaves. It is thus meaningful to make full use of fallen leaves to create added value in the garden. Accordingly, this study uses A. buergerianum as a trial harvest plant and analyzes the presence and contents of corilagin in different varieties, harvest periods, and tree parts. The aim is to explore the potential of A. buergerianum as a raw material for corilagin extraction and to provide a scientific basis for the development of the medicinal value of A. buergerianum.

Plant Materials
Sampling materials: Three A. buergerianum cultivars were used in this study: 'Xingwang', 'Qianlihong' and 'Qilujin'. The sampling timesinthe defoliation period were as follows: October 27 and November 6, 15, and 21, 2019. Three plants of each variety were selected andhealthy, disease-free mature leaves were sampled and transported to the laboratory for drying at 50°C, followed by milling. In the new leaf stage, shoots and tender leaves were collected twice, specifically, on April 15 and 20, 2020, and the samples from the two dates were mixed, dried, and ground. Collection from different parts of A. buergerianum tetraploid 'Xingwang': Samples of leaves, petioles, shoots, bark, pericarp, and seeds of 'Xingwang' were collected on August 25, 2020. Three plants were selected at random and all the materials were healthy and undamaged. The materials were brought to the laboratory, dried, and ground.

Solution Preparation
Reference substance solution. A total of 5.00 mg of corilagin standard product (purity ≥98%) was accurately weighed, dissolved in 50% methanol (chromatographically pure) in a 50 mL volumetric flask, and diluted to the mark and shaken well. In this way, a reference solution containing 0.10 mg/mL corilagin was prepared.
Test solution. In an extraction method modified by Li et al. (2018a), approximately 1.00 g of sample powder was accurately weighed and placed in a 100 mL conical flask. Then, 50 mL of 50% ethanol aqueous solution (analytical purity) with a volume fraction of 50%was added and ultrasonic extraction with an ultrasound instrument (kQ-250DE; power, 500 W; frequency, 40 kHz) was performed for 30 min. Next, the supernatant was collected and evaporated with a rotary evaporator (RE-2000A). The solid powder was dissolved in 50 mL of 50% methanol and the sample solution was passed through a 0.22 µm filter membrane for testing.

Linear Relationship Investigation
The reference solution was diluted with 50% methanol, a concentration gradient of 0.005, 0.01, 0.025, 0.05, 0.10 and 0.20 mg/mL solutions was prepared and sample injection and determination were performed under the chromatographic conditions described above. With the concentration of corilagin as the abscissa and the chromatographic peak area as the ordinate, a standard curve was drawn and regression analysis was performed.

Methodological Review
Precision test: From the reference solution, 6 consecutive sample injections under the chromatographic conditions of 'Chromatographic conditions' were performed, the peak area was recorded and the Relative Standard Deviation (RSD) was calculated.
Repeatability test: Six samples of the 'Qianlihong' test solution were analyzed under chromatographic conditions described in 'Chromatographic conditions'. The peak area was recorded and the RSD was calculated.
Stability test: Every 2 h, 2 μL of the reference solution was injected. The peak area was recorded 5 times continuously according to the chromatographic conditions described in 'Chromatographic conditions' and the RSD were calculated.
Sample recovery test: The powder of the 'Qianlihong' sample was accurately weighed and 1.410 mg of corilagin standard was added to a total of 5 parts; the samples were prepared according to the methods of 'Solution preparation' and measured under the chromatographic conditions described in 'Chromatographic conditions'. The chromatogram was recorded and the sample recovery rate and RSD were calculated.

Sample Determination
Three replicates of each sample powder, each approximately 1.0 g, were precisely weighed to prepare a sample solution and measured according to the chromatographic conditions described in 'Chromatographic conditions'.

Determination of A. Buergerianum Leaf Corilagin
The UPLC method was used to detect corilagin in the leaves of A. buergerianum. Chromatograms are shown in Fig. 1. A comparison of the retention times of the sample and corilagin standard revealed that the leaves of A. buergerianum contained corilagin.  The linear relationship under the test conditions was investigated and the external standard method was used to quantitatively analyze the corilagin (Fig. 2). The regression equation of the standard curve is Y = 50415X + 28.168, the correlation coefficient R is 0.9993 and the linear range is 0.005-0.250 mg. In the linear range, the correlation coefficient is greater than 0.999, indicating that the corilagin standard curve has a good linear relationship.
The RSDs of the precision test, repeatability test, and stability test were 2.12, 0.92, and 0.42%, respectively, indicating that the method has good repeatability, stability and precision. The sample recovery rate reached 95.31% and the RSD was 1.45% (Table 1), which confirms that this method has good accuracy in detecting corilagin. The UPLC method established in this experiment can be used to determinethe corilagin content in A. buergerianum leaves.

Changes in Leaf Corilagin Content in Different A. Buergerianum Cultivars at the Deciduous Stage
The leaf corilagin contents of different varieties of A. buergerianum at the deciduous stage are shown in Table 2. The corilagin content ranged from 1.2371 to 6.8887 mg/g, with an average of 4.3791 mg/g. In 'Xingwang', the highest corilagin content was observed on November 6, at 6.4435 mg/g and the average content over the deciduous period was 5.4707 mg/g; in 'Qianlihong' the highest content was found on October 27, at 6.8887 mg/g and the average content over the deciduous period was 5.6951 mg/g. The highest content in 'Qilujin', at 3.3204 mg/g, was found on October 27, with lower contents detected for later dates and the average content in the defoliation stage was 1.9714 mg/g. The results showed that the contents of corilagin in 'Xingwang' and 'Qianlihong' were significantly higher than that in 'Qilujin'. The CV ranged from 27.99~65.90%, indicating that the content of corilagin was quite variable during the deciduous period.

Corilagin Content in the Leaves of Different Varieties of A. Buergerianum at the New Leaf Stage
The leaf content of corilagin at the new leaf stage was significantly different among the three A. buergerianum cultivars, with 'Xingwang' having the highest value, 11.2441 mg/g, followed by 'Qiluhong', with 10.2752 mg/g and 'Qianlihong', with the lowest value of 8.7452 mg/g (Fig. 3). Compared with that in the deciduous stage (Table 2), the content of the new leaf stage was significantly higher. The new leaf stage of A. buergerianum is thus more suitable for harvesting for the extraction of corilagin. Of course, we must also pay attention to the differences among varieties and select high-content germplasms.

Corilagin Content in Different Parts of 'Xingwang'
Taking the higher content of 'Xingwang' as a trial harvest,the corilagin content in different plant parts of thisvariety was measured. The results showed that the amount significantly differed among the different parts (Fig. 4). The petiole had the highest content, 6.3878 mg/g, which was similar to the leaf content (6.2267 mg/g), so it is recommended that the leaves be harvested with the petiole for corilagin extraction.  The content in the peel was 2.6472 mg/g, which indicates that the peel can also be used for extracting corilagin. Generally, the peel is treated as waste during oil extraction. This experiment shows that the peel can be used to extract corilagin, which can save resources and fully exploit the medicinal value of the peel. The twigs, bark, and seed kernels had low corilagin contents. As these parts are important parts for plant growth or oil extraction, they should not be used as raw materials for corilagin extraction.

Discussion
In this study, a suitable UPLC method was established to determine corilagin contents in f A. buergerianum and it was demonstrated for the first time that the leaves, petioles, twigs, bark, and seeds of this plant contain corilagin. This method provides a basis for the identification and determination of corilagin in other species of the genus Acer. The method provides only a preliminary determination of the corilagincontent in A. buergerianum. In actual production applications, extracted corilagin needs to be separated and purified. In addition, it is necessary to optimize the method to achieve a higher extraction rate. Rangsriwong et al. (2009) used subcritical water extraction to extract corilagin from Terminalia chebula Retz. The extraction technology of this method is green, the extraction rate is high, the cost is low and selective extraction can be achieved. Prasad et al. (2009) used high-pressure extraction technology to extract longan fruit corilagin under conditions of 30℃, 50% ethanol, 1:50 (w/v) solidto-liquid ratio and 500 MPa for 2.5 min, and the extraction rate was increased by 22% compared with that using ultrasonic extraction. Therefore, different processes to extract corilagin from A. buergerianum as the raw material need to be explored.
The corilagin content in the deciduous stage was quite different among the three A. buergerianum cultivars in this study and the contents of 'Xingwang' and 'Qianlihong' were significantly higher than that of 'Qilujin'. The content ranged between 1.2371 and 6.8887 mg/g across the three varieties, with an average of 4.3791 mg/g;this value is much higher than that reported for other species from the same genus, A. nikoense and A. amoenum (Okabe et al., 2001;Honma et al., 2010), as well as forGeranium sibiricum (0.59 mg/g) of Geranium (Liu et al., 2020) and Dimocarpus longan (1.1726 mg/g) of Dimocarpus (Li et al., 2018b). However, the corilagin content of A. buergerianumis lower than that of Phyllanthus emblica leaves (13.16-28.76 mg/g) (Liu et al., 2020) and the young fruit of Terminalia chebula (10.36-12.70 mg/g) (Avula et al., 2013). Rambutan (3.80 mg/g) (Rakariyatham et al., 2020), Phyllanthus urinaria L. (2.288 mg/g) (Fan, 2016), Geranium wilfordii Maxim (2.0 mg/g) and Geranium carolinianum (4.10 mg/g) (Bastian et al., 2018), Erodium cicutarium (6.5 mg/g) (Fecka and Cisowski, 2015) and Terminalia catappa (6 mg/g) (Kinoshita et al., 2007) exhibit similar corilagin contents as A. buergerianum. The results of this study show that it is feasible to extract corilagin from the autumn leaves of A. buergerianum. In addition, although the leaf corilagin content of A. buergerianum is higher in the new leaf stage than in the deciduous stage, the new leaf stage is not suitable for a large number of leaf picks since the leaves are very important for the growth of the tree; thus, an appropriate numberof young leaves can be harvested in early spring. In industrial corilagin production using A. buergerianum leaves as raw materials, the leaves can be harvested in spring and autumn, with autumn as the main season and spring as a supplement.
The determination of corilagin content in different parts of 'Xingwang' showed that the leaves and petioles had the highest content, above 6 mg/g, followed by 210 husks; the lowest contents were in kernels. In other plants that have been reported to contain corilagin, the contentsare usually highest in leaves. For example, in longan, leaves contain the highest corilagin contents, followed by twigs, seeds, pericarp, bark, and pulp. Rambutan, Jatropha, Phyllanthus, and geranium also have very high leaf corilagin contents (Li et al., 2018c;Fan, 2016;Manpong et al., 2011;Rakariyatham et al., 2020). Whole plants and fruits are alsoused as the main raw materials for corilagin extraction; examples include wild geranium (whole plant), celery leaf ox seedlings (whole plant), and Terminalia chebula (fruit) (Fecka and Cisowski, 2015;Avula et al., 2013;Li et al., 2018a). In general, leaves are the main raw material used to extract corilagin.
According to a recent report (Li et al., 2018b), the plants containing corilagin are mainly concentrated in the families Euphorbiaceae (19 species), Geraniaceae (10 species), Combretaceae (7 species), and Sapindaceae (5 species). There are many plants in these families; there are more than 200 species of Acer in Sapindaceae alone. Therefore, we believe that these families and related plants have great potential as raw materials for the extraction of corilagin, which is the direction of our next work.

Conclusion
A. buergerianum leaf has potential as a material for the extraction of corilagin because of its high corilagin content. Petiole and pericarp of this plant could also be developed as raw material for extracting corilagin. There were great differences among the different varieties in this study. The contents of 'Xingwang' and 'Qianlihong' were higher than those of 'Qilujin' in the deciduous stage, whereas the contents of 'Xingwang' and 'Qiluhong' were higher than those of 'Qianlihong' in the new leaf stage. To increase the output value and efficiency, it is necessary to further screen high-content varieties for industrial production. There was a great difference in corilagin content between the different seasons, with that in the deciduous stage being significantly lower than that in the new leaf stage (4.3791 mg/g and 10.0881 mg/g, respectively). The harvest period can be divided into spring and autumn, with autumn as the main season and spring as a supplement.

Practical Application
Many parts of Acer buergerianum contain corilagin and the leaves contain the highest content, with a considerable amount, which can be developed into raw material for extracting corilagin.