Jujube leaf tea, which is made from the young leaves of Ziziphus jujuba, is a novel functional herb tea or infusion that inhibits the central nervous system. In the current study, the effects of iminodisuccinic acid (IDS), as a metal complexing agent, on mineral element content, oxidative damage, antioxidant enzyme activities, and antioxidant accumulation in the young and mature leaves of Z. jujuba were investigated. Results demonstrated that foliar fertilization with ionic (FeCl2 and ZnCl2) and chelated (Fe-IDS and Zn-IDS) fertilizers could drastically enhance iron and zinc contents, coupled with increased vitamin C level, glutathione accumulation, total phenolic content, and total antioxidant capacity (evaluated based on the Fe3+ reducing power of leaf extracts), compared with the control, particularly in young leaves. However, chelated fertilizers considerably reduced the chlorophyll level, H2O2 content, and lipid peroxidation rate than ionic fertilizers, particularly in young leaves. Compared with the control, chelated fertilizers induced greater superoxide dismutase and catalase activities, particularly in young leaves. Moreover, decreased enzyme activities were observed in the ionic fertilizer-treated leaves compared with the control-treated leaves. Thus, using a chelating agent could improve the accumulation of mineral elements and antioxidants in young leaves by reducing metal-mediated reactive oxygen species toxicity.
Humans consume vegetables and fruits daily because they provide abundant antioxidants, such as vitamin C (Vc), glutathione (GSH), polyphenols, and minerals [1,2]. Moreover, herbal infusions or tea have similar functions [3,4,5,6]. Chinese jujube (Ziziphus jujuba Mill.) is a thorny rhamnaceous plant, which has been found in China for more than 7,000 years. Because its fruit has a high nutritional value, it has been used as a traditional medicine for centuries [7,8,9]. Furthermore, jujube leaves have similar functions and are used to prepare bedtime infusions or jujube leaf tea . Most tea aficionados have to refrain from drinking tea infusions at night to avoid insomnia. However, a jujube leaf tea can be beneficial for aiding sleep because it nourishes the heart, soothes the nerves, and controls blood pressure . Therefore, jujube leaf tea has recently gained much attention in China and has occupied a substantial market share in the health tea market [11,12,13]. Jujube leaves have high phenolics and mineral elements (e.g. Fe) [14,15].
Vc and GSH are two well-known antioxidants beneficial for human health [16,17]. A correlation was observed between the antioxidant activity and the phenolic acid content of the leaves in various plants . Moreover, polyphenols are the most abundant antioxidants present in foods and beverages sourced from plants, such as tea and coffee . Their total dietary intake could be as high as 1 g per day, much higher than all other known dietary antioxidants . Furthermore, polyphenols were the major bioactive components in jujube leaves . In addition, mineral elements are indispensable for human nutrition. For example, iron and zinc are the two most abundant trace minerals in the human body, with 3–4 g of Fe and 1.5–2.5 g of Zn present in an average adult [22,23]. However, mineral elements (e.g. Fe and Zn) are present in low crop quantities during their growth and development .
Jujube leaf tea is made from young leaves . However, young leaves contain low amounts of minerals (e.g. Ca and K) and phytochemicals than mature ones [26,27]. Iminodisuccinic acid (IDS), a biodegradable chelator, has an excellent heavy metal ion complexation property and has low environmental toxicity, which can profoundly enhance heavy metal accumulation in plants [28,29]. Chelators can promote metal accumulation and enhance antioxidant response in plants [30,31]. For example, the inhibition of superoxide dismutase (SOD) and catalase (CAT) activities and induction of hydrogen peroxide (H2O2) production by excess Cu can be significantly reversed by ethylenediaminetetraacetic acid (EDTA) in Brassica napus L. . However, the mechanism by which foliar fertilizers in ionic (e.g. FeCl2) and chelate (e.g. Fe-IDS) forms affect the accumulation of mineral elements and antioxidants in the young and mature jujube leaves is unknown. Furthermore, the mechanism of IDS affecting the antioxidant response in jujube leaves after foliar mineral fertilization is unknown. The current study would help us improve the quality of jujube leaf tea in terms of its antioxidant content.
2 Materials and methods
2.1 Reagent preparation
All chemical reagents used in the current study were of analytical grade and were purchased from the Zhengzhou Biochemical Reagent Co., Ltd. (Zhengzhou, China). Fe-IDS was prepared by mixing an FeCl2 solution (10 mM) with an IDS-Na4 solution (50 mM) in a 1:1 molar ratio of Fe to IDS. Similarly, Zn-IDS was prepared by mixing ZnCl2 solution (10 mM) with IDS-Na4 solution (50 mM) in a 1:1 molar ratio of the metal to the chelator.
2.2 Material treatment
Winter jujube (Z. jujuba Mill., cv. “Dongzao”) leaves used in the current study were collected from the Jujube Cultivation Garden at Luoyang Normal University (N34°37′9.01″ and E112°26′50.37″), Henan Province, China. The jujube plants used were 3 years old and 150 ± 10 cm in height. The experiment was performed during the years 2020–2021. All the plants were randomly divided into groups 1 and 2. According to our preliminary experiment, all plants were sprayed with a 10 mM mineral element fertilizer. Group 1 plants were sprayed with an ionic fertilizer (10 mM FeCl2 or ZnCl2), whereas group 2 plants were sprayed with a chelate fertilizer (10 mM Fe-IDS or Zn-IDS) for 7 days. All plants were watered with tap water during this period. After 7 days, leaf samples of different age groups (second leaf from the top, young leaves; and ninth leaf from the top, mature leaves) were collected from the branches on the sunny and shady sides of the jujube tree early morning. Then, they were mixed well and transported to the laboratory within 2 h. Leaves that were uniform in shape and appearance and lacked visible defects were chosen. Each replicate contained 10 g of leaves. The leaves were then stored at −70°C for subsequent analyses of Vc, GSH, total phenolics, and chlorophyll; total antioxidant capacity; SOD and CAT activities; H2O2 content; and lipid peroxidation.
2.3 Iron and zinc assays
Jujube leaves were collected and washed with distilled water. They were then air-dried and ground into a powder using a mortar and pestle. For heavy metal extraction, digestion tubes were properly acid-washed and dried. Thereafter, 0.5 g of dry powdered samples were placed in the digestion tubes, and a 20 mL solution consisting of HClO4, H2SO4, and HNO3 (1:1:5) was added to each tube. These tubes were left for 12 h  and then placed in a digestion block at 70°C for approximately 60 min. Then, the temperature was increased slowly to 135–140°C. When digestion was complete, the solutions were cooled and then filtered with 0.45 μm filters before being diluted to 200 mL with doubly deionized water. The Fe and Zn contents of the filtrates were measured using an atomic absorption spectrometer (Analyst 700 from PerkinElmer, Waltham, MA, USA).
2.4 Total chlorophyll level assay
The total chlorophyll concentration of jujube leaves was determined spectrophotometrically using 0.1 g of the fresh sample ground with 8 mL of 80% acetone (v/v) in a prechilled mortar. After the complete extraction of the sample, the mixture was filtered, and the volume was adjusted to 10 mL with cold acetone. The absorbance of the extract was read at 663, 647, and 470 nm, and pigment concentrations were calculated according to the method of Lichtenthaler .
2.5 H2O2 and lipid peroxidation assays
The H2O2 content in fresh jujube leaves was determined as follows: 1 g of leaves was cut into pieces and homogenized in an ice bath with 10 mL of 0.1% (m/v) trichloroacetic acid. The samples were centrifuged for 15 min at 12,000g and 4°C, and 1 mL of the supernatant was added to 1 mL of 10 mM potassium phosphate buffer and 1 mL of 1 M KI. The absorbance was recorded using a spectrophotometer at 390 nm (Shimadzu, Kyoto, Japan). The H2O2 content of the sample was measured using a standard curve according to the method of Okuda et al. .
Lipid peroxidation was measured in young leaves as thiobarbituric acid reactive substances (TBARS) using Davenport et al. . The leaf extract was mixed with an equal volume of 0.5% (w/v) thiobarbituric acid containing 5% (w/v) trichloroacetic acid, and the mixture was heated at 98°C for 15 min; the absorbance was measured at 532, 600, and 450 nm after centrifugation (10 min at 12,000 rpm).
2.6 SOD and CAT activity assays
Approximately 1 g of jujube leaves was ground to powder in liquid nitrogen and homogenized in a 20 mL of ice-cold extraction buffer that consisted of 0.1 M tris-HCl (pH 7.5), 1% polyvinylpyrrolidone, 10 mM KCl, 10 mM MgCl2, and 1 mM EDTA. After homogenization and centrifugation (10,000g at 20 min) of the samples, the supernatants were collected and used for SOD and CAT assays.
For SOD activity detection, the reaction buffer, containing 13 mM methionine, 100 μM EDTA, 75 μM nitrobenzenethiol (NBT), and 2 μM riboflavin, was mixed with different volumes of an enzyme extract in 50 mM phosphate buffer (pH 7.8). Then, the mixture was exposed to light for 15 min. The increase in absorbance due to formazan formation was read at 560 nm. One unit of SOD activity was defined as the amount of enzyme that inhibits NBT photoreduction by 50% .
Each 3 mL reaction mixture was added with 150 mM potassium phosphate buffer (pH 7.0), 15 mM H2O2, and 50 μL of enzyme extract for CAT activity detection. The reaction was initiated by adding 15 mM H2O2. The activity was determined by determining H2O2 (E = 39.4 mM−1 cm−1) consumption at 240 nm for 3 min .
The soluble protein content was determined according to the method of Bradford  using bovine serum albumin as a standard.
2.7 Vc and GSH assays
The titrimetric method with 2,6-dichloro-phenol-indophenol (DCPI) with some modifications was used to assess the Vc content in jujube leaves. Briefly, 1 g of homogenized jujube leaves was mixed with 20 mL of 2% oxalic acid solution. The mixture was homogenized and then diluted to 100 mL with 2% oxalic acid solution before filtration. In total, 10 mL of the filtered solution was titrated with DCPI (0.01%, w/v). The final point was considered to be the appearance of pink colour for 15 s. Ascorbic acid solution (0.05%, w/v) was used to calibrate DCPI. The results were expressed as milligrams of ascorbic acid equivalents per gram of dry weight (mg g−1 of dry weight). The assays were completed between 10 and 15 min .
Reduced GSH was determined through the subtraction of oxidized glutathione (GSSG) from the total glutathione (GSH + GSSG) content. Total glutathione (GSH + GSSG) was measured using an enzymatic cycling assay method of Nagalakshmi and Prasad . GSSG was determined after the removal of GSH through 2-vinylpyridine derivatization. The results were expressed as milligram of GSH equivalents per gram of dry weight (mg g−1 of dry weight).
2.8 Total phenolic content assay
The total phenolic content of jujube leaf extracts was spectrophotometrically measured according to the method of Singleton and Rossi by using the Folin–Ciocalteu reagent as the reactive reagent . Homogenates were prepared as described in antioxidant activity determination, and the clear supernatant was collected and used to measure the total phenolic content. The absorbance was recorded at 760 nm by using an ultraviolet-visible (UV-vis) spectrophotometer (Shimadzu, Kyoto, Japan), and the results were expressed as gallic acid equivalents per gram of dry weight (mg g−1 of dry weight).
2.9 Total antioxidant capacity assay
Leaf extracts were prepared before determining the total antioxidant capacity (evaluated by Fe3+ reducing power). The jujube leaves were weighed and immediately quenched in liquid nitrogen. Approximately 1 g of jujube leaves was ground into a powder in liquid N2 using a mortar and pestle and then transferred to 100 mL of 70% (w/v) ethanol–water solution. The samples were maintained at room temperature for 24 h under dark conditions. The filtrates of each replicate were collected, and the solvent was discarded using a rotary evaporator under vacuum at 45°C. The obtained leaf extracts were stored at 4°C until the total antioxidant capacity analysis was used. The absorbance of the reaction mixture was recorded at 593 nm through UV-vis spectroscopy (Shimadzu, Kyoto, Japan) for the total antioxidant capacity measurement using the ferric reducing ability of the plasma assay .
2.10 Data analysis
The experiments were conducted in a completely randomized design. Three replicates were analysed for each treatment. All data were analysed using Duncan’s multiple range test (p < 0.05) in SPSS 13.0 software (IBM Corp., Armonk, NY, USA).
3 Results and analysis
3.1 Iron and zinc accumulation in jujube leaves
Foliar spraying with mineral element fertilizer significantly enhanced Fe and Zn accumulation, particularly in young leaves (p < 0.05; Table 1). Compared with the control, 10 mM FeCl2 and ZnCl2 increased Fe and Zn accumulation by approximately 66 and 108% in young leaves and 24 and 44% in mature leaves, respectively (Table 1; p < 0.05). However, no significant difference was observed between the ionic and chelated fertilizers of iron or zinc in mineral element accumulation in jujube leaves.
|Young leaves (mg 100 g−1)||Mature leaves (mg 100 g−1)|
|Control||24.3 ± 1.8a||2.5 ± 0.4a||63.2 ± 2.1a||5.2 ± 0.5a|
|FeCl2||40.4 ± 2.5b||—||78.6 ± 2.3b||—|
|Fe-IDS||38.6 ± 1.8b||—||74.7 ± 1.8b||—|
|ZnCl2||—||5.2 ± 0.3b||—||7.5 ± 0.3b|
|Zn-IDS||—||4.8 ± 0.5b||—||6.9 ± 0.4b|
Foliar spraying with mineral element fertilizer in the ionic (FeCl2 and ZnCl2) and chelate (Fe-IDS and Zn-IDS) forms on Fe and Zn content (mg 100 g−1 dry weight) were monitored in young and mature leaves of winter jujube after treatment for 7 days.
a,bMean values assigned with the same letter are not significantly different (n = 3; p < 0.05).
IDS, iminodisuccinic acid.
3.2 Oxidative damage in jujube leaves
Treatment with iron and zinc fertilizers significantly decreased the chlorophyll levels and enhanced H2O2 and TBARS (evaluated for lipid peroxidation) accumulation in jujube leaves (p < 0.05; Figure 1). Compared with the water control, foliar spraying with 10 mM FeCl2 decreased chlorophyll levels by approximately 45 and 8% in young and mature leaves, respectively (Figure 1a; p < 0.05). Similar change patterns were observed for ZnCl2. By contrast, treatment with Fe-IDS and Zn-IDS reduced the chlorophyll level by approximately 14 and 6%, respectively, compared with the young leaf water control (Figure 1a). Moreover, 10 mM ZnCl2 enhanced H2O2 and TBARS contents by approximately 44 and 107%, respectively, compared with the young leaf water control (Figure 1b and c; p < 0.05). However, treatment with a mineral element fertilizer in the chelate form can slightly alleviate oxidative damage. For example, foliar spraying with FeCl2 and Fe-IDS enhanced the TBARS content by approximately 138 and 51% in young leaves, respectively, compared with the water control (Figure 1c; p < 0.05). Similar change patterns were observed for Zn-IDS fertilizer (Figure 1; p < 0.05).
3.3 SOD and CAT activities in jujube leaves
Iron and zinc fertilizers significantly affected the SOD and CAT activities, particularly in young leaves (p < 0.05; Figure 2). For example, 10 mM FeCl2 and ZnCl2 decreased SOD activities by approximately 31 and 16%, respectively, compared with the young leaf water control (Figure 2a; p < 0.05). Similar change patterns were observed in CAT activities (Figure 2b; p < 0.05). However, treatment with Fe-IDS increased CAT activities by approximately 35 and 29% in young and mature leaves, respectively, compared with the water control (Figure 2b; p < 0.05). Similar change patterns were observed for Zn-IDS (Figure 2; p < 0.05).
3.4 Antioxidant accumulation in jujube leaves
Compared with the water control, foliar spraying with FeCl2 and ZnCl2 significantly reduced the Vc level, GSH accumulation, total phenolic content, and total antioxidant capacity of jujube leaves (Figure 3; p < 0.05). For example, foliar spraying with 10 mM FeCl2 decreased the Vc content by approximately 34 and 12% in young and mature leaves, respectively (Figure 3a; p < 0.05). Similarly, ZnCl2 treatment reduced GSH accumulation and total antioxidant capacity by approximately 37 and 8%, respectively but enhanced the total phenolic content by 6% compared with the young leaf water control (Figure 3b–d; p < 0.05). However, treatment with Fe-IDS and Zn-IDS significantly enhanced all these parameters. For example, foliar spraying with 10 mM Fe-IDS increased GSH accumulation by approximately 40 and 15% in young and mature leaves, respectively, compared with the water control (Figure 3b; p < 0.05). Similar change patterns were observed in Zn-IDS-treated jujube leaves. For example, the Zn-IDS application enhanced the total antioxidant capacity by approximately 26 and 17% in young and mature leaves, respectively, compared with the water control (Figure 3d; p < 0.05).
Foliar fertilization can efficiently improve fertilizer utilization efficiency in crops . In the present study, four mineral fertilizers (FeCl2, Fe-IDS, ZnCl2, and Zn-IDS) were foliar sprayed on jujube leaves. The mature leaves contained a higher accumulation (approximately two-fold) of Fe and Zn than did the young leaves (p < 0.05; Table 1). However, foliar spraying with 10 mM Fe and Zn fertilizers profoundly enhanced iron (by ∼60 and ∼20% for young and mature leaves, respectively) and zinc (by ∼100 and ∼40% for young and mature leaves, respectively) accumulation compared with the water control in jujube leaves after 7 days of treatment (p < 0.05). Thus, foliar fertilization can efficiently enhance mineral elements (e.g., Fe and Zn) in the young leaves of jujube. The assimilation of Fe and Zn is potentially more in the young leaves than in the mature leaves. This phenomenon could be partly attributed to the low accumulation of mineral elements in young leaves before treatment (Table 1). However, no significant difference was observed between the ionic (FeCl2 and ZnCl2) and chelate (Fe-IDS and Zn-IDS) fertilizers in the promotion of mineral element accumulation in jujube leaves (Table 1; p < 0.05). This is partly following a previous report, which showed that foliar fertilization with EDTA failed to enhance Fe accumulation compared with FeSO4 treatment in rice . However, numerous studies have shown that IDS can improve heavy metal accumulation in plants in polluted soil [29,30]. This phenomenon can be partly attributed to heavy metal exposure time differences. Thus, foliar spraying with a chelated metal of a large size cannot be assimilated by plant leaves within a short period. However, the mechanism by which a chelated fertilizer affects the antioxidant defence response in jujube leaves is unknown. Thus, oxidative damage (evaluated based on the chlorophyll level, H2O2 accumulation, and lipid peroxidation) and two key antioxidant enzyme activities (namely, SOD and CAT) were measured here .
Compared with the control, both ionic (FeCl2 and ZnCl2) and chelated (Fe-IDS and Zn-IDS) fertilizers can significantly reduce the chlorophyll level and enhance peroxide accumulation to different extents, particularly in young leaves (Figure 1; p < 0.05). For example, foliar spraying with 10 mM FeCl2 (or Fe-IDS) decreased chlorophyll accumulation by approximately 45% (or 14%) in young and 11% (or 6%) in mature leaves (Figure 1a; p < 0.05). Similar effects were observed in H2O2 and TBARS accumulation after foliar spraying (Figure 1b and c; p < 0.05). For example, foliar spraying with 10 mM ZnCl2 (or Zn-IDS) enhanced TBARS accumulation (evaluated for lipid peroxidation) by approximately 106% (or 30%) in young and 39% (or 13%) in mature leaves (Figure 1a; p < 0.05). Ionic fertilizer exhibited greater toxicity to the young leaves than the chelated fertilizer. Thus, chelated fertilizers were always applied to crop leaves [46,47]. Furthermore, the effects of ionic and chelated fertilizers on antioxidant enzyme (e.g. SOD and CAT) activities were measured, particularly in young leaves (Figure 2). Foliar spraying with FeCl2 profoundly decreased SOD (∼30%) and CAT (∼55%) activities in young leaves compared with the control (p < 0.05; Figure 2). SOD and CAT inactivation can be triggered by certain free radicals, which transition metals can produce in the presence of H2O2 . Thus, the foliar fertilizers used in the current study (FeCl2 and ZnCl2) resulted in the decline of SOD and CAT activities partly due to the metal-mediated free radical overproduction. By contrast, Fe-IDS treatment increased SOD and CAT activities by approximately 68 and 35%, respectively, compared with the control, in young leaves (Figure 2; p < 0.05). This is partly in accordance with previous reports [44,49], which showed that a chelated iron fertilizer significantly enhanced SOD and CAT activities in plants compared with the control. However, the mechanism by which the chelated fertilizer affects the nonenzymatic antioxidant (e.g. Vc and phenolics) accumulation in jujube leaves remains unknown.
The ionic and chelated fertilizers exhibited contrasting effects in regulating nonenzymatic antioxidant accumulation, particularly in young leaves (p < 0.05; Figure 3). Compared with the control, treatment with FeCl2 and ZnCl2 significantly reduced antioxidant (Vc, GSH, and phenolics) accumulation and the total antioxidant capacity, particularly in the young leaves of jujube (Figure 3; p < 0.05). This is partly in accordance with previous reports, which showed that copper and cadmium could induce oxidative damage and rapidly decrease low-molecular-compound (e.g. ascorbate and GSH) accumulation in plants [50,51]. In addition, foliar application of FeSO4 can significantly reduce the total phenolic content and total antioxidant capacity in the juice of pomegranate fruit . However, the chelated foliar fertilizer enhanced all these parameters above compared with the control, particularly in young leaves (Figure 3; p < 0.05). Fe-ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) fertilizer enhanced ascorbic acid profoundly and the total phenolic content in the purslane plant compared with the total phenolic content in the control . Furthermore, foliar fertilization with chelated fertilizers profoundly enhanced antioxidant nutrient (e.g. Vc, GSH, and phenolics) accumulation in the young leaves of jujube. Based on the data mentioned above, we can conclude that chelated iron/zinc fertilizers can profoundly alleviate metal ion toxicity in young leaves by enhancing antioxidant enzyme activities and nonenzymatic antioxidant content compared with the ionic fertilizer.
In the present experiment, no significant difference was observed between ionic (e.g. FeCl2) and chelated (e.g. Fe-IDS) fertilizers in improving mineral element accumulation (Table 1; p < 0.05). However, the young leaves exhibited higher sensitivity than the mature leaves to the toxicity of ionic fertilizers (Figure 1; p < 0.05). This phenomenon can be partly attributed to the low antioxidant defence capacity of young tissues compared with the mature ones in plants . A higher content of antioxidant nutrients (e.g. Vc, GSH, and phenolics) was observed in mature leaves than in young leaves without fertilizer application (Figure 3; p < 0.05). Interestingly, foliar application with chelated fertilizer reduced oxidative damage and enhanced antioxidant accumulation in the young leaves of jujube (Figures 1 and 3). Compared with the nondegradable chelating agents, IDS is a low-toxic chelator and is widely used in industrial, domestic, and agriculture applications  because most of it gets biodegraded within 1 week . In the current experiment, a low concentration of IDS–metal complex was foliar sprayed on jujube leaves collected after 7 days for subsequent jujube leaf tea production.
Moreover, the IDS would further be degraded during the jujube leaf tea production period. Thus, the toxicity from IDS residues could be negligible. Regarding using young leaves to produce jujube leaf tea , foliar spraying with chelated mineral fertilizer (Fe-IDS or Zn-IDS) may be an appropriate method for mineral element and phytochemical enhancement jujube leaves.
Some interesting conclusions were drawn from the current study. First, spraying with chelated fertilizers (Fe-IDS and Zn-IDS) profoundly enhances iron and zinc accumulation compared to controls; however, ionic fertilizers (FeCl2 and ZnCl2) do not show the observed effect in the young leaves of jujube. Second, IDS can alleviate the toxicities of FeCl2 and ZnCl2 by increasing SOD and CAT activities, coupled with reducing H2O2 and TBARS accumulation. Third, IDS profoundly enhances Vc, GSH, phenolic accumulation, and total antioxidant capacity compared with the ionic fertilizer. Overall, these results suggest that the IDS could be used as a cheap, biodegradable, and readily available reagent suitable for widespread application in agriculture to improve the mineral element and antioxidant nutrient accumulation in the edible parts of young tissues in plants.
The authors thank Benliang Deng for his valuable advice.
Funding information: The current work was supported by grants from the Natural Science Foundation of Henan Province (grant number 162300410198) and the Science and Technology Project of Henan Province (grant number 172106000049).
Author contributions: Hongxia Liu: conceptualization, investigation, funding acquisition, writing, and reviewing; Hui Liu and Mingyue Xu: investigation, project administration, visualization, and resources; Hui Liu: validation, formal analysis, writing, reviewing, and editing; and Xusheng Zhao: supervision.
Conflict of interest: The authors declare no conflicts of interest in the current study.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: All data analysed during the current study are included in this published article. The detailed data can be provided on reasonable request.
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