Purification and antioxidant properties of triterpenic acids from blackened jujube (Ziziphus jujuba Mill.) by macroporous resins

Abstract In order to investigate the purification process and antioxidant activity of triterpenic acids from blackened jujube, the macroporous resin was applied to purify the crude extract from blackened jujube. The adsorption and desorption characterizations of five different macroporous adsorption resins (AB‐8, D‐101, X‐5, HPD‐100, S‐8) for triterpenic acids of blackened jujube were compared, the optimum purification resins were screened, and the purification parameters were optimized. The antioxidant activity of crude extracts and purified products from blackened jujube was analyzed. The results showed that D‐101 resin possessed the best effect on the purification of blackened jujube triterpenic acids. The optimum purification parameters were as follows: sample concentration 25.5 μg/ml, 130 ml of the sample volume was with a flow rate of 2.0 ml/min, eluted with 95% ethanol, and speed flow was 1.0 ml/min. The purity of triterpenic acids was increased by 2.49 times after purification with a recovery rate of (78.58 ± 0.67)%. Furthermore, the IC50 values of hydroxyl radical scavenging capacity from triterpenic acids crude extract and purified substances were 0.900 and 0.850 mg/ml, respectively, and the IC50 values of superoxide anion radical were 0.745 and 0.594 mg/ml, respectively, indicating that the antioxidative capacity of the purified product was stronger than the crude extract. The purified triterpenic acids (PTA) groups at different doses had excellent protective effects on H2O2‐induced damage HUVEC cells. Results have revealed that triterpenic acids of blackened jujube have good antioxidant function and utilization and development prospects.

Therefore, the research on the active components of blackened jujube can not only increase the economic benefits of jujube but also provide technical support for the development and utilization of its products.
Triterpenoids dominate the bioactive components of jujube fruit, which are widely distributed in the plant kingdom (Song et al., 2020), mostly in the form of free state or ester-forming glycosides. Studies have indicated that triterpenic acids with antioxidative (Qiao et al., 2014), anti-inflammatory and antimicrobial (Yu et al., 2012), antitumor (Masullo et al., 2015), hepatoprotective , and hypoglycemic activities (Khanra et al., 2017), combined with low toxicity, which has emerged as one of the significant focuses of research in domestic and foreign. For example, betulinic acid isolated from jujube fruit inhibited the growth of human breast cancer MCF-7 cells . Triterpenes 9 and 10 were proved to be strong free radical scavenging activities in sour jujube (Qiao et al., 2014). However, the crude triterpenoid acid extract in jujube usually contains protein, polysaccharide, and other impurities, which affected the study of its physiological activity. Therefore, a high-efficient separation and purification technology were needed to acquire triterpenoids with high purity. Macroporous adsorption resins have been extensively used as the separation and purification of natural products, attributing to their excellent chemical stability and adsorption selectivity, large surface area, renewable utilization, and insoluble in acid, alkali, and organic solvents (Wu et al., 2018;Xi et al., 2015). Researches like pentacyclic triterpenes from Centella asiatica leaves (Yingngam et al., 2020), total flavonoids from Pteris ensiformis Burm. , and cocoa polyphenols from cocoa bean husks (Zhong et al., 2019) have been reported.
In vitro antioxidant activities of plants triterpenoids mainly focus on radical scavenging antioxidant assessment, while few studies focus on cell level antioxidant evaluation. The results showed that H 2 O 2 had become a commonly used reagent in the construction of cell oxidative pressure model (Bhakkiyalakshmi et al., 2014), and the model cells induced by H 2 O 2 can more accurately reflect the antioxidant effect of active ingredients. However, to date, no studies have focused on the cellular antioxidant activity of triterpenic acids from blackened jujube.
Therefore, in this research, five macroporous resins were used to enrich triterpenic acids from the blackened jujube. The resin screening and adsorption kinetics were first investigated, and then, the purification parameters were optimized through static and dynamic adsorption and desorption experiments. Additionally, to better utilize the triterpenoid acids from blackened jujube, the antioxidant activity of the triterpenoids-purified extracts and crude extracts was analyzed by radical scavenging experiments. Then, to further explore antioxidant activity, the protective effects of purification products on H 2 O 2 -induced HUVEC cells were investigated. This work would demonstrate that it is worth studying the biological activity of triterpenoids from blackened jujube and provide the basis for the development and utilization of functional foods and pharmaceuticals.

| Materials and reagents
The raw material of jujube was produced from Xinjiang, China, and was purchased from Tai'an Market. The jujube with neat fruit shape was first soaked in water for 1 hr, then bagged and sealed, and then processed in a drying oven with a temperature of 75°C and a humidity of 80% for about 55 hr to produce blackened jujube, and the blackened jujube with a water content of about 26%, a color of dark brown with a sweet and sour taste. After blackening, they were cut into a fine flap, dried in the oven at 60°C, then ground to fine powders and sifted 40 mesh, put into the sealed bag for standby.

| Extraction of triterpenic acids
An appropriate amount (100 g) of blackened jujube powder was subjected to triterpenoids extraction with 60% (v/v) ethanol solution at a ratio of 1:20 (w/v) for 45 min twice using ultrasonic assistance. The supernatant was filtered and concentrated by a rotary evaporator at 50°C. After concentration, the obtained crude triterpenoid solutions were added diatomite to remove macromolecular protein and sugar and then defatted with petroleum ether and extraction with ethyl acetate. The extraction solution in the upper layer was concentrated under a partial vacuum and stored at −20°C for further use .

| Determination of triterpenoids content
The content of triterpenoids was monitored based on the vanillinperchloric acid assay method (Cai et al., 2019) with slight modification.
Briefly, sample solution (0.1 ml) in a water bath to evaporate solvent firstly and then added 0.2 ml vanillin-acetic reagent (5:95 w/v, currently prepared) and perchloric acid (0.8 ml). The mixture was shaken well and reacted at 60°C for 15 min, followed by 5.0 ml glacial acetic acid. After 10 min, the absorbance value at 574 nm was measured using a UV spectrophotometer (Shanghai Oppel Instrument Co., Ltd.). The regression equation was expressed as y = 0.0409x − 0.01, (R 2 = 0.9989) with oleanolic acid as the reference, where y was absorbance and x was total triterpenoids concentration (μg/ml).

| Pretreatment of macroporous resins
The resins were first soaked in two times volume 95% (v/v) ethanol for 24 hr to fully swell the resins, filtered out the ethanol, and rinsed with deionized water until it had no ethanol flavor. Then, the washed resins were immersed with 5% HCl and 5% NaOH for 4 hr, respectively. Lastly, they were washed with deionized water until the pH of filtrate reached a neutral and soaked in deionized water at room temperature for standby (Bao et al., 2015).

| Static adsorption and desorption experiments
Screening of macroporous resins 5.0 g of weighed prepared resins were placed in a 100 ml Erlenmeyer flask with a stopper, and 40 ml crude triterpenic acids solution was added. The flasks were continually shaken for 12 hr at 120 rpm and 25°C to reach the adsorption equilibrium, filtered, and took the filtrate to determine the concentration of triterpenic acids.
Subsequently, the remaining resin was washed with deionized water until eluents were colorless, followed by desorption with 40 ml of 95% (v/v) ethanol, which was shaken (120 rpm) in a thermostated vibrator (Changzhou Kaihang Instrument Co., Ltd.) at 25°C for 24 hr.
After desorption, the triterpenic acids contents of the supernatant were measured. The adsorption/desorption capacity and the ratio of each resin were calculated with the formulas below.
where Q e and Q d are the adsorption and desorption capacity at equilibrium (μg/g), respectively; C o and C e represent the original and adsorption equilibrium concentrations of triterpenic acids in the solutions (μg/ml), respectively; C 2 is the concentration of triterpenic acids in the desorption solutions (μg/ml); V 1 and V 2 are the initial sample and desorption solutions volume (ml), respectively. A and D stand for the adsorption and desorption ratio (%); W is the dry weight of resin (g).

Adsorption kinetics experiment
According to adsorption and desorption capacities, the initially selected D-101 resin was employed in the adsorption kinetics study.
5.0 g of accurately weighed pretreated resin was conducted in 100 ml conical flasks containing 50 ml of the extract solution.
Then, the flask was continually shaken (120 rpm) for 10 hr at 25°C. Supernatants were taken out at specific time intervals till equilibration, and the concentrations of triterpenic acids in the solution were determined to plot the static adsorption kinetics curve.

| Dynamic adsorption and desorption experiments
The dynamic experiments of adsorption and desorption were performed in a chromatographic column (300 × 16 mm) wet-packed with the D-101 resin as selected. The crude triterpenic acids solution of blackened jujube was filtered through a 0.45 μm membrane firstly and then applied to the glass columns at a suitable flow rate.
A tube of effluent was collected every 10 ml to measure the triterpenic acid concentration of the effluent. The dynamic leakage curve of resin was drawn to determine the best feed volume. In order to study the influence of various flow rates and sample concentrations on dynamic adsorption, sample solutions containing different concentrations (10.5, 15.5, 20.5, 25.5, and 30.5 μg/ml) were loaded onto the column at the flow rates (1, 2, 3, and 5 ml/min). The effluent liquid was collected for detecting the triterpenic acids contents with UV spectrophotometry.
After reaching adsorption equilibrium, the deionized water was first used for washing the resin column and then eluted with stepwise gradient ethanol solution (40%-100%, v/v) at a flow rate of 1.0 ml/min for the desorption process. The desorption results were analyzed at different ethanol concentrations. Subsequently, the optimized desorption solution was used to elute resin at gradient flow rates (1, 2, 3, 4, and 5 ml/min) to investigate the effect of desorption flow rate. The triterpenoid acids eluents were collected and monitored by the UV spectrophotometry method.
Moreover, the elution curve was drawn as follows: the optimal loading conditions and elution processes were set on the basis of the above experiments of dynamic adsorption and desorption, then the elution was performed under the optimized conditions, the eluent was collected at 5 ml intervals, and the triterpenic acids concentration in each desorbed part was measured using UV spectrophotometry to plot the dynamic elution curve. (1)

| Preparative purification with optimal conditions
According to the optimized purification conditions, D-101 resin was applied for dynamic adsorption and desorption of crude triterpenic acids solutions. After the eluant was collected and concentrated by a rotary evaporator at 50°C, followed by frozen-dried, the dried products were weighed and dispersed in 80% ethanol to obtain sample solutions. The sample solution concentration was measured, and the formulas below were used for calculating triterpenic acids purity and recovery rate.
where P is the triterpenic acids purification level (%); m is the triterpenic acids content of the sample solution (mg); M is the sample weight (g); C 1 is the concentration of prepurified triterpenic acids solution (μg/ ml); V 1 is the total volume of prepurified triterpenic acids solution (ml); C 2 is the concentration of purified triterpenic acids solution (μg/ml); and V 2 is the total volume of purified triterpenic acids solution (ml). After washing gently three times with PBS and fluorescent microscope was used to examine the fluorescent.

| Statistical analysis
All experiments were repeated three times, and the data expressed as mean value ± standard deviation (SD). The significance of differences was assessed using one-way analysis of variance (ANOVA) combined with Duncan' test by SPSS 20.0 software at p < .05 and p < .01.

| Research on purification process
3.1.1 | Screening of macroporous resins for the purification of triterpenic acids The adsorption of macroporous resins depends on van der Waals force and hydrogen bonding between adsorbate and adsorbent, and the interaction between them is a physical process. Besides, chemical structures, polarities, surface area, and pore diameter are also the indicators that affect the adsorption and desorption of resins (Yang et al., 2016;Yu et al., 2020). Hence, in order to enrich black-

| Adsorption kinetics of D-101 resin
The adsorption kinetics of D-101 resin for triterpenic acids of blackened jujube was shown in Figure 1a. The unit adsorption capacity changed greatly within the first 1.5 hr. After 5 hr, the adsorption amount changed slowly and tended to be stable basically, indicating that the resins reached the adsorption saturation state. The adsorption rate was faster in the initial stage, which possibly ascribed to the D-101 resin having more adsorption surface area and sorption sites.
Subsequently, triterpenic acids molecules increased gradually in the resin, the repulsive force between the molecules on resin surface and the mass transfer resistance within the particles made it more difficult for triterpenoid acid molecules to occupy (Jin et al., 2015), resulting in slow adsorption. Therefore, the adsorption time of 5 hr was appropriate.

Dynamic leakage curve
In the process of resin adsorption, normally, the leakage point was considered as the concentration of target compound in the effluent reached 1/10 of the initial concentration . In the leakage curve (Figure 1b), the triterpenic acid concentration of the effluent gradually increased when loading sample volumes increased.
At 60 ml, the mass concentration of the effluent was 15.73 μg/ml, which was close to 1/10 of the upper column solution concentration, reaching the leakage point. When the effluent reached 130 ml, the concentration of triterpenic acids in the effluent was 25.23 μg/ml, which was close to that of the sample solution (25.50 μg/ml), indicating that the resin reached adsorption saturation. So, the loading volume was determined to be 130 ml.

Effect of concentration of sample solution on the adsorption capacity
As observed in Figure 2a, the adsorption ability for triterpenic acids increased first and then decreased with the increase in sample concentration, which reached a maximum of 25.5 μg/ml sample concentration with 65% adsorption rate of triterpenic acids. This phenomenon might be attributed to the fact that as the concentration increased, the resin had not reached the adsorption saturation, and there was enough space to adsorb triterpene acids in the solution, which led to the adsorption rate rose. However, when sample concentration was too high, the impurities contained also increased and the free active site on the resin reduced; the target substance and impurities compete for the active site, resulting in the adsorption capacity dropped (Sun et al., 2016). Thus, the mass concentration of loading sample was chosen as 25.5 μg/ml.

Effect of flow rate on the adsorption capacity
When the sample loaded at different speeds, the contact time between the target and the resin would be different, so the adsorption rate would be different. Figure 2b showed the adsorption ratio reduced for triterpenoic acids gradually as the sample flow rate increased. This reason could be explained by the fact that with the increase in sample flow rate, the contact time between the target object and the resin could be shortened, and the object flowed out of the column before it was adsorbed, resulting in overload leakage and reducing the adsorption effect of the resin (Huang et al., 2017).
Therefore, a lower flow rate was conducive to the full adsorption of resin, but in practice, a lower feed flow rate would lead to too long adsorption time and extend the experimental cycle. Finally, the proper flow rate of sample loading was 2 ml/min.

Effect of ethanol concentration on desorption ratio
Different concentrations of ethanol were used to establish the proper desorption conditions to select the appropriate desorption ethanol concentration. The desorption rate of target compound was the highest using 95% (v/v) ethanol eluted the resin, and the desorption rate declined when the ethanol concentration was increased or decreased (Figure 2c). This phenomenon might be related to the polarity of triterpenes. Ethanol reduced water polarity, and triterpenes were highly soluble in low-polar eluent, while the impurities rose in high concentration ethanol (Aalim et al., 2018;Hou et al., 2020).
Considering that the elution effect of triterpenes, 95% ethanol was proposed for elution.

Effect of elution speed on desorption ratio
The effect of elution speed on desorption ratio was summarized in Figure 2d. The desorption rate of triterpenic acids from blackened jujube decreased as elution flow rate rose. The possible reason for this was that when the elution speed was fast, the contact time between the eluent and the resin was short, the target substance in the resin was not replaced in time, and the desorption was incomplete.
Therefore, the higher the elution velocity, the worse the desorption effect. When the elution speed was low, the resin micropores were easier to allow ethanol to enter, and the triterpene molecules were dissolved entirely and eluted (Jia & Lu, 2008). Hence, the triterpenoids were more thoroughly desorbed at lower elution rate.
However, if the elution speed was too slow and the desorption time was prolonged, the experimental progress would be affected.
Considering comprehensively, the optimal flow rate was determined to be 1.0 ml/min. Dynamic elution curve Figure 1c showed the elution curve of triterpenes under optimum desorption conditions. The elution was carried out at a rate of 1.0 ml/min with 95% ethanol concentration, and triterpenic acids were basically eluted when the eluent was connected to the fifth tube, the volume of eluent was 25 ml. The absorbance value of triterpenic acids reached a peak at the 7th tube of eluent, and the triterpenic acid compounds were basically eluted when the eluent was collected to the 16th tube. The peak shape and width of the elution peak were sharp and small, and there was no tailing phenomenon.
Therefore, 5-9 tubes were selected as eluent of triterpenoid acid, and the amount of eluent was 80 ml.

| Determination of triterpenic acids purity in blackened jujube after purification
The validation experiments were carried out in accordance with the process conditions determined above. The 95% ethanol eluent was freeze-dried and weighed. The result showed that the triterpenic acids of blackened jujube were purified by dynamic adsorption and desorption of D-101 resin, the content was increased from (23.55 ± 0.60)% to (58.77 ± 0.52)%, which was about 2.49 times than before purification, and the recovery was (78.58 ± 0.67)%. This study confirmed that the method had high efficiency and good separation effect and was suitable for the purification of triterpenic acids in blackened jujube.

| Antioxidant activity in vitro
3.3.1 | Comparative antioxidant activities assay of crude and purified extract Different concentrations of triterpenic acids crude extract and purified product exhibited moderate •OH radical scavenging activity in a dose-dependent manner, as can be seen in Figure 3a. Before the mass concentration of 0.5 mg/ml, the scavenging effect of crude triterpenoid acids was higher than that of purified product.
Increasing the concentration of samples from 1.0 mg/ml to 3.0 mg/ ml enhanced the scavenging capacity of the purified triterpenic acids compared with the crude extract. At the concentration of 3.0 mg/ ml, the hydroxyl radical scavenging rates of V C , triterpenoid acid crude extract, and purified product reached the maximum value of The cells were subjected to PTA and H2O2 (300 μM) for 24 hr. Cell viability was measured via MTT assays. All data were expressed as mean ± standard deviation. *p < .05 and **p < .01 versus H2O2 group. ##p < .01 versus control group followed by the purified product (87.93%), and the crude extract was the weakest (50.73%). The growth trend of V C tended to be stable, while the purified triterpenic acids showed a steep upward trend and higher than that of crude extract during 0.5 ~ 1.5 mg/ml (Figure 3b).
This result indicated that the purified triterpenic acids had a better scavenging effect on superoxide anion.

The PTA inhibits H 2 O 2 -induced cell cytotoxicity in HUVEC cells
We

Effect of the PTA on MMP reduction in HUVEC cells
Studies have shown that the vital indicator of mitochondrial dysfunction and cell apoptosis was taken for loss of mitochondrial membrane potential (Song et al., 2017). Therefore, the changes in mitochondrial membrane potential in HUVEC cells were detected in this work. As presented in Figure 6,

-induced ROS generation in HUVEC cells
The generation of cellular ROS was adopted to investigate oxidative stress . After cells were treated using H 2 O 2 and three concentrations of PTA (0.5, 1.0, and 2.0 mg/ml) for 24 hr, the generation of ROS in HUVEC cells was explored by fluorescent probe DCFH-DA. As shown (Figure 7), the ROS level in HUVEC cells treated by H 2 O 2 was significantly rose (p < .01), and the fluorescence intensity reached 100.00% ± 6.13%, which was about 2.5 times that of the control group (249.77% ± 11.18%), implying that H 2 O 2 treatment could promote the increase in intracellular ROS levels. When the PTA was added to protect the H 2 O 2 -damaged HUVEC cells, the relative intracellular ROS production was declined significantly (p < .01) with increasing sample concentration. These results suggested that the PTA provided protection against H 2 O 2 stress in HUVEC cells by decreasing ROS generation.

| CON CLUS IONS
In this work, the results confirmed the purification method of blackened jujube triterpenoid acid established was practicable. The experiment was carried out to screen macroporous resins, and D-101 was identified as the best resin for the crude triterpenoid acid enrichment of blackened jujube. Through static and dynamic experiments, the optimum process parameters of resin were obtained.
The mass concentration of the sample solution was 25.5 μg/ml, the maximum loading volume was 130 ml, and the loading flow rate was 2.0 ml/min, the amount of eluent was 80 ml, the elution flow rate was 1.0 ml/min, and 95% ethanol solution was used for elution.
Under these conditions, the purity of triterpenic acids in blackened jujube was increased from 23.55% to 58.77%, and the recovery was

CO N FLI C T S O F I NTE R E S T
The authors declare that there are no conflicts of interest.

E TH I C A L R E V I E W
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.