**Optimized Extraction of Total Triterpenoids from Jujube (***Ziziphus jujuba* **Mill.) and Comprehensive Analysis of Triterpenic Acids in Di**ff**erent Cultivars**

**Lijun Song <sup>1</sup> , Li Zhang <sup>2</sup> , Long Xu <sup>1</sup> , Yunjian Ma <sup>1</sup> , Weishuai Lian <sup>1</sup> , Yongguo Liu <sup>3</sup> and Yonghua Wang 1,\***


Received: 19 February 2020; Accepted: 23 March 2020; Published: 27 March 2020

**Abstract:** Triterpenoid compounds are one of the main functional components in jujube fruit. In this study, the optimal process for ultrasound-assisted extraction (UAE) of total triterpenoids from jujube fruit was determined using response surface methodology (RSM). The optimal conditions were as follows: temperature of 55.14 ◦C, ethanol concentration of 86.57%, time of 34.41 min, and liquid-to-solid ratio of 39.33 mL/g. The triterpenoid yield was 19.21 ± 0.25 mg/g under optimal conditions. The triterpenoid profiles and antioxidant activity were further analyzed. Betulinic acid, alphitolic acid, maslinic acid, oleanolic acid, and ursolic acid were the dominant triterpenoid acids in jujube fruits. Correlation analysis revealed a significant positive correlation between the major triterpenic acids and antioxidant activities. The variations of triterpenoid profiles and antioxidant activity within the jujube fruits and the degree of variation were evaluated by hierarchical cluster analysis (HCA) and principal component analysis (PCA), respectively. The results provide important guidance for the quality evaluation and industrial application of jujube fruit.

**Keywords:** hierarchical cluster analysis; principal component analysis; ultrasound-assisted extraction; triterpenic acid; *Ziziphus jujuba*

#### **1. Introduction**

Jujube (*Ziziphus jujuba* Mill.), belonging to the Rhamnaceae family, is widespread in Asia, Europe, and America [1]. In China, jujube has been cultivated for 4000 years and there are more than 700 cultivars of the fruits [2]. More than four million tons of jujube fruits are harvested in China per year, which represents 90% of the total yield globally [3]. The fruit has been commonly used in Traditional Chinese Medicine (TMC) for its various pharmacological activities, such as its anticancer, antiepileptic, anti-inflammatory, anti-insomnia, and neuroprotective effects [4,5]. In general, the beneficial effects of health are derived from a variety of bioactive compounds, such as triterpenes, alkaloids, flavonoids, and polysaccharides [6].

Triterpenes, belonging to the Phytosterol family, are naturally occurring bioactive components that are commonly found in cereals and vegetables [7]. Modern studies have shown that triterpenes and triterpenic acids, derivatives of pentacyclic triterpenes, have a variety of biological effects, such as antioxidative, anti-inflammatory, anticancer, hepatoprotective, and anti-microbial activities, combined with low toxicity [8–10]. Triterpenic acids in jujube fruit have been demonstrated to be a group of major bioactive compounds [11–13]. For example, triterpenic acids have been reported to be the most active part in jujuba for the inhibitory effects on inflammatory cells [14]. Alphitolic acid and 3-O-trans-coumaroyl alphitolic acid in jujube can significantly reduce nitric oxide (NO) release and the inducible nitric oxide synthase (iNOS) expression in macrophages [15]. Furthermore, betulinic acid isolated from jujube can cause apoptosis of human breast cancer cell line MCF-7 cells through the mitochondria transduction pathway [16].

The discovery of new natural and safe health products in the form of plant extracts represents a real challenge today. Thus, efficient extraction and further utilization of bioactive triterpenes of jujube and its products have been attracting attention in recent years. [17,18]. To obtain the highest recovery of triterpenoids, it is vital to select the best extraction method and optimize the parameters [19,20]. Compared to conventional extraction methods, such as maceration and Soxhlet extraction, ultrasonic-assisted extraction (UAE) is a green and efficient technology used for its short extraction time, reduced consumption of solvents and energy, and higher extraction yield of bioactive compounds [19–21]. This technique has been successfully used to extract triterpene acids from olive pomace [20], pomegranate flowers [22], and *Rosmarinus o*ffi*cinalis* leaves [12]. However, to the best of our knowledge, the application of UAE processes for extracting triterpene compounds from jujube has not been reported before.

Previous research has proved that many factors, such as solvent concentration, extraction temperature, time, and liquid/solid ratio, can affect the extraction efficiency from plant materials. Considering all of these factors and their levels, it is a tedious task to optimize extraction conditions, during which not only does the number of experimental run increase but also the interactive effect cannot be determined [23]. Response surface methodology (RSM) is a statistical method that uses multifactorial modeling to optimize complex processes. It gives a free space wherein the experimental terms can be defined based on the response value, and the levels of factors can be adjusted according to the requirement of the experiment [23,24]. Therefore, this method may be an ideal strategy for the optimization of triterpenoid extraction from jujube.

In addition, the differences in contents of the triterpenes in the materials also affects the composition of the extracts. The compositional profile of bioactive compounds presented in jujube has been found to be influenced by factors, such as cultivar, geographical environment, processing conditions, and storage conditions [23–25]. However, because of the difference between the chemical compositions of different cultivars, there are some difficulties in the breeding and planting of jujube varieties, as well as in the quality evaluation and standardization of the developed products. Therefore, it is of great significance for customers and the industry to explore the profiles of triterpenic acids of different jujube cultivars without regional disparity.

The aims of this study were: (1) to optimize the UAE conditions for triterpenoids from jujube fruit using RSM. The effects of extraction temperature, ethanol concentration, time, and the solvent-to-solid ratio on the total triterpenoid yield were studied. (2) To analyze the antioxidant activities and major triterpenic acids profiles in the extracts of different jujube samples. (3) To study the differences in the contents of triterpenic acids and antioxidant activities among different cultivars using principal component analysis (PCA) and hierarchical cluster analysis (HCA). This study provides a comprehensive triterpenoid acid profile of different jujube cultivars, irrespective of the origin differences, and the results provide substantial information on the understanding and utilization of the phytochemical properties of these jujube cultivars for further research.

#### **2. Results and Discussion**

#### *2.1. UAE Process Optimization*

#### 2.1.1. Model Fitting

The merits of RSM include the use of a lower number of experimental measurements, the provision of a statistical interpretation of the data, and also the identification of the interaction amongst variables [23,24]. In this study, the Box–Behnken design (BBD) was employed to determine the interactions among *X*<sup>1</sup> (temperature), *X*<sup>2</sup> (ethanol concentration), *X*<sup>3</sup> (time), and *X*<sup>4</sup> (liquid-to-solid ratio), as well as to optimize the UAE conditions. Table 1 shows the experimental results, and Table 2 summarizes the results of the analysis of variance (ANOVA).



**Table 2.** Analysis of variance (ANOVA) for the response surface quadratic model.



**Table 2.** *Cont.*

ANOVA can fully reflect the significance and reliability of the response surface quadratic regression model [23,24]; as indicated in Table 2, the model was highly significant (*F* = 229.64, *p* < 0.0001). The *p*-value for the lack of fit was not significant (*F* = 2.67, *p* = 0.1785), which indicates the adequate predictive relevance of the model to explain the associations of independent variables with dependent variables. The linear coefficients (*X*1, *X*4), quadratic coefficients (*X*<sup>1</sup> 2 , *X*<sup>2</sup> 2 , *X*<sup>3</sup> 2 , and *X*<sup>4</sup> 2 ), and interaction coefficients (*X*<sup>1</sup> *X*3, *X*<sup>1</sup> *X*4) were significant (*p* < 0.05). The R<sup>2</sup> value of 0.9957 indicates a reasonable fit of the model to the experimental data. An *R* <sup>2</sup> value (multiple correlation coefficient) closer to one denotes better correlation between the observed and predicted values. In this study, the values of *R* 2 (0.9957), Pred *R* 2 (0.9774), and Adj *R* 2 (0.9913) indicate a good correlation between the experimental and predicted values, which shows that the model was significant. In addition, "Adeq Precision" (a measure of the signal-to-noise ratio) of 56.589 indicates an adequate signal. It can be concluded that the model was statistically credible and reliable.

By using multiple regression analysis, the correlation between the response and the tested independent variables was established by Equation (1), and the X<sup>i</sup> demonstrates the coded variables in the formula.

$$\begin{array}{l} Y = 19.14 + 0.86 \, X\_1 + 0.054 \, X\_2 - 0.020 \, X\_3 + 0.94 \, X\_4 - 0.083 \, X\_1 \, X\_2 - 0.13 \, X\_1 \, X\_3 - 0.16 \, X\_1 \, X\_4\\ - 0.075 \, X\_2 \, X\_3 + 0.065 \, X\_2 \, X\_4 - 1.81 \, X\_1^2 - 0.12 \, X\_2^2 - 0.31 \, X\_3^2 - 1.07 \, X\_4^2 \end{array} \tag{1}$$

#### 2.1.2. Model Validation

The contour plot and the three-dimensional (3D) surface plot response surfaces described by the regression model are represented in Figure 1, and the maximum yield total triterpenoid was recorded under follow conditions: temperature of 55.14 ◦C, ethanol concentration of 86.57%, time of 34.41 min, and liquid-to-solid ratio of 39.33 mL/g. In order to check if the model was valid, extraction was carried out in triplicate under the optimal conditions. Further, the measured values (19.21 ± 0.25 mg/g) were in the range of the 95% confidence interval (95% CI) of the predicted value (19.44 mg/g), which verifies the predictability of the proposed model.

**Figure 1.** *Cont.*

**Figure 1.** Contour plot (**a**,**c**) and three-dimensional (3D) surface plot (**b**,**d**) showing the interaction effects of the process variables on the total triterpenoid yield. (**a**,**b**): the interaction between temperature (*X*<sup>1</sup> ) and time (*X*<sup>3</sup> ) on total triterpenoid yield (*Y*); (**c**,**d**): the interaction between temperature (*X*<sup>1</sup> ) and liquid-to-solid ratio (*X*<sup>4</sup> ) on total triterpenoid yield (*Y*).

#### *2.2. Triterpenic Acid Contents in the 99 Jujube Samples*

The triterpenic acids extracted at optimal conditions from 99 cultivars of jujube samples were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC–MS). The typical chromatograms of the 99 jujube samples are shown in Figure 2.

**Figure 2.** Ultra-performance liquid chromatography (UPLC) chromatograms of mixed standards (**a**) and sample (**b**). 1: Maslinic acid isomer-1 (Ma1); 2: Maslinic acid isomer-2 (Ma2); 3: Maslinic acid isomer-3 (Ma3); 4: Maslinic acid isomer-4 (Ma4); 5: Alphitolic acid (Aa); 6: Maslinic acid (Ma); 7: 2α-hydroxy ursolic acid (2αHa); 8: Maslinic acid isomer-5 (Ma5); 9: Oleanolic acid isomer-1 (Oa1); 10: Maslinic acid isomer-6 (Ma6); 11: Maslinic acid isomer-7 (Ma7); 12: Betulinic acid (Ba); 13: Oleanolic acid (Oa); 14: Ursolic acid (Ua); 15: Betulonic acid (Ba'); 16: Oleanonic acid + Ursonic acid (Oa' + Ua').

Sixteen peaks were observed on the chromatogram, including alphitolic acid, maslinic acid, 2α-hydroxy ursolic acid, betulinic acid, oleanolic acid, ursolic acid, betulonic acid, oleanonic acid, and ursonic acid, which were then identified and quantified. The other peaks were preliminarily identified as maslinic acid isomers and one ursolic acid isomer (Table 3). The quantitative results are shown in Figure 3 (detailed data are shown in Table S2).

**Table 3.** The retention time, mass spectrum (MS) parameters, and regression equations of standards and isomers.


The triterpenes, secondary metabolites of plants, are distributed in several peels, leaves, stems and barks of plants, such as birch bark, olive leaves, mistletoe sprouts, clove flower, apple pomace, *Camellia sinensis*, etc. [26]. However, jujube is one of the few fruits with a high content of triterpenes. In this study, a significant difference in the total triterpenic acid content was observed among the jujube cultivars. The total triterpenic acid content ranged from 1082.775 to 7915.451 µg/g dry weight (DW), with a mean value of 3730.970 µg/g DW. Meanwhile, cultivar Jing39 (C41) had the highest content and Wanshuyuanling (C9) the lowest content of triterpenic acid. These results are consistent with previous results (166–6126 µg/g DW) [15].

Until now, more than 15 triterpenoid acids were found in the fruit of jujube [18]. A previous study reported that the identified triterpenic acids, including alphitolic acid, ceanothic acid, maslinic acid, 2a-hydroxyursolic acid, betulinic acid, ursolic acid, betulonic acid, oleanonic acid, and ursonic acid, showed large variations at different stages of growth [15]. In the present study, betulinic acid (516.409–4097.962 µg/g DW), alphitolic acid (198.195–3282.203 µg/g DW), maslinic acid (13.905–751.855 µg/g DW), oleanolic acid (36.696–837.463 µg/g DW), and ursolic acid (5.267–685.325 µg/g DW) were the dominant triterpenoid acids in jujube. Other triterpenoid acids, such as betulonic acid (9.417–304.731 µg/g DW), 2α-hydroxy ursolic acid (0.005–438.165 µg/g DW), and oleanonic acid + ursonic acid (9.834–244.797 µg/g DW), were relatively low. Notably, preliminary findings revealed seven isomers of maslinic acid; however, the detailed structure and chemical formula need to be investigated.

Some by-products of plant raw materials are good sources of triterpenoid acids. For example, olive pomace, a valuable by-product, contains maslinic acid and oleanolic acid in the proportions of 381.200 mg/g and 29.800 mg/g, respectively [20]. The outer bark of birch contains 11.600 mg/g of triterpenoid acids (betulinic + oleanolic) [27]. The extraction of ursolic, oleanolic, and rosmarinic acids in rosemary leaves reached a maximum of 15.800, 12.200, and 15.400 mg/g, respectively [12]. The ursolic acid content in other plant raw materials are as follows: *Calendula o*ffi*cinalis* flowers (20.530 mg/g DW), *Lamii albi flos* (110.400 mg/g DW), *Malus domestica* fruit peel (14.300 mg/g DW), and *Silphium* sp. flowers (17.950–22.050 mg/g DW) [19].

The 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ferric reducing antioxidant power kit (FRAP) assays were carried out to differentiate the antioxidant properties of extracts from different jujube (Figure 4). The ABTS assay is based on hydrogen-donating antioxidants against nitrogen radicals, while the FRAP assay reflects the ferric ion-reducing antioxidant power of antioxidant. These methods have been widely used to evaluate the antioxidant capacity of food extracts [28]. In the present study, the ABTS and FRAP values ranged from 0.753 to 5.421 mM TE/100 g (C24, *Junzao*) and 0.968 to 5.529 mM FE/100 g (C86, *Huizaobianzhongyihao* ), respectively.

In order to explore the effect of the antioxidant capacity in jujube, correlations among the triterpenic acids and the antioxidant activities were also analyzed (Table 4). As documented in Table 4, a significant positive correlation was observed between ABTS<sup>+</sup> radical scavenging activity and the contents of alphitolic acid, maslinic acid, betulinic acid, ursolic acid, betulonic acid, and total triterpenic acids (*p* < 0.05). Meanwhile, alphitolic acid, maslinic acid, betulinic acid, oleanolic acid, ursolic acid, betulonic acid, and total triterpenic acids also showed a positive correlation with the FRAP value (*p* < 0.05).

Obviously, the major triterpenic acids that widely exist in jujube are one of the main antioxidants with various important physiological and pharmacological properties. Previous researchers have proved that pentacyclic triterpenes, such as maslinic acid, alphitolic acid, maslinic acid, oleanolic acid, ursolic acid, glycyrrhetinic acid, betulinic acid, and lupeol, contribute various important physiological and pharmacological properties [29]. For example, ursolic acid and its isomer, oleanolic acid, have been reported have many beneficial effects, such as antioxidative, antimicrobial, anti-inflammatory, anticancer, anti-hyperlipidemic, analgesic, hepatoprotectory, gastroprotective, anti-ulcer, anti-HIV, cardiovascular, antiatherosclerotic, and immunomodulatory effects [19]. Betulinic acid has been reported to have anti-inflammatory, anti-cancer, anti-leukemia, anti-viral, and antihelmintic activities [30]. Due to its selective cytotoxicity against tumor cells and favorable therapeutic index, betulinic acid is considered a promising chemotherapeutic agent against HIV infection and cancers [31]. Maslinic acid has been shown to have antioxidant, anti-inflammatory, antimalarial, and antiprotozoal activities [29]. Therefore, the results of this study provide important guidance for health product development based on jujube.

**Figure 3.**Contents (µg/g dry weight (DW)) of triterpenic acids in different jujube samples.

**Figure 4.** The antioxidant activities of the extracts of di fferent jujube samples. ABTS = 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) FRAP = ferric reducing antioxidant power kit.


**Table 4.** Correlation coefficients (r) of the studied triterpenic acids and the antioxidant activity of jujube cultivars.

a Significant at*p*<0.05. b Significant at*p*<0.01.

#### *2.3. HCA and PCA*

HCA and PCA are effective tools for multivariate analysis, which can be used to explore the existing differences among groups. HCA indicates the similarity among different cultivars, while PCA indicates the significant differences among the cultivars, thus reducing the dimensionality and increasing the interpretability of large datasets [24]. In this study, HCA and PCA were carried out based on the triterpenic acid content and antioxidant activities.

As shown in Figure 5, the 99 cultivars were divided into five clusters. The mean values of the detected compounds and antioxidant activities in each cluster are listed in Table 5.

**Figure 5.** Hierarchical cluster analysis (HCA) of 99 cultivars of jujube samples. Cultivar lines with the same color are in the same cluster.


**Table 5.** The mean values of the detected compounds in different clusters.

Extreme values are in bold; <sup>a</sup> the element with the lowest mean value among the five clusters; <sup>b</sup> the highest mean value.

Cluster 1 contained 21 samples, which were generally clustered together according to the higher values of alphitolic acid (mean of 1535.713 µg/g DW), maslinic acid (mean of 449.873 µg/g DW), FRAP (mean of 3.931 mM TE/100 g), and lower content of ursolic acid (mean of 92.502 µg/g DW). Cluster 2 consisted of 22 samples, which were the cultivars with the highest content of betulonic acid (mean of 109.488 µg/g DW) and relatively low levels of ursolic acid (mean of 92.296 µg/g DW), respectively. It is noteworthy that cluster 3 consisted of five samples (C15, C31, C40, C41, and C78), in which the mean contents of 2α-hydroxy, betulinic acid, oleanolic acid, ursolic acid, and total triterpenic acids were the highest among those five clusters. The mean values were 216.845, 3158.536, 568.121, 530.525, and 6814.528 µg/g DW, respectively. Accordingly, these samples also had relatively high level of ABTS and FRAP, with mean values of 3.066 and 3.172 mM TE/100 g, respectively. On the contrary, cluster 3 contained 26 samples, which had lower concentrations of most of the major triterpenic acids. For example, the mean levels of alphitolic acid, maslinic acid, 2α-hydroxy ursolic acid, betulinic acid, oleanolic acid, and total triterpenic acids, as well as the antioxidant activities (ABTS and FRAP assay), were the lowest in the five clusters. Meanwhile, the 25 samples in cluster 5 also had relatively lower contents of most compounds.

According to the above results, all of the studied variables might contribute to sample classification. Typically, clusters 3 represented the groups with higher contents of triterpenic acids and higher antioxidant activity, while cluster 4 was indicative of the groups with lower levels.

PCA was carried out to analyze the differences among the 99 cultivars of jujube. The extraction sums of squared loadings are listed in Table 6.


**Table 6.** Total variance explained by principal component analysis (PCA).

The ellipses of the constant distance of the PCA method were calculated with a 95% confidence interval. Four principal components (PCs) were extracted, and the accumulative contribution rate of the four principal components was 73.53%. PC1, PC2, and PC3 explained 31.79%, 20.46%, and 14.23% of the total variance, together accounting for 66.49% of the total variance (Table 6). From the component matrix of the four principal components (Table S3), it can be inferred that the weight occupied by different triterpenic acids showed significant differences among the different main components. Overall, almost all of the compounds may contribute to the classification of the samples. A reduction in date dimension was successfully achieved. These four principal components, to a large extent, are indicative of the original 18 variables.

In addition, the scatter plot produced by PCA is very important and powerful, since it displays all samples in two- or three-dimensional graphs, and comparisons can be carried out among samples on the basis of the response variables applied in the study [28].

The interrelationship between different cultivars is clearly shown in the 3D score plot of PCA (Figure 6a). A significance of differences between groups can be observed in Figure 6b, which shows the profiles of the 95% confidence ellipse for different groups. If there is no intersection between two ellipses, it means that those two groups have significant differences [24]. In this case, since groups 1, 2, and 5 had an intersection between them, they could not be completely distinguished from each other. In other words, because all of the samples were collected from the same origin with similar cultivation conditions, the variability between the samples was not sufficient enough to classify all of the groups accurately. However, groups 3 and 4 were completely separated from each other, which indicates that there is a significant difference between these two groups. This result is consistent with

HCA, which revealed that groups 3 and 4 were the cultivars with highest and lowest triterpenic acids contents, respectively.

**Figure 6.** The 3D plots (**a**) and biplots (**b**) of PCA. Groups 1, 2, 3, 4, and 5 are the classified clusters of jujube by HCA. The ellipses with different colors represent the 95% confidence ellipse for different clusters.

Similar results have also been reported by previous researchers when PCA was used to distinguish between different clusters of jujube [24], jujube leaves [32], and finger millets [28]. The results provide important support for cultivation and breeding, quality evaluation, and product development of jujube. Furthermore, research on the main mechanism behind these differences of the different cultivars is urgently needed by molecular biological techniques, such as genomics and enzymology.

#### **3. Materials and Methods**

#### *3.1. Plant Materials*

Jujube of 99 cultivars (red maturity stage) (Table S1) were picked from the Germplasm Resources Base of Tarim University at Alaer City of Xinjiang Province, China, by the end of October 2019. Fruits without disease and mechanical injury and uniformly shaped were collected randomly from each side of the trees. After harvesting, all samples were lyophilized and then ground to fine powders and stored below –18 ◦C before analysis.

#### *3.2. Chemicals*

The following standards were purchased from ANPEL Co., Ltd (Shanghai, China): alphitolic acid (SPR01052), maslinic acid (Lot 67050010), betulinic acid (B330270), oleanolic acid (Lot Y4430050), and ursolic acid (Lot 40920050). Chemicals, such as acetonitrile, methanol, and ammonium formate, were all of HPLC grade and were purchased from Merck (Darmstadt, Germany). A 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) kit (Art. No. A015-2) and a ferric reducing antioxidant power kit (FRAP, Art. No. A015-3) were provided by Jiancheng Biology Engineering Institute (Nanjing, Jiangsu, China). Other reagent solutions were of analytical grade (Solarbio Life Sciences Co., Ltd., Beijing, China).

#### *3.3. Determination of Total Triterpenoid Content (TTC)*

The TTC was measured using the vanillin–perchloric acid assay method [5]. The results were expressed as oleanolic acid equivalents (OAE, mg/g DW) through the standard calibration curve (*y* = 16.005*x* − 0.0256, *R* <sup>2</sup> = 0.9987). The total triterpene yield was measured using the following equation:

$$\text{Total triterpre yield } (\text{mg/g}) = \frac{\text{the mass of extracted triterpenes} (\text{mg})}{\text{the mass of dried sample } (\text{g})}. \tag{2}$$

#### *3.4. Analysis of Triterpenic Acids by UPLC–MS*

The extracts (extracted at optimum conditions) from the 99 cultivars of jujube were analyzed using a Waters ACQUITY UPLC H-CLASS system coupled with a Waters Xevo G2-XS QTof (Waters, Milford, MA) [15]. A Waters BEH C18 column (100 × 2.1 mm, 1.7 µm) operated at 30 ◦C was used. The injection volume was 2.0 µL, and the flow rate was 0.3 mL/min. The mobile phase was composed of A (3 mmol of ammonium formate) and B (methanol mixed with equal volume of acetonitrile) with a gradient elution of 0–2 min, 24–23% A; 2–18 min, 23% A. The parameters for the MS were set as follows: capillary voltage of 3.0 kV, source temperature of 110 ◦C, desolvation temperature of 450 ◦C, cone gas flow rate of 50 L/h, and desolvation gas flow rate of 800 L/h. Mass spectra in negative ion modes were recorded within the range of 100–1000 *m*/*z*. Concentrations of the compounds were calculated using the peak areas of the sample and the corresponding standards.

#### *3.5. Analysis of Antioxidant Activities*

The antioxidant activities were analyzed according to the methods reported by our lab [33]. Briefly, the samples were extracted according to the optimized extraction process. After centrifugation and lyophilization, the extract was then diluted to 10 mL for further antioxidant activity analysis. The antioxidant activities were evaluated using an ABTS kit and a FRAP kit, and the operational steps were performed in compliance with the instructions of the kits. The ABTS radical scavenging activity was expressed as millimoles of Trolox equivalent per 100 g (mM TE/100 g) of dry sample. The FRAP results were expressed as millimoles of ferrous sulfate equivalent per 100 g (mM FE/100 g) of dry sample.

#### *3.6. UAE Procedures*

Two grams (2.0 g) of dried jujube powder (C24, Junzao) was placed in an ultrasonic extractor (XY–2008; Xiyu Instruments Co., Ltd., Shanghai, China) at different influencing factors, including temperature (30–70 ◦C), ethanol concentration (55–95%), time (20–40 min), and liquid-to-solid ratio (15:1–55:1 mL/min). After extraction, the extracts were centrifuged at the speed of 5000 rpm for 5 min (Centrifuge 5804; Eppendorf AG, Germany). Then, the supernatants were evaporated, lyophilized, and stored below −18 ◦C until further analysis. Based on the results of single-factor experiments, the factors that have a major influence and the levels of those influences were determined and applied in the RSM design.

#### *3.7. RSM Experimental Design*

A four-factor three-level experimental RSM was employed to determine the optimal conditions. The yield of total triterpenoids (*y*, mg/g) was regarded as a dependent variable. Furthermore, the four-factor ranges were determined according to the previous single-factor experiments (data not shown). Table 7 shows the experimental design.


**Table 7.** Independent variable codes and levels in experimental design.

#### *3.8. Statistical Analysis*

Design Expert 8.0.5 (Stat-Ease Inc., Minneapolis, MN, USA) was used to analyze RSM optimization and regression. The values of the dependent parameters obtained from the experiments were fitted to the second-order polynomial model as shown below:

$$Y = \beta\_0 + \sum\_{i=1}^{k} \beta\_i X\_i + \sum\_{i=1}^{k} \beta\_{ii} X\_i^2 + \sum\_{i=1}^{k-1} \sum\_{j>1}^{k} \beta\_{ij} X\_i X\_j. \tag{3}$$

*Y* stands for the estimated response, *X<sup>j</sup>* and *X<sup>i</sup>* represent the independent variables, while *k* suggests variable number. Meanwhile, β*<sup>i</sup>* , β0, β*ij*, and β*ii* represent the regression coefficients of the linear, intercept, interaction, and quadratic terms, respectively.

All data were collected in triplicate and expressed as mean ± SD. The one-way ANOVA analysis, HCA, and PCA were carried out by SPSS 22.0 software (SPSS, Chicago, IL, USA).

#### **4. Conclusions**

In this study, the ultrasound-assisted extraction of total triterpenoids from jujube was optimized by RSM. The optimal conditions obtained were as follows: temperature of 55.14 ◦C, ethanol concentration of 86.57%, time of 34.41 min, and liquid-to-solid ratio of 39.33 mL/g. The triterpenoid yield was 19.21 ± 0.25 mg/g under the optimal conditions.

The triterpenoid acid profile of the extracts obtained from 99 cultivars of jujube were further analyzed by UPLC–MS. Betulinic acid (mean of 1602.008 µg/g DW), alphitolic acid (mean of 1017.060 µg/g DW), maslinic acid (mean of 265.568 µg/g DW), oleanolic acid (mean of 264.445 µg/g DW), ursolic acid (mean of 151.166 µg/g DW), betulonic acid (mean of 69.570 µg/g DW), and 2α-hydroxy ursolic acid (mean of 66.032 µg/g DW) were found to be the main triterpenoid acids in jujube of different cultivars. According to HCA and PCA, the 99 cultivars were categorized into five clusters, among which cluster 3 had relatively higher contents of most triterpenoid acids.

These results indicate that jujube is a potential natural source of triterpenic acids for the development of functional foods, and the differences in the compositional profile of cultivars may lead to their different applications. UAE is an efficient method to extract triterpenoids from jujube, and RSM is a useful method to optimize the UAE parameters of triterpenoid compounds from jujube. This study would be further contributable for the deep processing and utilization of jujube.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2223-7747/9/4/412/s1. Table S1. Summary of the information of jujube samples; Table S2. Contents (µg/g DW) of triterpenic acids in jujube fruit samples; Table S3. Component matrix of principal component analysis.

**Author Contributions:** Conceptualization, L.S. and L.Z.; methodology, Y.W.; software, L.X.; validation, Y.M.; formal analysis, W.L.; investigation, L.S.; resources, L.S. and L.Z.; data curation, Y.L. and Y.W.; writing—original draft preparation, L.S.; writing—review and editing, L.Z.; supervision, Y.W. All authors read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the National Natural Science Foundation of China (31560462), the Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU) (20171049), and the Projects of Innovation and Development Pillar Program for Key Industries in Southern Xinjiang of Xinjiang Production and Construction Corps (2018DB002).

**Acknowledgments:** The authors sincerely thank Minjuan Lin for her assistance during the sample collection.

**Conflicts of Interest:** The authors declare no conflicts of interest.

### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Underutilized Mexican Plants: Screening of Antioxidant and Antiproliferative Properties of Mexican Cactus Fruit Juices**

**Elda M. Melchor Martínez, Luisaldo Sandate-Flores, José Rodríguez-Rodríguez, Magdalena Rostro-Alanis, Lizeth Parra-Arroyo, Marilena Antunes-Ricardo, Sergio O. Serna-Saldívar, Hafiz M. N. Iqbal \* and Roberto Parra-Saldívar \***

> Tecnologico de Monterrey, School of Engineering and Sciences, Monterrey 64849, Mexico; elda.melchor@tec.mx (E.M.M.M.); a00812589@itesm.mx (L.S.-F.); jrr@tec.mx (J.R.-R.); magda.rostro@tec.mx (M.R.-A.); A00812589@exatec.tec.mx (L.P.-A.); marilena.antunes@tec.mx (M.A.-R.); sserna@tec.mx (S.O.S.-S.)

**\*** Correspondence: hafiz.iqbal@tec.mx (H.M.N.I.); r.parra@tec.mx (R.P.-S.)

**Abstract:** Cacti fruits are known to possess antioxidant and antiproliferative activities among other health benefits. The following paper evaluated the antioxidant capacity and bioactivity of five clarified juices from different cacti fruits (*Stenocereus* spp., *Opuntia* spp. and *M. geomettizans*) on four cancer cell lines as well as one normal cell line. Their antioxidant compositions were measured by three different protocols. Their phenolic compositions were quantified through high performance liquid chromatography and the percentages of cell proliferation of fibroblasts as well as breast, prostate, colorectal, and liver cancer cell lines were evaluated though in vitro assays. The results were further processed by principal component analysis. The clarified juice from *M. geomettizans* fruit showed the highest concentration of total phenolic compounds and induced cell death in liver and colorectal cancer cells lines as well as fibroblasts. The clarified juice extracted from yellow *Opuntia ficus-indica* fruit displayed antioxidant activity as well as a selective cytotoxic effect on a liver cancer cell line with no toxic effect on fibroblasts. In conclusion, the work supplies evidence on the antioxidant and antiproliferative activities that cacti juices possess, presenting potential as cancer cell proliferation preventing agents.

**Keywords:** Underutilized Mexican plants; Cactus fruits; Antioxidant activities; Antiproliferative properties

#### **1. Introduction**

According to a recent report by the World Health Organization (WHO), cancer is a collection of diseases that is the second leading cause of mortality worldwide. The condition starts in any organ/tissue of the body and spreads beyond its initial boundaries to invade adjoining tissues until eventually it reaches other organs and tissues. The uncontrolled stage of this collection of illnesses is called metastasis and is a major cause of death. Cancer disease cause of death globally accounted for an estimated 9.6 million deaths in 2018, which could have increased by 50% to 15 million by 2020 [1]. The tissues most commonly affected were lung, breast, colorectal, prostate, skin, and stomach. Liver cancer alone caused 782,000 deaths [1]. In order to decrease the mortality rate, efforts should focus on preventing or treating cancer at its early stages. Mexican plants have been proven to act as natural antioxidant products as well as providing anticancer activities [2]. Cactaceae plants are a group of uncommon species worldwide that grow in arid areas. Mexico is home of 518 species of which 47.7% are endemic [3]. Cacti fruits are highly valued in the region for their chemical composition which grants them their distinctive organoleptic properties such as color and taste [4]. Few studies detail the chemical characterization of cacti fruits, and their bioactive properties.

**Citation:** Martínez, E.M.M.; Sandate-Flores, L.; Rodríguez-Rodríguez, J.; Rostro-Alanis, M.; Parra-Arroyo, L.; Antunes-Ricardo, M.; Serna-Saldívar, S.O.; Iqbal, H.M.N.; Parra-Saldívar, R. Underutilized Mexican Plants: Screening of Antioxidant and Antiproliferative Properties of Mexican Cactus Fruit Juices. *Plants* **2021**, *10*, 368. https://doi.org/ 10.3390/plants10020368

Academic Editor: Stefania Lamponi

Received: 23 January 2021 Accepted: 10 February 2021 Published: 14 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Fruits from the *Stenocereus* genus (pitayas) can be harvested semi-annually from April to May and September to October. They are ovoid in shape, pigmented, and possess small thorns [5] (Figure 1). Partial characterizations of pitaya fruits have shown that they contain betalains, flavonoids, and phenolic compounds (caffeic, ferulic, and *p*-coumaric) [3,6]. Their uses have been reported against insect bites, rheumatism, hemorrhages, and gastrointestinal issues [3,7].

**Figure 1.** Cactus fruits (**a**) *Stenocereus pruinosus* yellow fruit (SY), (**b**) *Stenocereus pruinosus* red fruit (SR), (**c**) *Opuntia ficus-indica* yellow fruit (OPY), (**d**) *Opuntia ficus-indica* red fruit (OPR), (**e**) *Myrtillocactusgeomettizans* fruit (MG).

Prickly pears are oval shaped fruits commonly referred to as "tunas" (Figure 1) and are extensively distributed throughout Mexico [8]. They have been known to contain flavonoids, phenolic acids (caffeic, coumaric, vanillic, among others), betalains, ascorbic acid, fatty acids, lignans, and sterols [3,6]. Additionally, they possess a variety of health benefits, yet not restricted to a high antioxidant capacity [9], including cytotoxic activity [10,11], and an antiproliferative effect on cancer colon (HT29/Caco-2) and prostate cancer cell lines (PC-3) in vitro [12,13].

*Myrtillocactus geomettizan* produces cacti fruits called garambullo, also known as the berry cactus, which ripens from May to July. Garambullo fruits are 1.5 cm in length, globular, and purple (Figure 1) [14]. The berry cactus fruits contain flavonoids, betalains, ascorbic acid, and many phenolic acids (caffeic, gallic, vanillic) [3,6] with associated health properties such as the improvement of renal functions in rats as well as decreased glucose and cholesterol levels in blood [14].

There are only a few characterization studies about the Cactaceae family and their fruits. The exploration of nature as a source of novel compounds to treat chronic diseases, such as cancer, is growing. Considering the above properties of underutilized Mexican plants, herein, we report the chemical characterization and compositional analyses of five clarified juices extracted from different cacti fruits (*Stenocereus* sp., *Opuntia* sp. and *M. geomettizans*). The scientific rational behind this study was to test and report the correlation between antioxidant capacity and antiproliferative properties of Mexican cactus fruit juices. Furthermore, we studied the in vitro bioactive potential of in-house extracted juices from different cacti fruits against cancer cell lines and a normal cell line, with the aim of proposing their medicinal valorization.

#### **2. Results and Discussion**

#### *2.1. Total Soluble Solids, Betacyanin, Betaxanthin Content, and Antioxidant Activity*

There are few reports quantifying the chemical content of the cactus fruits here mentioned in order to establish a comparison. The clarified juices obtained had low values around 0.2 ◦Brix, of soluble solid content (Table S1). The juice of Pitaya (*Stenocereus pruinosus*) reported 9.8 ◦Brix [15], a higher value of the total solids than in SY (0.2 ◦Brix) and SR (0.2 ◦Brix). One possible reason for the discrepancy between the results from this paper and those in the literature is a difference in the procedure used to obtain the clarified juices, as showed in the Supplementary material.

The content of betalains is shown in Table 1. The OPR clarified juice had the highest concentration of betacyanins of the clarified juices (403.56 ± 1.41 µg/g of FS-fresh sample). The analysis of the different *Opuntia* fruits showed similar betacyanin compositions to that of *Opuntia robusta*, that goes by the common name of Tapón, reported in the literature (300.5 ± 8.8 µg/g FS) [12]. Regarding the betaxanthin concentrations, the SR clarified juice had the highest value (404.59 ± 2.33 µg/g of FS) followed by the OPR

juice (263.24 ± 36.36 µg/g of FS), and the *Opuntia rastrera* juice (86.2 ± 22.3 µg/g FS) [12]. Betalains are water soluble which is why a higher content was observed in the clarified juices than in the pulps. A similar phenomenon was observed in freeze-dried cherry [16]. The clarified juice of *Myrtillocactus geomettizan* had less betacyanins (103.50 ± 0.01 µg/g FS) and betaxanthins (45.76 ± 0.42 µg/g FS), and even betacyanins in the fruit pulp (36.9 ± 3.7 µg/g FS) [17]. This may be due to the stage of maturity and geographical origin of the cactus berries [18].

**Table 1.** Betacyanin and betaxanthin content and antioxidant activity of clarified juices.


Values represented as mean ± standard deviation (*n* = 3), different lowercase letters (**a–d**) indicate statistical significance difference (*p* < 0.05), FS = fresh sample, GA gallic acid equivalents, TE Trolox equivalents. ND = not determined *Stenocereus pruinosus* yellow fruit (SY), *Stenocereus pruinosus* red fruit (SR), *Opuntia ficus-indica* yellow fruit (OPY), *Opuntia ficus-indica* red fruit (OPR), and *Myrtillocactus geomettizans* fruit (MG). 2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), α-α-Diphenyl-β-picrylhydrazyl (DPPH), Ferric Reducing Antioxidant Power (FRAP.

> Using the Folin–Ciocalteu method, the MG clarified juice (138.38 ± 0.14 mg GA/ 100 mL of FS) had the highest antioxidant content followed by the OPR clarified juice (111.72 ± 0.36 mg GA/100 mL of FS). A similar behavior to that of the MG clarified juice has been reported in red fruits such as raspberry *Rubusidaeus L*. (148.9 ± 0.45 mg GA/100 mL FS) [19]. The above results could be explained by the resulting effect of a wide range of structures such as monophenols, catechols, pyrogallols, phloroglucinols, resorcinols, p-hydroquinols, naphthols, anthracenes, flavonoid aglycones, glycosides, hydroxycoumarins, aminophenols [20], and vitamins as dehydroascorbic acid [21] that are detected by this technology.

> The ABTS method corroborated the antioxidant results from the Folin–Ciocalteu method; the MG clarified juice (3123.77 ± 26.15 µmol TE/100 g FS) had the highest antioxidant content followed by the OPR clarified juice (1097.35 ± 20.20 µmol TE/100 g FS). Red fruits reported higher antioxidant activity through the DPPH procedure; OPR clarified juice being the highest (1115.25 ± 56.46 µmol TE/100 g of F) followed by SR juice (854.601 ± 17.60 µmol TE/100 g of FS). A similar trend was observed in the FRAP results of red fruits. However, the SR clarified juice had the highest antioxidant effect (2744.48 ± 42.16 µmol TE/100 g of FS). Antioxidants assays are classified in electron transfer (ET) methods such as ABTS, FRAP, and DPPH and hydrogen atom transfer (HAT) like methods such as ORAC [22]. The cacti fruits complex matrix contains a variety of chemical compounds that cause their antioxidant properties. In order to obtain an accurate understanding of the antioxidant capacity of cacti fruits and to be able to compare results across different labs with varying conditions, multiple assays with distinct mechanisms must be run. Nevertheless, juices from passion fruit (176.42 ± 23.40 µmol TE/100 mL FS) and lemon juice (56.75 ± 26.63 µmol TE/100 mL FS) [23], demonstrated a lower antioxidant capacity than all clarified juices analyzed here.

> Recent studies have found that the flowers also have phenolic compounds, such as *Artocarpus lakoocha Roxb* [24]. For this reason, it will be interesting to study the extract

of flowers of *Stenocereus pruinosus*, *Opuntia ficus-indica*, and *Myrtillocactus geomettizans* in future works.

#### *2.2. Phenolic Composition Analysis*

Due to their availability and cytotoxic activity, MG and OPY were analyzed by HPLC in order to detect the content of *p*-coumaric acid, gallic acid, caffeic acid, and vanillic acid in each clarified juice, using each phenolic acid's respective standard. Figures S2–S5 displays the HPLC profile of the MG and OPY clarified juices. The results indicated a high content of *p*-coumaric acid (60.60 ± 0.25 mg/L of FS) in MG clarified juice. The OPY clarified juice had a high amount of gallic acid (21.75 ± 0.75 mg/L FS) as well as *p*-coumaric acid (16.85 ± 1.02 mg/L FS) (Table 2).

**Table 2.** Phenolic acids' composition of *Myrtillocactus geomettizans* fruit (MG) and *Opuntia ficus-indica* yellow fruit (OPY) detected by HPLC.


Values represented as mean ± standard deviation (*n* = 3), different lowercase letters (**a,b**) indicate statistical significance difference (*p* < 0.05). FS = fresh sample.

The HPLC analysis of the phenolic acids previously mentioned in *Opuntia joconostle* reported significant amounts of protocatechuic and caffeic acid. Vanillic acid was not detected in the whole fruit [25]. Additionally, *p*-coumaric and caffeic acid derivatives were detected in *Opuntia ficus-indica* by chromatography coupled to high resolution time of flight mass spectrometry (UPLC-QTOF-MS) [26], as the present work demonstrated. In the literature, *Myrtillocactus* was reported to have a higher amount of gallic acid than caffeic acid [18], in agreement with the present work.

#### *2.3. Cell Viability Assay*

A fast screening of the cytotoxic assay was performed, using a single concentration of the clarified juices of 2% (*v*/*v*) in order to identify the species with the highest cytotoxicity on the cancer cell lines while affecting the normal cell line as minimally as possible. The results showed, HepG2 cells were more sensitive to the clarified juices of OPY, OPR, SR, and MG with cell viability percentages of (49.02 ± 1.32), (63.82 ± 1.08), (64.35.78 ± 2.84), and (69.75 ± 3.70) respectively, compared to the Caco-2 cell line which was only affected by the MG clarified juice with cell viability percentage of (57.50 ± 4.58). All clarified juices except the SR one had a similar effect on the PC3 cell line. The cell viability percentage of the SR clarified juice was not detected in the PC3 cell line, the blank had high values of absorbance, leading to negative cytotoxicity percentages. The antiproliferative effect of the five clarified fruit juices on MCF7 could not be demonstrated as the cell viability percentage was more than 100 percent. The NIH/3T3, normal cell line was used as a control and the SY, MG, and SR clarified juices diminished the cell line proliferation with percentages of (43.15 ± 3.27), (55.68 ± 2.09), and (58.59 ± 4.56) respectively (Figure 2). A future investigation may be performed in order to evaluate the dose and time-dependence of the cactus juices with the potential on cytotoxicity.

199

**Figure 2.** Cell viability of Caco-2, HepG2, NIH/3T3, PC3, and MCF7 cell-lines treated with cactus clarified juices at 2% *v*/*v*. Cell viability was expressed in terms of percentage of living cells relative to the non-treated control. Results were expressed as means of triplicate experiments and error was expressed as Standard error of mean (SEM). Different lowercase letters (a–c) indicate a statistical significance difference (*p* < 0.05). *Stenocereus pruinosus* yellow fruit (SY), *Stenocereus pruinosus* red fruit (SR), *Opuntia ficus-indica* yellow fruit (OPY), *Opuntia ficus-indica* red fruit (OPR), and *Myrtillocactus geomettizans* fruit (MG).

In vitro studies of juices extracted from fruits of *Opuntia ficus-indica* showed antiproliferative effects on hepatic cancer cells while no effect on normal fibroblast viability [12], corresponding with the findings of the present work. In order to understand if phenolic acids were responsible for the potential antiproliferative effect of clarified juices, the individual phenolic acids measured by HPLC analysis were calculated in µg/100 µL in the clarified juice at 2% in the cell proliferation assay. This calculation showed that the concentration of the phenolic acids previously mentioned was too low to have significantly contributed to the antiproliferative effect observed (Table S3). Further investigation to detect specific compounds such as quercetin, isorhamnetin, kaempferol, and betalains by HPLC is necessary to prove that the antiproliferative effect above described was due to the phenolic compounds. Evidence of these compounds has been reported in cactus juices. The fruit juices of *Stenocereus pruinosus* and *Stenocereus stellatus* were evaluated by HPLC-DAD-ESI/MS, to quantify the amounts of quercetin 3-*O*- rutinoside, kaempferol hexoside, isorhamnetin hexoside, isorhamnetin 3-*O*- glucoside, nine betacyanins, and two betaxanthins [6,15]. Isorhamnetin, quercetin, conjugated phenolic acids, indicaxan-

thin and coumarins were observed using UPLC-QTOF-MS in three *Opuntia ficus-indica* juices [26]. Quercetin was found in the cactus berry (*Myrtillocactus geometrizans*) fruit at different maturity stages before and after storage by HPLC-DAD [18]. The antiproliferative activities of flavonoids and betalains have been reported in extracts of *Opuntia robusta* and *Opuntia ficus-indica* fruit juices as they diminished human colorectal cancer cell line HT29 proliferation. The antiproliferative compounds identified were betacyanins, ferulic acid, and isorhamnetin derivatives [13]. In order to evaluate the therapeutic potential of the clarified juices for cancer, the molecular mechanism should be investigated and elucidated on normal and cancer cell lines of the same tissue. Previous studies have demonstrated the effect of plant extracts on proliferation, morphology, and cell death in MCF-7 breast adenocarcinoma and non-carcinogenic MCF-12A cell lines, where MCF-7 cell line was more susceptible to plant extracts exposure [27].

#### *2.4. Principal Component Analysis (PCA)*

In this study, PCA was used to correlate nine experimental variables: content of betacyanins and betaxanthins, antioxidant activity by both ABTS and DPPH methods, total phenolic composition by Folin–Ciocalteu assay according to cytotoxic activity of each clarified juice on HepG2, Caco-2, PC3, and NIH/3T3. The MCF7 cell line was not considered in the PCA analysis due to its high cell viability percentage and non-significant difference in all clarified juices. FRAP activity was not included in the PCA plot due to missing experimental results from MG fruit because it was unavailable during the analysis, due to the time at which it was collected. In Figure 3 we identified two components, principal component 1 (cell viability of cancer cell lines HepG2, Caco-2 which have a correlation with total phenolic composition by Folin–Ciocalteu assay and antioxidant activity by ABTS) and principal component 2 (betacyanins and betaxanthins which strongly correlated with antioxidant activity by DPPH and prostate cytotoxic activity on the PC3 cell line with red cactus fruits juices, SR and OPR). The variance of the data for the Principal Component 1 (PC1) was 36.81% and for the Principal Component 2 (PC2) was 31.89% of the variance in the data. The two principal components contributing to 68.7% of the total variance of the results. Based on PCA, the cell viability of the NIH/3T3 cell line was not correlated in either of the two groups. It is a normal cell line that was not observably influenced by the chemical content of the clarified juices in the grouping. Compared to cancer cell lines, PC3 was influenced by the chemical content of betalains and antioxidant compounds measured by DPPH; whilst Caco-2 and HepG2 were influenced by chemical content measured by ABTS and Folin–Ciocalteu methods. OPY, MG, and SY were excluded from principal component 2, due to their lower content of betalains. The PCA analysis evidenced that red cactus fruits showed a higher content of betalains which positively correlated with antioxidant activity by the DPPH method as shown in Table 1. The data is presented by two or three principal components defined as a linear combination and correlation between each other, therefore, it reveals clusters of the observed variables in terms of their similarities and dissimilarities [28]. However, additional investigation is needed in order to demonstrate SR antiproliferative activity on the PC3 cancer cell line. Previous investigations have used PCA to explain attributes of the sample and generate a global analysis of results; for example, to correlate the total phenolic content or flavonoids with antioxidant activity by hydrogen peroxide (H2O2), hydroxyl (·OH), peroxyl (ROO·) and ABTS radicals from *Opuntia elata* (Arumbeva) fruit extract [29]. Clear correlations were evidenced between total phenolics, fatty acids, phenolic compounds, and antioxidant activity of *Opuntia ficus-barbarica* A. Berger fruit pulp and seed oil harvested at different times [30].

248 249 250

252

242 clarified juices along principal components 1 and 2 using nine variables. Betacyanins (Betac), betaxan-**Figure 3.** Distribution of five clarified juices along principal components 1 and 2 using nine variables. Betacyanins (Betac), betaxanthins (Betx),antioxidant activity by ABTS method (ACABTS), antioxidant activity by DPPH method (ACDPPH), total phenolic composition (AFolin), fibroblast cell line (NIH/3T3), colon cancer cell line (Caco-2), hepatic cancer cell line (HepG2), prostate cancer cell line (PC3) *Stenocereus pruinosus* yellow fruit clarified juice(SY), *Stenocereus pruinosus* red fruit clarified juice (SR), *Opuntia ficus-indica* yellow fruit clarified juice (OPY), *Opuntia ficus-indica* red fruit (OPR) clarified juice, and *Myrtillocactus geomettizans* fruit (MG) clarified juice. The oranges circles are cancer cell lines, the blue circles are the analytical methods and green circles are the clarified juices.

#### **3. Materials and Methods**

#### *3.1. Chemical and Reagents*

251 ′ 253 ≥ ≥ ≥ 2,2-Diphenyl-L-picryl-hydrazyl and sodium carbonate were purchased from Sigma Aldrich (Steinheim, Germany). 2,2′ -Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Folin & Ciocalteu's Phenol Reagent, potassium persulfate, *p*-coumaric acid (concentration ≥ 98%), caffeic acid (concentration ≥ 98%), vanillic acid (concentration ≥ 97%) were purchased from Sigma-Aldrich (St. Louis, MO). Disodium phosphate and potassium chloride were acquired from Productos Químicos Monterrey S.A. de C.V. (Nuevo Leon, Mexico). was purchased from Productos Quimicos Monterrey S.A. de C.V. (Nuevo Leon, Mexico). Iron (lll) chloride hexahydrate and gallic acid monohydrate were obtained from Sigma Aldrich (Shanghai, China), Glacial acetic acid and methyl alcohol were purchased from Tedia High Purity Solvents (Fairfield, OH), hydrochloric acid was purchased from CTR Scientific (Nuevo Leon, Mexico) and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) was from Sigma Aldrich (Buchs, Switzerland). The milli-Q water purification system was used to filter the water that was used to perform the procedures (Q-POD, Darmstadt, Germany). Potassium phosphate monobasic was acquired from Sigma-Aldrich (Tokyo, Japan). Sodium chloride was bought from Desarrollo de Especialidades Químicas, S.A. de C.V. (Nuevo Leon, Mexico).

#### *3.2. Production of Clarified Juice*

3.2.1. Preparation of Pitaya, Garambullo, and Tuna Pulps

Around, 3 kg of *Myrtillocactus geometrizan* fruit (MG) and 5 kg of yellow prickly pear *Opuntia ficus-indica* (OPY) from Ahualulco (San Luis Potosí, México); 5 kg of yellow pitaya *Stenocereus pruinosus* fruit (SY) and 5 kg of red pitaya *Stenocereus pruinosus* from Ahuatlán (Puebla, México); and 5 kg of red prickly pear *Opuntia ficus-indica* fruits (OR) from San Nicolás (Nuevo León, México) were refrigerated, while ensuring not handling for longer than 48 hours after being gathered. The fruits were washed with tap water and Extran MA05 (Merck, Item 1400001403, Lot Mx1400005004, Estado de Mexico, Mexico). Afterwards, the spines and peels were detached manually. Ultimately, the pulp, and

seeds were separated using a juice extractor (Model TU05, Turmix ML, Estado de México). Seedless pulp was obtained from this procedure and its moisture was measured [31].

#### 3.2.2. Production of Clarified Juice

The following procedure was carried out in the dark. The previously acquired seedless pulp (30g) was centrifuged (4000 g, 4 ◦C, 10 min, Model SL 40R, Thermo Fisher Scientific, Langenselbold, Germany) in 50 mL polypropylene conical tubes (Corning®, Tewksbury, MA, USA). 30 g of pulp of each fruit (SY, SR, OPY, OPR, and MG) was prepared to obtain clarified juice as shown in Figure S1. The supernatant was strained though 150 mm of Whatman paper grade 4 (item 1009150, GE Healthcare Life Sciences, Little Chalfont, UK), the supernatant of this second filtration was considered the clarified juice. Water was not added to the clarified juices.

#### *3.3. Total Soluble Solids*

The total soluble solids (◦Brix) were determined in the clarified juices. One mL of each clarified juice was placed in the refractometer HI96811 (HANNA, Smithfield, RI, USA). Three samples of each clarified juice were used in this procedure.

#### *3.4. Betacyanin and Betaxanthin Content and Antioxidant Activity Assay*

#### 3.4.1. Quantification of Betacyanin and Betaxanthin

In order to determine the pulp's betacyanin and betaxanthin content (µg/g, fresh weight) the spectrophotometric method described in [32–34] was carried out on clarified juices using a Model DR 500 spectrophotometer (Hach Lange GmbH, Düsseldorf, Germany). The clarified juices samples were diluted as shown in Table S2 in 5 mL volumetric flasks using Milli-Q water. The extinction coefficients used were E1% = 60,000 L mol−<sup>1</sup> cm−<sup>1</sup> , λ = 540 nm for betacyanin, and E1% = 48,000 L mol−<sup>1</sup> cm−<sup>1</sup> , λ = 480 for betaxanthin.

3.4.2. Antioxidant Activity by 2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) Diammonium Salt Capacity (ABTS)

The ABTS, a single electron transfer (ET) reaction-based assay, was carried out following the method proposed by Re et al. [35]. The Phosphate buffered saline (PBS) used was created with 0.8 g of NaCl, 0.02 g of KH2PO4, 0.115 g of Na2HPO4, 0.02 g of KCl, and 0.02 g of NaN3. The volume was filled up to 100 mL, the difference being Milli-Q water. To create the ABTS reagent the following were used: 38.4 mg of ABTS 1 mM, 6.62 mg of potassium persulfate 2.45 mM, and 10 mL of the solution of PBS. The solutions were left mixing in the dark for 16 h. The absorbance was measured at 734 nm, with a spectrophotometer (Model DR 500, Hach Lange GmbH, Düsseldorf, Germany) and the dilution of the initial reagent that read 0.7 absorbance units was used (40 mL of PBS with 3 mL of ABTS solution). 20 µL of diluted clarified juice of each fruit and 2 mL of ABTS solution (with the absorbance of 0.7) were placed in a water bath at 30 ◦C for six minutes. Thereafter, the absorbance was read using Trolox as a standard in concentrations ranging from 5 to 200 ppm. The blank was created using Milli-Q water and the procedure was triplicated.

#### 3.4.3. Antioxidant Activity by α-α-Diphenyl-β-picrylhydrazyl (DPPH)

Based on Brand-Williams et al., protocol [36] 0.0148 g of DPPH were added to a 25 mL volumetric flask and filled to the mark with methanol (mother solution). One mL of this mother solution was added to a new 25 mL volumetric flask and filled to the mark with methanol, creating a diluted solution. The solutions were placed in 4.0 mL cuvettes (75 µL of clarified juices dilutions and 3 mL DPPH solution), and left to react for 16 min before being read by the spectrophotometer (Model DR 500, Hach Lange GmbH, Düsseldorf, Germany) at 515 nm [37]. All measurements were made in triplicates with a calibration curve of Trolox at varying concentrations from 5 to 200 ppm.

#### 3.4.4. Ferric Reducing Antioxidant Power (FRAP)

The antioxidant capacity of each clarified juice was determined through a modified method [38]. The FRAP reagent was prepared with acetate buffer 300 mM pH 3.6, which is a mixture of sodium acetate trihydrate, glacial acetic acid, and distilled water, a solution of iron triplicidyl triazine (TPTZ), concentrated HCl, and distilled water and finally a solution with FeCl3·6H2O and Milli-Q water. The solutions were mixed (10:1:1) respectively and incubated at 30 ◦C for 30 min in darkness. Then, 100 µL of each clarified juice was added to 3 mL of FRAP reagent. Concentrations of Trolox ranging from 10 to 200 ppm were used as standards in a calibration curve. Lastly, the absorbance was measured in a spectrophotometer (Model DR 500, Hach Lange GmbH, Düsseldorf, Germany) at 593 nm. All measurements were made in triplicate.

#### *3.5. Total Phenolic Composition*

In order to determine the overall phenolic composition, the Folin–Ciocalteu colorimetric procedure was used [39]. Twenty µL of the diluted clarified juices were added to a 96 well plate. Subsequently, 100 µL of 10% Folin reagent was added to each well and after a five minutes incubation an additional 80 µL 7.5% w/v of sodium carbonate was placed per well. The plates were incubated for 1.5 h in the absence of light at 37 ◦C. Once the incubation period had elapsed, microplates were read at 765 nm and 25 ◦C. In order to create the calibration curve, solutions of 50 to 200 mg/L of gallic acid were made in Milli-Q water. The blank was created with the same solvent and the procedure was replicated twice (in triplicate).

#### *3.6. HPLC-DAD Analysis*

After filtering the clarified juices through a 0.2 µm nylon filter (Waters, Milford, MA, USA), their chromatographic profile was analyzed through equipment from Altus Perkin Elmer with an autosampler, photodiode array detector (PDA) and a Zorbax Eclipse XDB C18 column (5 µm, 150 × 4.6 mm). In order to analyze the phenolic composition, a gradient method was achieved with Solvent A, consisting of a mixture of water and acetic acid (pH 2.5), and Solvent B, methanol. The mobile phase composition started at 100% solvent A for 3 min, followed by an increase of solvent B up to 30% from minutes 3 to 8 min, 50% from minutes 8 to 15 min, 30% from 15 to 20 min, and then returning the mobile phase composition back to 100% solvent A for the end of the run. Around, 20 µL of the clarified juices were injected at a flow rate of 0.8 mL/min at column temperature of 25 ◦C. The UV absorption spectra were documented of clarified juices and standards at 270 nm. The phenolic acids, caffeic, gallic, p-coumaric, and vanillic acids were used as standards to compare retention time and identified compounds in the clarified juices. The standards were dissolved in milli-Q water to prepare the calibration curves.

#### *3.7. Cell Viability Assay*

#### 3.7.1. Cell Culture

A normal fibroblast cell line (NIH/ 3T3) and four different mammalian cancer cell lines: mammary (MCF-7), prostate (PC3), colon (Caco-2), and hepatic (HepG2), were cultivated in DMEM-F12 medium containing 10% FBS (Fetal Bovine Serum) (Gibco, Grand Island, NY, USA) and kept in a 5% CO<sup>2</sup> atmosphere at 37 ◦C and 80% humidity.

#### 3.7.2. Cell Proliferation Assay

In order to determine viability, a cytotoxicity assay was carried out in 96-well plates with 100 µL of 5 × 10<sup>5</sup> cells/mL per well. Cancer cell lines (MCF-7, PC3, Caco-2 and HepG2) and NIH/3T3 were seeded as a control and incubated for 12 h to reach confluence. All the clarified juices were evaluated at a final concentration, 2% *v*/*v*. The plates were left in the incubator at 37 ◦C, with less than 5% CO<sup>2</sup> for 48 h. Subsequently, 20 µL of Cell Titer 96®AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) was added to each well, and the 96-well-plate was incubated at 37 ◦C, with 5% CO<sup>2</sup> for 1 h, then the

absorbance was read at 490 nm using a microplate reader (Synergy HT, Bio-Tek, Winooski, VM). The viability was determined through the calculation of average absorbance units per well and conveyed as a percentage of the untreated cell wells. The experiment was done in triplicate, culture medium without cells was used as a blank, and the cells that only had medium were a positive viability control [12].

#### *3.8. Principal Component Analysis (PCA)*

PCA is a tool used to highlight the relationships among a group of experimental variables based on multivariate statistical analysis, where a map is generated to show how variables are distributed. The correlation of antioxidant results and antiproliferative effect of the clarified juices of the cactus fruits was determined by inspection of the principal component analysis (PCA). The variables analyzed were percentage of cell viability on Caco-2, HepG2, PC3 and NIH/3T3 cell lines; antioxidant activity regarding DPPH (µmol TE/100 g FS), ABTS methods (µmol TE/100 g FS), total phenolic content obtained by the Folin–Ciocalteu method (µg GA/g of fresh sample) and betacyanin and betaxanthin (µg/g FS).

#### *3.9. Statistical Analysis*

All experiments were performed in triplicate. Results were analyzed by ANOVA and different means were compared using the Tukey test with a level of significance of *p* > 0.05. The computer software used was MINITAB 18. The multivariate analysis of Principal component analysis (PCA) above described was done by SPSS (Version 19, IBM Corp, Chicago, IL, USA.

#### **4. Conclusions**

The results presented in this paper prove the antioxidant properties of five Mexican native cactus fruits by ABTS, DPPH, FRAP and Folin–Ciocalteu methods, as well as the in vitro cell cytotoxicity on cell lines. Our findings exemplified the antiproliferative effect of *Myrtillocactus geomettizans* clarified juices through the diminished cell viability of Caco-2, HepG2, and PC3 as well as, on the normal cell line NIH/3T3. The red fruit *Stenocereus pruinosus* clarified juices affected the cancer cell line HepG2 as well as the NIH/3T3 cell line. *Opuntia ficus-indica* yellow fruit may potentially be used as a cancer preventing agent due to its selective cytotoxicity on only cancer cells, with demonstrated activity against HepG2 and no effect on the normal cell NIH/3T3. A future investigation may be performed in order to evaluate the time and dose-dependence of the cactus juices on cytotoxicity. The total phenolic compounds could be the main contributors of the bioactivity, although a synergistic effect between phenolic acids, flavonoids, coumarins, alkaloids, and vitamins could explain the cactus fruit juices' cancer potential therapeutic use. More research is needed in order to identify the bioactive compounds and mechanisms that give the juices these properties.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2223-7 747/10/2/368/s1, Table S1: Total soluble solids (◦Brix) in clarified juices, Table S2: Dilutions of the clarified juices in the different techniques, Table S3: Calculated concentration of phenolic acids composition of *Myrtillocactus geomettizans* fruit (MG) and *Opuntia ficus-indica* yellow fruit (OPY) detected by HPLC. Figure S1: General procedure to obtain clarified juices from cactus fruits Figure S2: HPLC detection of phenolic compounds (vanillic acid and coumaric acid) from *Myrtillocactus geomettizans* fruit (MG), Figure S3: HPLC detection of phenolic compounds (gallic acid and caffeic acid) from *Myrtillocactus geomettizans* fruit (MG), Figure S4: HPLC detection of phenolic compounds (vanillic acid and coumaric acid) from *Opuntia ficus-indica* yellow fruit (OPY), Figure S5: HPLC detection of phenolic compounds (gallic acid and caffeic acid) from *Opuntia ficus-indica* yellow fruit (OPY).

**Author Contributions:** Data curation, J.R.-R.; Formal analysis, E.M.M.M. and L.S.-F.; Funding acquisition, R.P.-S.; Investigation, M.A.-R. and S.O.S.-S.; Methodology, E.M.M.M. and L.S.-F.; Project administration, M.R.-A.; Software, L.S.-F. and J.R.-R.; Supervision, J.R.-R., M.R.-A. and R.P.-S.; Validation, L.S.-F.; Writing—original draft, E.M.M.M., M.R.-A., L.P.-A., S.O.S.-S., H.M.N.I. and R.P.-S.; Writing—review & editing, E.M.M.M., H.M.N.I. and R.P.-S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) Doctoral Fellowship No. 492030 awarded to author Luisaldo Sandate-Flores.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data, belongs to this work, is given and presented herein the manuscript. Additional data can be found in the supplementary material.

**Acknowledgments:** The authors appreciate the support from the FEMSA-Biotechnology Center and Latin America and Caribbean Water Center of Tecnologico de Monterrey, México. The authors would thank to J. Villela-Castrejón for supporting in cell viability experiments. The authors acknowledge to Opciones de vida.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Article*

### **Accumulation of Anthocyanins and Other Phytochemicals in American Elderberry Cultivars during Fruit Ripening and its Impact on Color Expression**

#### **Yucheng Zhou <sup>1</sup> , Yu Gary Gao 2,3,\* and M. Monica Giusti 1,\***


Received: 18 November 2020; Accepted: 3 December 2020; Published: 7 December 2020

**Abstract:** American elderberry (*Sambucus canadensis*) is a plant native to North America with anthocyanin-rich fruits. Our objective was to investigate the effects of cultivar and ripeness on the phytochemical characteristics of its fruits and the corresponding color performance. Cultivars 'Adams', 'Johns', 'Nova', 'Wyldewood', and 'York' were examined for their ◦Brix, pH, anthocyanin (pH-differential method), and phenolic content (Folin-Ciocalteau method). Extract composition were analyzed by uHPLC-PDA-MS/MS. Color and spectra were determined using a plate reader. All characteristics evaluated were significantly affected by ripeness and cultivar, except for ◦Brix and total phenolic content, which did not vary significantly among cultivars. Most anthocyanins (63–72%) were acylated with *p*-coumaric acid, with cyanidin-3-(trans) coumaroylsambubioside-5-glucoside the most predominant. The proportion of acylated anthocyanins was the only characteristic evaluated that decreased during ripening (from 80 to 70%). Extract from fully-ripened fruits exhibited red (*l*vis-max ~520 nm) and blue hues (*l*vis-max ~600 nm) at acidic and alkaline pH, respectively. Extracts from half-ripe fruit rendered yellowish tones and overall dull color. C-18 semi-purified extracts displayed higher color saturation (smaller L\* and larger C\*ab) than crude extracts. The vibrant and broad color expression of fully-ripened fruit extract, especially after C-18 purification, suggests this North American native plant as a promising natural colorant source.

**Keywords:** polyphenols; fruit development; natural food colorant; *Sambucus canadensis*

#### **1. Introduction**

Elderberry (*Sambucus* spp. L.) is a large perennial shrub or small deciduous tree in the Adoxaceae family with purplish-black small fruits that mature in late August to September in different parts of the USA [1]. It is distributed mostly in the temperate and subtropical regions of the Northern Hemisphere and includes 9 to 40 species depending on the taxonomy [1–3]. The most economically important species is European elderberry (syn. *S. nigra* L. subsp. *nigra*), which has been widely cultivated in the world from Europe to North America and East Asia [2]. Its fruits have been used as natural food colorants, and used to produce jam, jellies, yogurt, and wine [4]. In the USA, American elderberry (syn. *S. nigra* L. subsp. *canadensis* [L.] Bolli) is the most important domesticated species [3]. The first American elderberry cultivar 'Adams' was released in the 1920s, with few additional cultivars developed since then [4], mostly from the New York and Nova Scotia Agricultural Experiment Station breeding programs [1], and the Missouri State University and the University of Missouri Elderberry Improving Program [5].

American elderberry cultivars have shown to have higher yield in the midwestern states in the USA than their European counterparts [6]. A life cycle assessment of these American elderberry cultivars could reveal lower labor and pesticide input due to greater adaptability by following a similar assessment method on wine production [7]. This is because American elderberry can be completely pruned to the ground level in March for a more concentrated harvest in late summer to early autumn, thereby leading to a higher harvest efficiency and less carbon dioxide emission from tractors used for spraying and harvest. Complete removal of old shoots in American elderberry also help control elder shoot borer [8].

Elderberry has been traditionally used as a medicinal crop in many indigenous cultures [4]. The therapeutic effects of elderberry extracts, including flu symptoms alleviation, blood pressure regulation, diabetes and obesity control, and immune system enhancement, have been confirmed in a number of in vitro and in vivo studies [9,10]. Researchers attribute its medicinal and nutritional benefits to its abundant polyphenol content, composed mostly of anthocyanins, flavonols, phenolic acids, and proanthocyanidins [9]. Those polyphenols are known to be high in antioxidant capacity and possess antiviral, antibacterial, and antifungal activities [9]. The demand for valuable natural sources of antioxidant compounds has been going for decades, and this market is expected to gain an annual growth of ~6% globally during the period of 2019–2029 [11], forecasting a considerable growth potential of attention for elderberry in the coming years.

Anthocyanins contribute to both the high antioxidant capacity and the dark-violet color of elderberry fruits. These water-soluble natural pigments have been widely applied as food colorants since they are viewed as safer than synthetic colorants [12]. According to the FDA CFR, fruit (21CFR73.250) and vegetable (21CFR73.260) juice concentrates are approved for use as natural food colorants, but their practical application is restricted by their stability and color shades. Acylated anthocyanins is expected to have enhanced stability to pH, heat treatment, and light exposure than non-acylated anthocyanins through intramolecular copigmentation and self-association [13]. Vegetable sources, such as red cabbage and radish, are usually more abundant in acylated anthocyanins, but they may impart undesirable aromas or flavors to food products [13]. Anthocyanin profiles of European and American elderberry were previously compared, and acylated anthocyanins were only found in American elderberry in considerable quantity [1].

European elderberries have been evaluated for their anthocyanin and phenolic composition [1,14]; and the stability of their anthocyanin extract to heat [15,16], light [15,16], and processing [17]; as well as coloring strength [18]. Due to its high pigment content, European elderberry concentrate has been commercialized as a food coloring agent in the E.U. Despite its unique anthocyanin profile, American elderberry has not been as extensively studied as its European counterpart [18]. Our knowledge regarding American elderberry anthocyanins and phenolics is primarily related to their composition, with little research on their practical applications. Considering its high acylated anthocyanin content and milder flavor, American elderberry can potentially be a promising source of natural color. Its phytochemicals, mostly anthocyanins, may vary among cultivars and during ripening, and this variation could ultimately affect the colorimetric and spectrophotometric properties, therefore merit a more systematic examination.

Our objectives were to investigate the impact of cultivar and ripeness on the accumulation of anthocyanins and other phytochemicals in American elderberry, as well as the color performance of their anthocyanin extracts under a wide pH range. Our research aimed to reveal valuable information to help shape the potential application of American elderberry extract as a natural colorant for food industry.

#### **2. Results**

#### *2.1. Phytochemical Attributes of Di*ff*erent Cultivars*

Significant differences in pH, monomeric anthocyanin content, polymeric color, and anthocyanin/phenolic ratio were observed among five cultivars (*p* < 0.05) (Table 1). American elderberry had a pH between 4.5 and 4.9, with 'Wyldewood' having significantly lower pH than that of others. The monomeric anthocyanin content varied between 354 and 581 mg C3GE/100 g FW, and the anthocyanin/phenolic ratio was between 0.61 and 0.84, with 'Johns' and 'York' obtaining the highest and the lowest on both attributes, respectively. However, significantly higher percentage of polymeric color was detected in 'York' (10.8%) than in 'Johns' (5%), partially explained by their different monomeric anthocyanin content. The ◦Brix ranged from 12.0 (in 'Nova' and 'Wylderwood') to 13.1 (in 'York') and the total phenolic content was between 582 (in 'York') and 707 mg GAE/100 g FW (in 'Johns') with no significant differences among them.

**Table 1.** Fruits' phytochemical attributes comparison among different American elderberry cultivars. Results expressed as mean ± SD (*n* = 3). Cultivars with different superscripts were significantly different (*p* < 0.05).


<sup>1</sup> Monomeric anthocyanin (mg cyanidin-3-glucosides equivalent/100 g fresh weight); <sup>2</sup> Polymeric color (%); <sup>3</sup> Total phenolics (mg gallic acid equivalent/100 g fresh weight); <sup>4</sup> Anthocyanin/Total phenolic (%).

#### *2.2. Color Development and Phytochemicals Accumulation during Ripening*

Elderberry fruits reached a visibly darker surface color when fully ripe, as denoted by the significantly smaller L\* (lightness) value of the fully-ripened fruits (L\* = 19.1 ± 0.5) compared to the half-ripe counterpart (L\* = 23.5 ± 0.5). The mean a\* and b\* values of the fully-ripe fruits were determined to be 2.4 ± 0.2 and 0.3 ± 0.2, respectively, similar to those of the half-ripe fruits (a\* = 2.4 ± 0.2; b\* = −0.2 ± 0.1).

◦Brix, pH, anthocyanin, and phenolic content all significantly increased during fruit ripening except the pH of 'Wyldewood' (Figure 1). ◦Brix increased ~4% in both 'Wyldewood' and 'Nova' during ripening. Half-ripe berries were low in monomeric anthocyanin content, with means of 39 and 72 mg C3GE/100 g FW of 'Nova' and 'Wyldewood', respectively, consisting with the less intense redness of the fruits, but later greatly increased to 593 and 619 C3GE/100 g FW when fully ripe. Although the total phenolic content multiplied almost threefold during ripening, its rate of increase was not as high as that of the anthocyanin content. For this reason, the contribution of anthocyanins to total phenolic content was modest at the half-ripe stage, but accounted for almost 80% when ripe. Polymeric color proportion was the only monitored attribute that decreased during ripening.

#### *2.3. Major Anthocyanins in American Elderberry*

Major anthocyanins in the extract from fully-ripe fruits (FRFE) (listed in order of elution, Figure 2) were identified as cyanidin-3,5-diglucoside (peak 1), cyanidin-3-sambubioside-5-glucoside (peak 2), cyanidin-3-(*cis*)-coumaroylsambubioside-5-glucoside (peak 3), cyanidin-3-(*trans*)-coumaroyl-glucoside (peak 4), cyanidin-3-(*trans*)-coumaroylsambubioside-5-glucoside (peak 5) according to their visible spectra, MS data, and retention times. The extract from half-ripe fruits (HRFE) presented the same anthocyanins with the exception of peak 4. All anthocyanins identified were cyanidin-derivatives, with cyanidin-3-(*trans*)-coumaroylsambubioside-5-glucoside being the most predominant, representing 65–70%

of the total anthocyanin content. The *cis* isomers of cyanidin-3-coumaroylsambubioside-5-glucoside (peak 3) were present at both ripening stages, accounted for 7.6% (in HRFE) and 1.8% (in FRFE) of the peak area. The *cis* isomers eluted earlier than its *trans* counterpart showed lower absorption at 310–360 nm, and displayed a larger *l*vis-max (~525 nm). *p*-Coumaric acid was the only acylation found in American elderberry anthocyanins. Acylated anthocyanins constituted ~80% of the total pigments at the half-ripe stage, and ~70% when fully ripened. Nevertheless, ripened berries were much more abundant in both acylated and non-acylated anthocyanins due to their higher total anthocyanin content.

**Figure 1.** Comparison of 'Nova' and 'Wyldewood' fruits' phytochemical attributes (◦Brix, pH, monomeric anthocyanins, polymeric color, total phenolic content) at the half and fully-ripe stages. Results expressed as mean ± SD (*n* = 3).

**Figure 2.** HPLC chromatograms of half and full-ripe 'Nova' extracts detected at 520 nm, their peak identifications, and quantifications, and the UV-Vis spectra of peaks 3 and 5. Cy: Cyanidin; glu: glucoside; sam: sambubioside; coum: coumaroyl. \*ND: Not detected.

#### *2.4. Anthocyanin Profile of Di*ff*erent Cultivars*

Different cultivars had similar anthocyanin profiles with minor differences in the proportion of individual peak areas. Among all cultivars tested, 'Wyldewood' was overall higher in non-acylated pigments but contained the lowest proportion of the *trans*-isomers (*p* < 0.05) (Table 2). 'Adam' and 'York' had the highest levels cyanidin-3-(*cis*)-coumaroylsambubioside-5-glucoside, while 'York' contained a significantly lower percentage of cyanidin-3,5-diglucoside (*p* < 0.05). Nevertheless, a higher percentage of a certain compound in one cultivar does not necessarily represent higher amount since cultivars also varied on total anthocyanin content.

**Table 2.** Percentage of individual anthocyanin in different American elderberry cultivars. Anthocyanins are listed in the order of elution. Results expressed as mean±SD (*n*=3). Cultivars with different superscripts were significantly different (*p* < 0.05).


#### *2.5. Spectral Properties of American Elderberry Extract under Various pH*

All extracts showed similar*l*vis-max ~520 nm at pH 2, but some differences on *l*vis-max were observed at higher pH. Crude HRFE did not show a clear *l*vis-max at pH 4–6, while the *l*vis-max of its fully-ripe counterpart increased from 523 to 549 nm when the pH increased from 4 to 7, although its visible absorbance was weak in this pH range (Figure 3, Table 3). When the pH increased to alkaline region, the *l*vis-max of both HRFE and FRFE shifted to 580–600 nm region. Interestingly, FRFE always obtained larger *l*vis-max than HRFE. The HRFE generally showed higher absorbance at 400–490 nm, rendering orange-yellowish undertones at all pH.

**Figure 3.** Spectral characteristics of American elderberry 'Nova' extracts at two ripening stages before and after C-18 semi-purification in pH 2–9 buffers. Data was collected 1 h after mixing.


**Table 3.** Spectrophotometric (*l*vis-max, nm) and colorimetric (CIE-L\*, C\*, h\*) data of half and fully-ripe 'Nova' extracts before and after C-18 semi-purification in pH 2–9 buffers (*n* = 3). Data was collected 1 h after mixing.

<sup>1</sup> Crude extract. <sup>2</sup> Not detected.

Both HRFE and FRFE exhibited sharper peaks and higher absorbance at their *l*vis-max after C-18 semi-purification, particularly at low and alkaline pH, concurrent with a more vibrant color expression (Figure 3). The shape of the absorption spectra and the *l*vis-max of the HRFE more closely resembled those of FRFE after semi-purification. In spite of this, semi-purified HRFE still showed higher absorbance at 400–490 nm, and lower absorbance at *l*vis-max than FRFE at most pH. Semi-purification of FRFE had a negligible impact on *l*vis-max. However, it reduced the absorption between 400–440 nm and increased the absorbance at *l*vis-max notably, particularly under alkaline pH, showing bolder color expression.

#### *2.6. Colorimetric Properties of American Elderberry Extract under Various pH*

The FRFE expressed colors from red to colorless to purple and blue when the pH increased from 2 to 9 (Figure 3). Under pH 2–3, the FRFE displayed intense red hues with h\*ab between 0.4◦ and 7.3◦ and C\*ab between 28.6–52.6 (Table 3). As the pH increased to mildly acidic (4–6), the L\* increased by over 20 units and C\*ab were generally small (≤10) due to the deprotonation of flavylium cations, with extracts appearing almost colorless. When the pH further increased into the alkaline region, a purple-bluish color appeared due to the formation of quinonoidal bases, exhibiting h\*ab between 249.2–341.8◦ , and C\*ab increased back to 17.5 at pH 8.

The h\*ab of HRFE was between 31.9◦ (red)–103.1◦ (yellow) under the tested pH range, as predicted from our spectral data (Figure 3, Table 3). The HRFE generally had smaller L\* and larger C\*ab than its fully-ripe counterpart, especially under mildly acidic pH.

The color properties (L\*, C\*ab, h\*ab) of FRFE before and after semi-purification were similar at acidic pH, while they largely differed at alkaline pH (Table 3). At pH 7–9, the semi-purification resulted in a decrease in the L\* of 24.7–34.4 units and an increase in C\*ab of 16–31.7 units. Semi-purification also resulted in the shifts of h\*ab from 341.8◦ to 316.2◦ (purple hues) at pH 7 and from 249.2◦ to 270.4◦ (blue hues) at pH 8. The C-18 semi-purification greatly enhanced color intensity and saturation of the extract, rendering bluer color hues under neutral to alkaline pH.

At acidic pH, extracts from all cultivars except 'York' showed high similarities on their color properties (∆E < 5 in a pairwise comparison) (Table 4). At pH 3, the ∆E between the extracts from 'York' and other cultivars fell between 7.28 and 8.25, mainly resulting from their different L\* (lightness) value

as the L\* of York extracts (76.13) were significantly lower than others (82.56–83.93, data not shown). Larger ∆E (up to 14.4) was observed when comparing the color of these extracts under alkaline pH. At pH 8, most pairwise ∆E were larger than 5. Different from the findings at acidic pH where the variance mostly came from L\* value, the relatively large ∆E at alkaline pH was mainly contributed by ∆b\* (blue-yellow color) value. At pH 8, the a\* values were similar among cultivars, ranging between −5.98 ('York') and −7.35 ('Johns'), while the b\* value varied between −7.69 ('York') and −21.52 ('Adam'), leading to an overall large ∆E among cultivars.

**Table 4.** Mean (*n* = 3) color differences (∆E) of the extracts from different cultivars of fully-ripe fruits at pH 2–9.


#### **3. Discussion**

American elderberry fully-ripe fruits had a ◦Brix of 12.0–13.1 and a pH of 4.5–4.9, similar to the previously reported data [1,19]. The ◦Brix of American elderberry is comparable to those of European elderberry (8.9–14.6) [1,20] and blueberry (10.3–13.9) [21], but higher than those of blackberry (4.9–8.0) [22] and raspberry (9.4–11.5) [23]. The pH of most berries ranges between 2.8–4.2 [20–23]. High ◦Brix and low acidity are usually associated with a mild, pleasant taste. Nevertheless, American elderberry is seldom consumed as fresh fruit and used most extensively as processed food and beverages [2]. This would be ascribed to its rather small fruit size [24], and tart, tangy or bitter sensory attributes brought by its abundant polyphenols contained [25]. Most anthocyanins have negligible color expression at pH ~4.5 as they transited into the colorless hemiketal form, especially for 3,5-glycosides derivatives [26]. American elderberry was abundant in 3,5-diglycosides (~90%) with a fruit pH ~4.5 (Table 1, Figure 2). Its intense dark purplish-black coloration could be explained by the lower pH of the vacuoles, where anthocyanins are usually localized together with considerable organic acids [27]. The pH of grape vacuole has been reported to be ~1 unit lower than the pH of grape

pulp [27]. Similarly, the pH of American elderberry vacuoles is expected to be lower than the fruit pH, therefore protects the integrity of anthocyanins.

Relatively large variability in anthocyanin and phenolic content within the cultivar was observed in both this and previous studies, where berries were sorted by their surface color ahead of phytochemical analysis [20]. Surface color is usually used as a maturity indicator of fruits; nevertheless, a slight variation in pH might cause considerable changes on the intensity of fruit color [27]. Moreover, fruits may exhibit the same color but have varying anthocyanin content, as the quantification of anthocyanin content is related to fruit size and water content, as well as anthocyanin distribution.

Reports on the effect of cultivar on phytochemical content vary among different studies. For example, Mathieu et al. reported higher anthocyanin and phenolic content of 'Nova' than 'York' during a two-year observation [20], consistent with our findings, while an opposite observation was reported by Perkins-Veazie et al. [19]. Conflicting results may also occur within the same study, as Lee and Finn observed significantly higher anthocyanins content of 'Adams' than 'Johns' and 'York' in 2005, but not in 2004 [1]. Such variability suggests an interplay involving both genetics and environmental factors. Geographic, climatic, edaphic conditions, and even the position of the sampled fruits on the mother-plants can all be origins of the variance displayed [27]. American elderberry appeared to be highly responsive to these environmental factors; thus, cultivar selection with interested biochemicals should be in accordance with specific environmental conditions and cultivation approaches.

American elderberry fruits went through significant phytochemical changes during ripening, which was reflected by increased ◦Brix, pH, phenolic (including anthocyanins) content, and decreased percentage of polymeric color. During red fruit ripening, an increase in ◦Brix and pH is the most common, along with an accumulation of anthocyanins. The increased ◦Brix and pH are attributed to the acids in the fruit converting to sugars during the ripening process [2,27]. Although both increased significantly, the total phenolic content did not increase as much as the anthocyanin content did. This phenomenon was also observed during the development of other berries, like grape and raspberry [28,29]. The total phenolic content is an equilibrium between biosynthesis and metabolism, thus can ascend, decline, or stay flat during maturation depending on enzyme activities and precursor availability [27]. On the other hand, anthocyanins are continuously synthesized during fruit development, thereby the accumulation of anthocyanin and phenolic is not expected to be correlated.

Acylated anthocyanins are a group of more stable anthocyanins and more commonly found in vegetable sources like red radish, black carrot, and red cabbage [13]. Conventional fruit sources, such as cranberry, blackberry, blueberry, or European elderberry usually only contain non-acylated anthocyanins [13]. Red grape may contain about 30% anthocyanins acylated with aromatic or aliphatic acids [30]. Black goji berry was reported to be abundant in anthocyanins acylated with *p*-coumaric acids [12], but it is mainly distributed in central and east Asia with limited availability. The high acylated anthocyanin content as well as its accessibility in North America make American elderberry an excellent candidate as a natural colorant with many attractive traits.

Both exogenous and endogenous factors can initiate anthocyanin composition evolution. For example, in blueberry, cyanidin derivatives were more abundant in ripe fruits, whereas malvidin derivatives were predominant in overripe fruits [27]; the stink bug infestation decreased malvidin-derivatives and increased other aglycone derivatives [31]. Our study revealed a higher ratio of acylated anthocyanins in American elderberry at the half-ripe stage (Figure 2). Similar fluctuation has been reported in some vegetables, such as colored waxy corn [32] or red cabbage [33], both featuring a higher acylated anthocyanin ratio at an earlier ripening stage. Fruit sources, like blueberry, usually express variation on anthocyanin aglycone.

Cyanidin-3-(*cis*)-coumaroylsambubioside-5-glucoside made up 7.6% and 1.8% of the total pigments in HRFE and FRFE, respectively (Figure 2). This isomer differed from its *trans* counterpart only on the spatial configuration of the acyl group but occurs much more rarely in nature with different UV-Vis spectral characteristics. An absorption band is generally observed in the 310–360 nm range for anthocyanins acylated with aromatic acids, giving a higher ratio of A310–360/Avis-max than for aliphatic acylated or non-acylated anthocyanins [13]. However, our UV-Vis spectra of *cis* isomers revealed a much lower absorption in that range, being approximately half of A310/Avis-max ratio of *trans* isomers. Apart from that, the *cis* isomers displayed a larger *l*vis-max (525 nm) than their *trans* counterparts (521 nm) in the same solvent. The coexistence of *cis* and *trans* isomers are seldom found in edible sources, and current studies about the impact of *cis-trans* configuration on the color of these pigments are few and limited only to petunidin or delphinidin derivatives [34]. Therefore, our research provided novel information about the impact of acyl group spatial configuration on cyanidin derivatives.

Different anthocyanin/polyphenol ratios between the HRFE and FRFE can explain their spectrophotometric and colorimetric differences. The anthocyanin/phenolic ratio increased from ~20% to 85% from half- to fully-ripened. Therefore, when both extracts were standardized by their anthocyanin concentration, the HRFE contained significant higher polyphenol levels. The main phenolics in the extracts besides anthocyanins were hydroxycinnamic acids and flavonols (data not shown). These compounds can affect the extract color expression via producing yellow colors on their own (*l*vis-max ~360 nm), or anthocyanin copigmentation. Though the copigmentation may enhance anthocyanin stability, such interaction is only efficient with non-acylated anthocyanins at a low acidic pH [35], and may alter the color properties simultaneously. With the removal of these interfering compounds, the American elderberry pigments expressed more vivid colors and obtained a sharper spectrum.

Due to the high similarities of their anthocyanin profiles, different cultivars produced similar color with most pairwise ∆Es < 5. All of the extracts were able to express blue hues at pH 8 with h\* between 233.1–253.6◦ , resembling that of FD & C Blue No.2 [33]. About 90% of pigments in American elderberry were cyanidin-3,5-diglycosylated (Figure 1). This glycosidic pattern is characterized by a larger *l*vis-max than cyanidin-3-glycosides at all pH, therefore is capable of expressing blue hues at alkaline pH [26]. Common fruit-based anthocyanin sources like European elderberry and chokeberry lack this glycosidic pattern and do not express blue hues at any pH. Currently, natural sources of blue colorants are very limited, thus the glycosidic pattern along with the high acylated anthocyanin content make American elderberry a desirable natural colorant candidate.

#### **4. Materials and Methods**

#### *4.1. Reagents*

ACS or HPLC grade acetone, chloroform, methanol, trifluoroacetic acid (TFA), ammonium hydroxide (NH4OH), acetonitrile, potassium hydroxide (KOH), and sodium phosphate dibasic (Na2HPO4) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). ACS grade formic acid was purchased from Honeywell (Morris Plains, NJ, USA).

#### *4.2. Collection of Plant Materials*

American elderberry fruits of cultivars 'Adams', 'Johns', 'Nova', 'Wyldewood' and 'York' were harvested from plants grown at the South Center, The Ohio State University, near Piketon, Ohio, USA, during two summers in 2015 and 2017. Fruits harvested between mid-August and early-September 2015 were used to investigate the impact of cultivar. 'Nova' and 'Wyldewood' fruits were further harvested in late-August 2017 to determine the impact of ripeness.

Fruit samples were harvested from three plants of each cultivar. After the harvest, samples were placed in polyethylene bags, labeled, and transported to the lab. Fruits were stored at −20◦C until further analysis.

#### *4.3. Determination of Maturity Stage*

Elderberry fruits on a branch do not mature at the same time (Figure 4); thus, individual fruits were sorted into one of three maturity categories according to their appearance and surface color before analysis: (1) Immature stage: fruits were entirely green or with minimal red color shown on the surface; (2) Half-ripe stage: fruits were overall red with minimal green color shown on the surface; (3) Fully-ripe stage: fruits were entirely dark purple-violet on the surface (Figure 4). Surface color properties (Hunter CIE L\*, a\*, b\*) of samples in categories (2) and (3) were measured by a Minolta handheld colorimeter (Konica Minolta, Osaka, Japan). The L\* value indicated the level of lightness with 0 representing the darkest black and 100 representing the brightest white; the a\* value indicated the redness (+) and greenness (−) of the object and b\* value indicated the yellowish (+) and bluish (−) of the objective, according to the International Commission on Illumination (CIE) [36]. To determine the impact of cultivar, only berries in category (3) were retained to minimize the variance in maturity. To determine the impact of ripeness, berries in category (2) and (3) were stored separately for further analysis.

**Figure 4.** American elderberry fruits on the same branch at (**1**) Immature, (**2**) Half-ripe, and (**3**) Fully-ripe stages.

#### *4.4. Fruit Extracts and Their Quality Attributes*

◦Brix and pH were measured during sample extraction. Each sample was weighed (~30 g) and blended with liquid nitrogen for 2 min. ◦Brix was quantified using a handheld refractometer (Atago Co., Ltd., Tokyo, Japan), and pH was measured using a pH meter (Mettler Toledo, Inc., Columbus, OH, US) after the powder was thawed into puree.

Anthocyanins and other phenolics were extracted with acidified acetone and partitioned with chloroform [37]. About 30 mL of acetone acidified with 0.01% HCl was added to the puree and homogenized for 2 min. The blend was then vacuum filtered using a Buchner funnel with Whatman #4 filter paper (Whatman Inc, NJ, US). After filtration, the slurry was re-extracted with 70%(*v*/*v*) aqueous acetone with 0.01% HCl until a pink color was barely visible. The anthocyanin extract was placed in a separatory funnel with 2 volumes of chloroform, and the mixture was gently mixed and left to sit at room temperature until a good separation was achieved. The top layer (anthocyanin and phenolic concentrate) was collected into a flask, while the bottom layer (chloroform and polar solvents) was discarded. Residual acetone was evaporated using a Buchi rotary evaporator at 40 ◦C. The final volume of sample was documented for quantification purposes.

#### *4.5. Quantification of Anthocyanin and Phenolic Content*

*λ*

*λ* Monomeric anthocyanin content was estimated using the pH differential method [38]. The absorbance of the anthocyanin extracts at pH 1 and pH 4.5 was measured using a SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at 520 nm (λmax) and 700 nm with automated 1-cm pathlength correction. The molecular weight (449.2 g mol \*\*) and molar extinction

coefficient (29,600 L cm \*\* mol \*\*) of cyanidin-3-glucoside (C3G) were used for calculation. The total monomeric anthocyanin content was expressed as mg C3GE per 100 g of FW.

Polymeric color was determined by measuring the absorbance of the extracts at 420 nm, 520 nm (λmax), and 700 nm after being treated with sodium bisulfite [38]. The percent polymeric color was expressed as the ratio between polymerized color and overall color density.

Total phenolic content was quantified using the Folin-Ciocalteau method and expressed as gallic acid equivalents [39]. Absorbance was read at 765 nm using the SpectraMax 190 Microplate Reader. Total phenolic content was calculated and expressed as mg gallic acid equivalents (GAE) per 100 g of FW.

#### *4.6. Sample Purification*

Anthocyanin extracts were semi-purified using solid phase extraction with a C-18 cartridge. The C-18 cartridge was activated by methanol before loading the elderberry crude extract. The crude extract was then washed with acidified water (0.01% *v*/*v* HCl) and ethyl acetate to eliminate acids, sugars, and less polar phenolics. The semi-purified anthocyanins were eluted with acidified methanol (0.01% *v*/*v* HCl) and evaporated until dryness to remove all methanol. Anthocyanins were re-dissolved in acidified water (0.01% *v*/*v* HCl) for further analysis.

#### *4.7. Anthocyanin Identification*

Anthocyanin identification was conducted using a Shimadzu ultra-High-Pressure Liquid Chromatography (uHPLC) system equipped with LC-2040C pumps coupled to a triple-quadrupole Shimadzu LCMS-8040 mass spectrometer using LC-2040 PDA detector (Shimadzu, Columbia, MD, USA). A Restek reverse phase C-18 column (50 × 2.1 mm) with 1.9 µm particle size was used (Restek Corporation, Bellefonte, PA, USA). Samples were filtered through a 0.2 µm RC membrane filter (Phenomenex, Torrance, CA, USA) before injection (10 µL). Samples were run using a flow rate of 0.25 mL/min and solvent A: 4.5% formic acid in HPLC water and solvent B: 100% acetonitrile, at 60 ◦C. Anthocyanin separation was achieved using a linear gradient from 1% to 3% B in 2 min; 2 to 3 min, 3% to 4.5% B; 3 to 7.5 min, 4.5% to 8.5% B; 7.5 to 13 min, 8.5% to 40% B. Spectra (200–700 nm) was collected. The mass spectrometer was set for positive ion mode, with total ion scan from 100–1000 m/z and precursor ion scan at 271, 287, 301, 303, 317, and 331 m/z. MS data, order of elution, and comparison to literature were used for the anthocyanin identification.

#### *4.8. Bu*ff*er and Sample Preparation*

Buffer solutions of pH 2–9 were prepared as follows: 0.025 M KCl for pH 2, 0.1 M sodium acetate for pH 3–6, 0.2 M Na2HPO<sup>4</sup> and 0.2 M NaH2PO<sup>4</sup> for pH 7–9 [12,40]. The final anthocyanin concentration was adjusted to 100 mM with buffer and kept at 4 ◦C in dark.

#### *4.9. Spectrophotometric and Colorimetric Analysis*

The initial spectral measurement (400–700 nm, 1 nm interval) of each extract at pH 2–9 was taken 1 h after mixing with buffers, when sufficient equilibration was achieved. A 300 mL aliquot was pipetted into a poly-D-lysine coated polystyrene 96-well microplate and read on the SpectraMax 190 Microplate Reader. The spectral data was converted into colorimetric data (L\* (lightness), a\*, b\*, C\*ab (chroma), h\*ab (hue angle)) using the ColorBySpectra software according to CIE 1964 standard observer, D65 illuminant spectral distribution and 10◦ observer angle [41]. The color difference (∆E) was calculated using the following equation:

$$\sqrt{(\Delta \mathbf{a}^{\*\*} + \Delta \mathbf{b}^{\*\*} + \Delta \mathbf{L}^{\*\*})}$$

#### *4.10. Statistical Analysis*

One-way ANOVA (two-tailed, *a* = 0.05) and post hoc Tukey test were conducted to determine the impact of cultivar. A *t*-test was conducted to evaluate the impact of ripeness. All of the statistical analysis was conducted using Prism software (GraphPad, La Jolla, CA, USA).

#### **5. Conclusions**

American elderberry differed on most phytochemical attributes, including pH, anthocyanin content and profile, as well as anthocyanin/phenolic ratio. Those differences led to small but visible color and spectral differences under various pH environments, particularly under alkaline pH. Johns' berries exhibited overall higher anthocyanin content and acylated anthocyanin proportion with relatively low non-anthocyanin phenolic content, possessing more favorable attributes for potential application as natural colorants. All these attributes increased during fruit ripening, except the percentage of polymeric color and acylated anthocyanin. The acylated anthocyanin proportion dropped from 80% at the half-ripe stage to 70% when fully ripened. All the major anthocyanins in American elderberry were cyanidin-derivatives, with both *cis* and *trans*-configured *p*-coumaric acid acylation co-existing. FRFE exhibited a "red-colorless-purple-blue" color expression pattern at pH 2–9 and expressed more vibrant colors and sharper spectra after C-18 semi-purification.

Our results contribute to the selection of proper cultivars and ripeness for specific applications of this North American native plant and expand the potential scientific and industrial applications. The high proportion of acylated anthocyanins, along with blue hues (λvis-max ~600 nm) expression of its extracts under alkaline pH, make it a promising alternative to synthetic dyes and expands the natural color spectrum. This is particularly attractive for food applications as most fruit sources contain little to no acylated anthocyanins and tend to be less stable. On the other hand, vegetable sources with high levels of acylated anthocyanins typically possess stronger flavor. Moreover, *trans* and *cis*-configured cyanidin-derivatives co-exist in American elderberry, which is rare in nature. Thus, this material can be further utilized to explore the impact of acyl group spatial configuration on anthocyanin color properties.

**Author Contributions:** Investigation, formal analysis and writing—original draft preparation, Y.Z.; plant selection and growing, sample collection and writing—review and editing, Y.G.G.; writing—review and editing, supervision, project administration and funding acquisition, M.M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by a Specialty Crop Block Grant from USDA Agricultural Marketing Service through Ohio Department of Agriculture, the USDA National Institute of Food and Agriculture, Hatch Project OHO01423, Accession Number 1014136.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


### **References**


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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Comparative Study of Anti-Gouty Arthritis Effects of Sam-Myo-Whan according to Extraction Solvents**

**Yun Mi Lee, Eunjung Son and Dong-Seon Kim \***

Herbal Medicine Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Korea; candykong@kiom.re.kr (Y.M.L.); ejson@kiom.re.kr (E.S.)

**\*** Correspondence: dskim@kiom.re.kr; Tel.: +82-42-868-9639

**Abstract:** Sam-Myo-Whan (SMW) has been used in Korean and Chinese traditional medicine to help treat gout, by reducing swelling and inflammation and relieving pain. This study compared the effects of SMW extracted by using different solvents, water (SMWW) and 30% EtOH (SMWE), in the treatment of gouty arthritis. To this end, we analyzed the main components of SMWW and SMWE, using high-performance liquid chromatography (HPLC). Anti-hyperuricemic activity was evaluated by measuring serum uric acid levels in hyperuricemic rats. The effects of SMWW and SMWE on swelling, pain, and inflammation in gouty arthritis were investigated by measuring affected limb swelling and weight-bearing, as well as by enzyme-linked immunosorbent assays, to assess the levels of proinflammatory cytokines and myeloperoxidase (MPO). In potassium oxonate (PO)-induced hyperuricemic rats, SMWW and SMWE both significantly decreased serum uric acid to similar levels. In monosodium urate (MSU)-induced gouty arthritis mice, SMWE more efficiently decreased paw swelling and attenuated joint pain compare to SMWW. Moreover, SMWE and SMWW suppressed the level of inflammation by downregulating proinflammatory cytokines (interleukin-1*β*, tumor necrosis factor-*α*, and interleukin-6) and MPO activity. HPLC analysis further revealed that berberine represented one of the major active ingredients demonstrating the greatest change in concentration between SMWW and SMWE. Our data demonstrate that SMWE retains a more effective therapeutic concentration compared to SMWW, in a mouse model of gouty arthritis.

**Keywords:** Sam-Myo-Whan; traditional medicine; gouty arthritis; inflammation; monosodium urate

#### **1. Introduction**

Gout is a metabolic disease caused by increased blood uric acid levels (hyperuricemia) and the deposition of monosodium urate (MSU) crystals in the joints, bone, and subcutaneous tissues. Moreover, gout is closely associated with chronic hyperuricemia, which can markedly reduce patient quality of life due to the severe associated pain [1,2]. Currently, a number of anti-gout agents, including anti-inflammatory drugs (colchicine and indomethacin) as well as urate-lowering drugs (allopurinol and benzbromarone) are often selected as primary therapies for gout. Although these agents are generally effective, they are also associated with various adverse effects, including gastrointestinal, hepatic, and renal toxicity and hypersensitivity [3]. Therefore, it is critical to develop novel agents with fewer associated adverse effects while retaining, or improving, their clinical efficacy. Existing evidence suggests that several natural agents exhibit beneficial efficacy and produce fewer side effects in the treatment of gouty arthritis [4,5]. We have, therefore, focused our research on these candidate natural products.

Sam-Myo-Whan (SMW) has been a common prescription for the treatment of gout and is recorded in traditional Eastern medicine, such as Donguibogam and Chinese Pharmacopoeia. It has good therapeutic efficacy in reducing dampness (edema), decreasing heat and swelling (inflammation), and alleviating pain [6,7]. Moreover, SMW and modified SMW, which is combined with other herbal medicines, are commonly used clinically for the treatment of gouty and rheumatoid arthritis in China [8,9]. SMW is composed of

**Citation:** Lee, Y.M.; Son, E.; Kim, D.-S. Comparative Study of Anti-Gouty Arthritis Effects of Sam-Myo-Whan according to Extraction Solvents. *Plants* **2021**, *10*, 278. https://doi.org/10.3390/ plants10020278

Academic Editor: Stefania Lamponi Received: 29 December 2020 Accepted: 28 January 2021 Published: 1 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Phellodendri cortex (*Phellodendron chinense* Schneider)**,** Atractylodes rhizome (the rhizome of *Atractylodes chinensis* Koidzumi), and Achyranthes radix (the root of *Achyranthes japonica* (Miq.) Nakai) in a compatible ratio of 2:3:1. SMW has been shown to inhibit lipopolysaccharide (LPS)-induced inflammatory responses by reducing nitric oxide (NO), tumor necrosis factor-α (TNF-α) production, and inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells and BV2 cells [7]. SMW produced dual hyperuricemic actions by downregulating hepatic XOD to reduce uric acid production and inhibiting renal mURAT1, to decrease urate reabsorption and enhance urate excretion in hyperuricemic mice [10]. In addition, SMW effectively treats osteoarthritis by suppressing chondrocyte apoptosis, cartilage matrix degradation, and the inflammatory response [11]. SMW also modifies the expression of matrix metalloproteinases (MMPs)-3 and aggrecanases (ADAMTSs)-4, which are considered key enzymes in cartilage matrix degradation, and enhances the expression of gouty arthritis-reduced tissue inhibitors of metalloproteinases (TIMPs)-1 and -3, resulting in the effective inhibition of cartilage matrix degradation in gouty arthritis [12]. Several recent studies have also reported that SMW may exhibit therapeutic synergy in gouty arthritis by regulating numerous biological processes and pathways. These include the lipopolysaccharide-mediated signaling pathway, positive regulation of transcription, Toll-like receptor, Janus kinase–signal transducer and activator of transcription (JAK–STAT), nucleotide binding and oligomerization domain (NOD)-like receptor, and mitogen-activated protein kinase (MAPK) signaling pathways [13–15]. In addition, SMW used a modified SMW, adding herbal medicines, to maximize the efficacy of patients with gouty arthritis and to alleviate various symptoms of patients with different phases of gouty pathology. Furthermore, modified SMW has exhibited good results on patients with gout characterized by swelling and edema (dampness-heat type in Chinese medicine) and has been shown to inhibit inflammatory factors in the joint fluid of rats with acute gout arthritis [16–19]. However, according to these previous reports, the SMW was pulverized to a fine powder and suspended in distilled water, or extracted by refluxing with water. While several studies have reported on the efficacy of SMW as a treatment option, there have been no investigations into the differences in composition and efficacy according to the extraction solvent used. Although traditional Chinese and Oriental herbal medicines have used water extracts, ethanol or ethanol/water mixture has recently been introduced as an extraction solvent for pharmaceuticals and dietary supplements. Moreover, the Korea Food and Drug Administration exempts or requires minimum toxicity test data for drug approval of Oriental herbal medicine when using ethanol content up to 30% in mixture with water as an extraction solvent. Thus, this study investigated the differences and changes in the ingredients and efficacy of SMW according to the extraction solvent, namely water (SMWW) and 30% ethanol (SMWE). The quantities of index components and the anti-gouty arthritis activities of two kinds of SMW extract were compared in rat and mouse models.

#### **2. Results**

#### *2.1. Chemical Profiling Analysis of SMWW and SMWE*

Based on their UV–Vis absorption spectra and retention times, palmatine, armepavine, and berberine, protoberberine groups with quaternary ammonium salt structures, were identified as major components of SMW. SMWW contained 15.2 ± 0.09 mg/g of palmatine, 18.7 ± 0.17 mg/g of armepavine, and 21.1 ± 0.23 mg/g of berberine; while SMWE contained 14.2 ± 0.40 mg/g of palmatine, 21.2 ± 0.26 mg/g of armepavine, and 27.9 ± 0.16 mg/g of berberine. We also identified small amounts of atractylenolides I and III, which are part of the sesquiterpenoid group with three isoprene units, by comparing their retention times and UV–Vis absorption spectra with their reference standards (Figure 1).

**Figure 1.** Representative UPLC chromatogram at 200 nm: (**A**) Sam-Myo-Whan (SMW) water extract and (**B**) SMW 30% ethanol extract. (1) Palmatine, (2) armepavine, (3) berberine, (4) atractylenolide III and (5) atractylenolide I.

#### *2.2. Serum Uric Acid Levels of Hyperuricemic Rats Treated with SMHW or SMHE*

 The effects of SMWW and SMWE on serum uric acid levels in potassium oxonate (PO)-induced hyperuricemic rats are shown in Figure 2. Serum uric acid levels in the PO group rats were significantly increased, compared to those in the Con group (*p* < 0.0001). Treatment with SMWW or SMWE at a 400 mg/kg dose significantly reduced serum uric acid levels by 34.3% and 35.6%, respectively, compared with the PO group (both *p* < 0.01); however, there was no significant difference in efficacy between the two extracts. Rats treated with allopurinol (10 mg/kg) as a positive control showed a 60.4% decrease in their serum uric acid levels (*p* < 0.0001).

#### *2.3. Anti-Inflammatory Effects of SMWW and SMWE on Paw Swelling in MSU-Induced Gouty Arthritis*

MSU crystals led to a significant increase in paw thicknesses of injected mice compared with the controls (Figure 3B,C). Meanwhile, treatment with SMWW (100 and 200 mg/kg) or SMWE (50, 100, and 200 mg/kg) significantly suppressed MSU-induced paw swelling compared with the MSU group. At the same dose (200 mg/kg), SMWE caused a greater decrease in paw thickness than SMWW, while the 100 mg/kg SMWE dose showed similar anti-inflammatory effects on paw swelling as the 200 mg/kg SMWW dose.

**Figure 2.** Effects of SMW extracted with water (SMWW) and SMW extracted with 30% EtOH (SMWE) on serum uric acid levels in PO-induced hyperuricemic rats. Con, normal control mice; PO, POinduced hyperuricemic rat; SMWW, PO rats treated with SMWW; SMWE, PO rats treated with SMWE; AP, PO rats treated with 10 mg/kg of allopurinol. Data are expressed as the mean ± SEM (n = 6). #### *p* < 0.0001 (compared with control group) and \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001 (compared with PO group).

**Figure 3.** Effect of SMWW and SMWE on paw swelling in mice with monosodium urate (MSU)-crystal-induced gouty arthritis. Con, normal control mice; MSU, MSU-crystal-injected mice; SMWW, MSU mice treated with SMWW; SMWE, MSU mice treated with SMWE; Col, MSU mice treated with 1 mg/kg of colchicine. (**A**) Experimental design. (**B**) Representative images of the right leg from mice in each group. (**C**) Quantification of changes in the thickness of each mouse paw recorded 3 days after the induction of MSU. Data are presented as the mean ± SEM (*n* = 5). #### *p* < 0.0001 (compared with control group); \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 (compared with MSU group); <sup>a</sup> *p* < 0.05 (compared with 200 mg/kg of SMWW group); <sup>b</sup> *p* < 0.01 (compared with 1 mg/kg of colchicine group).

#### *2.4. Effect of SMWW and SMWE on Hind Paw Weight-Bearing Distribution*

In the weight-bearing test, indicating the progressive pain of gouty arthritis, the mice injected with MSU exhibited a clear reduction in weight-bearing on the affected paw, as compared with the control mice (Figure 4). Although hind-paw weight distribution showed no change with a 100 mg/kg SMWW treatment dose, the 200 mg/kg SMWW dose and 50 mg/kg SMWE dose, increased weight distribution to levels similar to the Col treatment group. In particular, the 100 and 200 mg/kg SMWE doses significantly elevated hind-paw weight distribution.

**Figure 4.** Effect of SMWW and SMWE on hind-paw weight-bearing distribution in mice with MSUcrystal-induced gouty arthritis. The relative right/left hind paw weight-bearing distribution was measured by using a dynamic weight-bearing (DWB) device, compared to that of the MSU-crystalinjected group. Con, normal control mice; MSU, MSU-crystal-injected mice; SMWW, MSU mice treated with SMWW; SMWE, MSU mice treated with SMWE; Col, MSU mice treated with 1 mg/kg of colchicine. Data are presented as the mean ± SEM (*n* = 5). ### *p* < 0.001 (compared with control group); \* *p* < 0.05 (compared with MSU group); <sup>a</sup> *p* < 0.05 (compared with 100 mg/kg of SMWW group).

#### *2.5. Effects of SMWW and SMWE on Proinflammatory Cytokines*

β α β α β α α We investigated the anti-inflammatory effects of MSU-injection by assessing the levels of IL-1β, IL-6, and TNF-α, using ELISA. The results showed that MSU-injected mice had significantly elevated IL-1β, IL-6, and TNF-α levels (Figure 5). However, SMWW and SMWE treatment significantly downregulated IL-1β production by at least 43.9%, at all treatment concentrations, with the 200 mg/kg SMWE dose displaying the greatest efficacy (68.7% reduction), compared with the Col positive control (66.2% reduction). In addition, the 200 mg/kg SMWE dose effectively reduced TNF-α levels by 52%, while the 200 mg/kg SMWW dose and the 100 mg/kg SMWE dose reduced TNF-α to similar levels (29.2% and 30.3%, respectively). Both SMW extracts exhibited a weak dose-dependent decrease in IL-6 production, however, these results were not statistically significant.

#### *2.6. Effects of SMWW and SMWE on MPO Activity*

To evaluate the possible cellular infiltration induced by MSU, MPO activity was used as an index of neutrophil accumulation. As shown in Figure 6, MSU injection was found to markedly increase MPO activity in affected paw tissue, compared to the controls (*p* < 0.0001). Meanwhile, SMWW and SMWE both reduced MPO activity, with the highest effect observed following administration of SMWE at a dose of 200 mg/kg (*p* < 0.01). The positive control group, treated with 1 mg/kg Col (which inhibits neutrophil recruitment and activation), also exhibited a significant reduction (*p* < 0.05) in MPO levels, compared to the MSU group.

β α **Figure 5.** Effects of SMWW and SMWE on proinflammatory cytokines expression in MSU-crystal-injected paw tissue. Con, normal control mice; MSU, MSU-crystal-injected mice; SMWW, MSU mice treated with SMWW; SMWE, MSU mice treated with SMWE; Col, MSU mice treated with 1 mg/kg of colchicine. (**A**) IL-1β, (**B**) IL-6, and (**C**) TNF-α levels measured by ELISA. Data are presented as the mean ± SEM (*n* = 5). # *p* < 0.05, ## *p* < 0.01, #### *p* < 0.0001 (compared with control group); and \* *p* < 0.05, \*\* *p* < 0.01. \*\*\* *p* < 0.001 (compared with MSU group).

**Figure 6.** Effects of SMWW and SMWE on myeloperoxidase (MPO) activity in MSU-crystal-injected paw tissue. Con, normal control mice; MSU, MSU-crystal-injected mice; SMWW, MSU mice treated with SMWW; SMWE, MSU mice treated with SMWE; Col, MSU mice treated with 1 mg/kg of colchicine. Data are presented as mean ± SEM (*n* = 5). #### *p* < 0.0001 (compared with control group); \*\*\* *p* < 0.001(compared with the MSU group); <sup>a</sup> *p* < 0.01 (compared with 200 mg/kg of SMWW group).

#### **3. Discussion**

Gout is a common disease characterized by the deposition of MSU crystals in the joints or subcutaneous tissues, causing acute inflammatory flares or chronic arthritis [20]. Hyperuricemia (high blood uric acid concentration) occurs above the saturation point of MSU, at which point the risk of crystallization increases [21]. MSU crystals result in acute gout attacks characterized by IL-1β-driven acute inflammation, fever, and intense pain caused by neutrophil accumulation and activation in joints. [22]. Therefore, control of hyperuricemia and treatment that reduces inflammation represent the major therapeutic approaches against gouty arthritis [23]. In the present study, we compared the compositional changes as well as treatment efficacy of SMW extracted with water or 30% ethanol. The anti-hyperuricemic effects of SMWW and SMWE in the hyperuricemic animal model, in which serum uric acid levels were increased by intraperitoneal PO injection (to induce hyperuricemia), and the anti-gouty arthritis effects of SMWW and SMWE, were assessed

in a gouty arthritis model induced by MSU-crystal injection. In addition, we analyzed the phytochemical contents of SMWW and SMWE, using HPLC.

The ability of SMW to reduce blood uric acid concentration has been demonstrated previously in many animal experiments and clinical studies [10,24,25], and it was confirmed in our study. Moreover, SMWE and SMWW exhibited similar efficacies.

The identification of MSU crystals in joint fluid or synovium is the basis for a clinically definitive diagnosis of gout arthritis, as these crystals have been shown to cause strong inflammatory reactions, leading to acute gout arthritis [26,27]. The most significant symptom of gouty arthritis is swelling and pain, which is observed in the mice injected with MSU [26,28]. In the present study, the MSU-injected mice showed a clear increase in swelling, compared with the controls, and markedly reduced weight-bearing on the affected hind paw, indicating pain. Meanwhile, SMWE treatment markedly prevented the MSU-crystal-induced elevation in paw swelling, compared with that of the SMWW or Col groups. Moreover, the 200 mg/kg SMWE dose elicited excellent pain relief, with hind-paw weight-bearing returning to that similar of the Con group. These results demonstrated that SMWE reduced swelling and pain at dosages of 100–200 mg/kg more effectively than did SMWW at 200 mg/kg.

MSU crystals are one of the most effective proinflammatory stimuli, through their ability to trigger, amplify, and sustain a strong inflammatory reaction in the joint cavity [29]. MSU crystals stimulate the synthesis and release of IL-1β, a key inflammatory cytokine that regulates the differentiation, proliferation, and apoptosis of cells in gout arthritis [30]. In addition, IL-1β induces the expression of a wide range of cytokines, including TNF-α and IL-6, resulting in a large influx of neutrophils into the synovium [31]. In turn, neutrophil interactions with MSU crystals stimulates the synthesis and release of a large variety of pro-inflammatory signals, such as reactive oxygen species, leukotrienes, prostaglandin E2 (PGE2), TNF-α, IL-1, IL-6 and IL-8. This response promotes the vasodilation, erythema and pain associated with acute gout attack [23,32]. Thus, inhibiting MSU-induced recruitment of neutrophils and blocking secretion of inflammatory mediators may prove beneficial for the control and management of acute gouty arthritis [29].

Our results further demonstrated that the levels of IL-1β and TNF-α in the paw tissue were significantly increased in response to MSU, however, became markedly downregulated, in a dose-dependent manner, following SMWW or SMWE treatment. Furthermore, MPO activity was significantly elevated in mice with gouty arthritis, compared to the control group (indicating an influx of neutrophils and acute inflammation), while both SMWW and SMWE effectively decreased MPO activity. Again, SMWE treatment resulted in superior inhibition of MPO activity caopared to SMWW, at a level similar to that of the positive control, colchicine, which is a known regulator of neutrophil activity [33]. These results suggest that SMWE relieves acute gout symptoms caused by MSU crystals by inhibiting the major inflammatory cytokines and suppressing MPO activity, which is a key feature in the initiation and progression of gouty arthritis. Furthermore, our data indicates that SMWE treatment is more effective than SMWW.

Extraction solvents have different abilities to solubilize various biologically active compounds, which can have a significant effect on the content and biological activity of the extract [34,35]. Although SMW has long been used to water extract from herbal medicines consisting of a ratio as 2:3:1 (Phellodendri cortex, Atractylodes rhizome and Achyranthes radix), no studies have reported the specific composition of these compounds. For the single medicinal herb, *Atractylodes japonica*, the extract is reported to contain stigmasterol, hinesol, eudesmol, atractylenolides, atractylon, atractylodin, and sitosterol [36], while methanol extract was reported to contain 0.08% hinesol, 0.09% eudesmol, and 0.02% atractylodin [37]. Moreover, Chikusetsusaponin IVa methyl ester, separated from *Achyranthes japonica* 80% methanol extract, reportedly elicits an anti-inflammatory effect, however, no report has been made on quantity [38]. Additionally, *Phellodendron amurense* is reported to contain alkaloids, such as phellodendrine, magnoflorine, tetrahydropalmatine, columbamine, jatrorrhizine, 8-oxyepiberberine, berberine, palmatine, and bis-[4-(dimethylamino)phenyl]

methanone [39]. While most studies of such ingredients are conducted by using nonpolar extraction solvents (methanol and ethanol) for a single herb, the only traditional method used includes water extraction in a complex of these three herbs (Phellodendri cortex, Atractylodes rhizome and Achyranthes radix). Alternatively, water and ethanol are commonly used as solvents for the extraction of herbs for preparation of traditional decoction, food ingredients, dietary supplements, etc. Thus, we conducted a study using a 30% ethanol extract, which offers the best efficacy in the range of acceptable ethanol concentrations used in traditional methods.

In this study, SMW was extracted with 30% ethanol or water, and the main ingredients were identified as palmatine, armepavine, and berberine. When SMWE was compared to SMWW, the palmatine content was slightly lower and the armepavine content slightly higher than that of SMWW. However, the berberine content of SMWE was 32.2% higher than that of SMWW. Berberine has been reported to possess a wide range of pharmacological activities, including anti-inflammatory, antimicrobial, antioxidant, hypoglycemic, hypolipidemic, and hepatoprotective properties [40]. Additionally, berberine has been shown to downregulate NLR family pyrin domain-containing protein 3 (NLRP3) and IL-1β expression in MSU-crystal-induced inflammation [41]. Other compounds, such as atractylenolide III (a known anti-inflammatory agent), were only detected in SMWE, albeit in small quantities [42]. It has been shown that extraction using an alcohol/water mixture (versus water alone) increases the content of active components that are insoluble in waterwhile also extracting water-soluble active ingredients, thus optimizing the extraction of relatively small amounts of active ingredients present in natural products [34]. Therefore, it is suggested that small amounts of compound, atractylenolide III, and 32.2% increased, berberine, are characteristic components of SMWE and are bioactive compounds that may affect the mouse gouty arthritis model. The compounds may contribute to synergistic or additional effects, and our results suggest that SMWE is more effective in reducing swelling, pain, and inflammation in MSU-induced gouty arthritis mouse model than SMWW.

#### **4. Materials and Methods**

#### *4.1. Preparation of SMW*

The SMW preparation used in this study was purchased from Kwangmyoungdang Pharms (Ulsan, S. Korea). The voucher specimen was deposited at the Korean Herbarium of Standard Herbal Resources of Korea Institute of Oriental Medicine (2-20-0354~2-20- 0356, Daejeon, S. Korea). According to Donguibogam, Phellodendri cortex (Phellodendron chinense Schneider) was stir-fried with Makgeolli (1:10, w/v) for 2 h. The Atractylodes rhizome (Atractylodes chinensis Koidzumi) was soaked in rice-washed water for 3 h and then dried. Each sample was ground into a powder. The mixture was prepared with 60 g of Achyranthes radix (Achyranthe japonica Nakai), 180 g of rinsed Atractylodes rhizome, and 120 g of stir-fried Phellodendri cortex, and was extracted with 2 L of water (SMWW) or 30% ethanol (SMWE), for 3 h, by reflux. These extracts were then concentrated under reduced pressure and freeze-dried.

#### *4.2. Components Analysis of SMW*

Reference standards, palmatine, armepavine, berberine, atractylenolide III, and atractylenolide I, were purchased from Chemfaces (Hubei, China). After confirming compounds by comparing the retention time and absorption profile of the reference material, each component was quantified through the area comparison.

HPLC analysis was performed on an Acquity UPLC system (Waters, MA, USA) equipped with a quaternary pump, auto-sampler, and photodiode array detector with Acquity UPLC®BEH C18, 100 × 2.1 mm, 1.7 µm. A gradient elution with solvent A (0.1% phosphoric acid) and solvent B (acetonitrile), at a flow rate of 0.5 mL/min, was conducted as follows: 0–2 min, 2–2% B; 2–32 min, 2–50% B; 32–42 min, 50–100% B; 42–45 min, 100–100% B; 45–47 min, 100–2% B; and 47–50 min, 2–2% B. The detection wavelength was

set to 200 nm. The column temperature was maintained at 40 ◦C, and the injection volume was 2 µL.

#### *4.3. Animals*

Male Sprague Dawley (SD) rats (7 weeks) and male C57BL6 mice (7 weeks) were purchased from Orient Bio (Seongnam, Korea) and housed at a temperature of 22 ± 2 ◦C in a 50 ± 10% humidity-controlled room under a 12 h light/dark cycle. The animals were allowed *ad libitum* access to a laboratory diet and water. At the end point of the experiment, the rats were anesthetized using zoletil and sacrificed by cervical dislocation. No systemic adverse effects were observed following treatment with SMWW or SMWE, in any study group. The experimental design was approved by the Committee on Animal Care of the KIOM (approval No. 20-016), and the study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Bethesda, MD, United States).

#### *4.4. Hyperuricemia Induction and Sample Treatment.*

The uricase inhibitor PO was injected into rats, to induce hyperuricemia [43]. The rats were divided into the following seven groups (*n* = 6/group): (1) Controls (Con), (2) POtreated controls, (3) PO+200 mg/kg SMWW, (4) PO+400 mg/kg SMWW, (5) PO+200 mg/kg SMWE, (6) PO+400 mg/kg SMWE, and (7) PO+10 mg/kg allopurinol (AP). Rats in groups (2-7) were injected intraperitoneally with 150 mg/kg PO prepared in 0.5% carboxymethyl cellulose (CMC) with 0.1 M sodium acetate (pH 5.0) to induce hyperuricemia, while the normal control (1) rats were treated with 0.5% CMC with 0.1 M sodium acetate. SMWW, SMWE, and AP were dispersed in 0.5% CMC and administered by oral gavage, 1 h prior to PO injection.

#### *4.5. Analysis of Uric Acid in Serum*

Blood samples were collected via cardiac puncture, under anesthesia, 2 h after PO treatment. Serum was obtained by centrifugation at 3000× *g* for 10 min at 4 ◦C, after allowing the blood samples to clot for 2 h, at room temperature. The separated serum uric acid levels were determined, using an enzymatic-colorimetric method, using commercial assay kits (Biovision, Milpitas, CA, USA) according to manufacturer's protocols.

#### *4.6. Induction of Gouty Arthritis with MSU Crystals in Mice*

MSU was synthesized as previously described [44]. After acclimation, C57BL6 male mice (8 weeks old, 20-22g body weight) were divided into the following eight groups (*n* = 5/group): (1) normal controls, (2) MSU-crystal-treated, (3) MSU+100 mg/kg SMWW, (4) MSU+200 mg/kg SMWW, (5) MSU+50 mg/kg SMWE, (6) MSU+100 mg/kg SMWE, (7) MSU+200 mg/kg SMWE, and (8) MSU+1 mg/kg colchicine (Col). The right hind paw of each mouse in groups (2–8) was injected intradermally with MSU crystal suspension (4 mg/50 µL) in PBS with 0.5% Tween 80, while the normal control (1) mice were treated with PBS with 0.5% Tween 80. SMWW, SMWE, and Col were dispersed in 0.5% CMC and administered by oral gavage, 1 h before the MSU crystal injection, and then once daily, for 3 days. The experimental design is shown in Figure 3A.

#### *4.7. Assessment of Inflammatory Paw Swelling and Pain*

Inflammatory paw swelling was quantified by measuring the thickness of the MSUinjected paw, using a Vernier scale, 3 days after the induction of MSU. The change of thickness (mm) was calculated as follows: Change of thickness (mm) = MSU-treated paw thickness - normal control paw thickness [45]. The pain was measured by right and left hind-limb weight distribution, using a dynamic weight-bearing device (Bioseb, Boulogne, France), which was developed to measure the weight borne by each limb in freely moving animals [44,46]. The mice were placed in a small Plexiglas chamber (11.0 × 19.7 × 11.0 cm) with a floor sensor containing pressure transducer, for 2 minutes, and the analyzer recorded

the average weight in grams, for each limb put on the floor. All movements were filmed and validated according to the position of the mouse on the device, and the results were analyzed for the weight of the paw, which touches the floor in grams [47]. The relative right/left hind paws weight-bearing distribution was calculated by using the following equation: (weight on right hind limb / weight on left hind limb) × 100.

#### *4.8. Measurement of Inflammatory Cytokines and Mediators*

The levels of IL-1β, IL-6, TNF-α, and myeloperoxidase (MPO) were measured by using ELISA kits from R&D Systems (Minneapolis, MN, USA) and MyBioSource (San Diego, CA, USA) according to the manufacturers' protocols.

#### *4.9. Statistical Analysis*

The results were expressed as the mean ± standard error of the mean (SEM) and analyzed, using a one-way analysis of variance (ANOVA), followed by Dunnett's tests for multiple comparisons or unpaired Student's *t*-tests for two-group comparisons. Normality was performed by using Shapiro–Wilk's test. All analyses were performed, using Prism 7.0 (GraphPad Software, San Diego, CA, USA), and *p*-values < 0.05 were considered significant.

#### **5. Conclusions**

In conclusion, this study demonstrated that SMWW and SMWE equally reduced serum uric acid levels in PO-induced hyperuricemic rats. However, in a gouty arthritis animal model, SMWE more efficiently downregulated MSU-crystal-induced swelling and pain, and it exerted anti-inflammatory effects by suppressing proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and MPO activity. Moreover, berberine was found to be one of the most differentially abundant main active ingredients between SMWW and SMWE, while atractylenolide III was identified only in SMWE, both of which are known to elicit antiinflammatory effects. These observations show that 30% ethanol is an efficient solvent for SMW extraction with anti-gouty arthritis efficacy at the concentrations reduced compared with water extracts. Further studies should be conducted to determine whether SMWE has similar efficacy in clinical trials at lower doses than SMWW.

**Author Contributions:** Conceptualization, Y.M.L.; methodology, Y.M.L.; software, Y.M.L. and E.S.; validation, Y.M.L. and E.S.; formal analysis, Y.M.L.; investigation, Y.M.L. and E.S.; writing—original draft preparation, Y.M.L.; writing—review and editing, Y.M.L. and E.S.; visualization, Y.M.L. and E.S.; supervision, D.-S.K.; project administration, D.-S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was financially supported by grants from the Korea Institute of Oriental Medicine (KSN2012330). The funders have no role in designing the experiment and publication of the manuscript.

**Institutional Review Board Statement:** The experimental design was approved by the Committee on Animal Care of the KIOM (approval No. 20-016), and the study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Bethesda, MD, United States).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study is contained within the article.

**Acknowledgments:** The authors thank all of the colleagues who contributed to this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


#### **References**


*Article*
