Antioxidant and Anti-Obesity Potentials of Korean-Native Wild Vegetables (Allium species)

Four Korea-native wild vegetables belonging to the Allium genus (Allium tuberosum Rottl., Allium senescens L., Allium thunbergii G. Don., and Allium sacculiferum Maxim.) were investigated for their antioxidant and anti-obesity potential, in order to promote the utilization of wild edible plants for the development of potential new crops. In this study, we compared the antioxidant activity of the plant extracts by ABTS+ and DPPH scavenging method. We then compared the anti-adipogenic effects of the extracts in adipocytes. We quantified the phenolics in the extracts using high-performance liquid chromatography (HPLC) to identify the bioactive compounds. A. tuberosum and A. sacculiferum had a higher total phenolic content (19.0 ± 0.3 and 19.6 ± 0.7 mg GA/g extract, respectively) than A. senescens and A. thunbergii (16.0 ± 0.2 and 14.5 ± 0.7 mg GA/g extract). Caffeic acid, an antioxidant and anti-adipogenic compound, was detected in A. tuberosum and A. sacculiferum, but not in A. senescens and A. thunbergii. A. tuberosum, and A. sacculiferum showed superior antioxidative and anti-adipogenic activities compared to A. senescens and A. thunbergii. Therefore, A. tuberosum and A. sacculiferum, both of which contain antioxidant and anti-adipogenic phenolics, such as caffeic acid, present a therapeutic potential against obesity. Various phenolic compounds, which have been found to differ among Allium spp., may partially account for differences among the Allium spp. with respect to their antioxidant and anti-obesity effects.


Introduction
Globally, obesity has emerged as a major cause of various metabolic diseases, such as diabetes, cardiovascular disease, and osteoporosis. It is known that high-fat or highcalorie diets can cause hyperlipidemia, obesity, increased oxidative stress, and increased inflammation [1]. Oxidative stress disrupts the balance between reactive oxygen species (ROS) and antioxidant defense systems [2]. Oxidative stress causes cellular DNA damage and the oxidation of enzyme cofactors, thus resulting in various diseases such as cancer, neurodegenerative diseases, and aging processes [3]. In addition, oxidative stress promotes adipogenesis through NADPH oxidase, a major source of intracellular lipid and ROS generation [4]. Oxidative stress, including free radicals and ROS, can be inhibited or eliminated by enzymatic and non-enzymatic antioxidants to maintain cell balance [5]. According to recent studies, maintaining hypoglycemia, regulating the body's antioxidant Horticulturae 2021, 7, 541 2 of 11 system and insulin, inhibiting the differentiation and proliferation of pre-adipocytes, and suppressing adipogenesis are all thought to suppress obesity [6].
Plants generate specialized metabolites in order to protect themselves against insects, viruses, and dispersing animals [7,8]. These metabolites include alkaloids, glycosides, amines, steroids, flavonoids, and related metabolites [7,8]. They possess biological activities and are mostly found in the leaves of various plants [7]. Among plant metabolites, phenolics have various biological effects, such as antioxidant, anti-microbial, and anti-diabetic properties [9]. In particular, phenolics are compounds directly linked to non-enzymatic antioxidants because of their reactivity as hydrogen or electron donors and reactivity with other antioxidants [10]. Phenolics are useful natural compounds capable of preventing oxidative stress and are considered important in food, pharmaceutical, and cosmetic industries [11].
Wild vegetables and flowers of the Allium spp. belong to the Amaryllidaceae family, with approximately 977 different spp. of garlic, onions, chives, shallots, and leeks [12]. The most important Allium spp. contain flavonoids, organosulfur compounds, and saponin. Allium tuberosum (A. tuberosum) grows in the wild in the central and northern parts of Asia, such as Japan, China, Korea, India, Nepal, Thailand, and the Philippines. Of these countries, only China, Korea, and Japan use A. tuberosum as a food source [12]. In Korea, A. tuberosum grows in the mountains, and its industrial cultivation has increased due to the development of greenhouses, pest control technology, and pharmaceuticals. A. tuberosum is a greenyellow vegetable that contains many nutrients, such as carotene, vitamin B2, vitamin C, calcium, and iron. It is known to have anti-cancer [13], anti-bacterial [14], anti-diabetic, hepatoprotective [15], and anti-obesity activities [16]. Other Allium spp. that are cultivated in Korea include A. anisopodium, A. cyanem, A. cyanem var. deltoides, A. deltoidefistulosum, A. senescens, A. thunbergii, and A. sacculiferum. [12]. Of these, A. senescens, A. thunbergii, and A. sacculiferum have anti-obesity, anti-cancer, and anti-inflammatory activities [16][17][18][19]. Although the classification and growth among Allium spp. in Korea are being studied, studies on their biological activity and phenolic profiling are scarce [20,21].
In this study, we analyzed the general growth characteristics of four Allium spp. (A. tuberosum, A. senescens, A. thunbergii, and A. sacculiferum) in identical environmental conditions and determined their antioxidant and anti-adipogenic activities. We also investigated their bioactive components by total phenol (TP) content analysis and phenolic profiling.

Plant Material and Growth Characteristics
The plant samples of Allium spp. (A. tuberosum, A. senescens, A. thunbergii, and A. sacculiferum) were collected from Korea. Voucher specimens were deposited at the Korea Medicinal Resources Herbarium, Eumseong, Korea (Table 1). Allium spp. seeds were sown in 200-cell plug trays (60 cm × 41 cm × 5 cm) containing commercial medium (Baroker medium, SeoulBio, Eumseong, Korea) and were maintained at 20 ± 5 • C in a greenhouse. After 15 days, the seedlings were cultivated in the field.
Allium spp. were harvested after 2 months (15 June 2018), and their growth characteristics were examined (Figure 1). For subsequent experiments, 50 g of Allium spp. was extracted with water (500 mL), repeated 3 times, at room temperature for 24 h and concentrated using an evaporator system under reflux (BÜCHI, Meierseggstrasse, Flawil, Switzerland). Allium spp. extracts were freeze-dried.

Cell Viability
Cell viability was measured with CellTiter 96 ® AQueous One Solution according to the manufacturer's instructions (Promega, Madison, WI, USA) [22]. The 3T3-L1 pre-adipocytes were cultured in 96-well plates at 1 × 10 5 cells/mL for 24 h and were treated with Allium spp. extracts. After 24 h, the absorbance was measured at 490 nm using a multi-plate reader (Synergy H1, Biotek, Winooski, VT, USA).

ORO Staining
Cultures were fixed for at least 1 h with 10% formalin diluted in water, in a 37 • C incubator, then washed twice with water and stained with a working solution of ORO (Sigma-Aldrich, St. Louis, MO, USA). To determine the extent of adipose conversion, 100% isopropanol was added to the stained tissue [24]. The absorbance of the extracted dye was read at 510 nm using a microplate reader.

ABTS Radical Scavenging Assay
The ABTS + scavenging assay was performed as described by Tanruean et al., with slight modifications [26]. The ABTS + solution was prepared with ABTS tablets (Sigma-Aldrich, St. Louis, MO, USA) and potassium persulfate (2.6 mM) at 4 • C for 16 h with the light blocked. Absorbance was measured at 734 nm using a multi-mode microplate reader. The radical scavenging activity was expressed as IC 50 value, a concentration that scavenges 50% of the radicals.

Determination of TP Content
The TP content was analyzed using the Folin-Denis method [27]. Briefly, 500 µL of each extract (3 mg/mL) was added to 50 µL of Folin-Ciocalteu reagent (Sigma-Aldrich, MO, USA). After 5 min, 100 µL of 20% sodium carbonate anhydrous saturated solution was added. After 1 h, the absorbance was measured at 725 nm using a multi-plate reader. The TP content was determined through a comparison with the standards. Gallic acid (Sigma-Aldrich, USA) was used as a standard, and the TP content (mg/g) was determined using the following equation (R 2 = 0.999): where Y is absorbance and X is measured concentration (mg/mL).

Statistical Analysis
All experimental results are presented as the mean ± standard deviation (SD) of three independent experiments. The statistical significance of the differences was calculated using a one-way analysis of variance (ANOVA) using Duncan's test, and correlations were calculated using Pearson's correlation coefficient (Statistical Package for the Social Sciences, ver. 21.0 for Window).

Growth Characteristic of Allium Spp.
In this study, the growth characteristics of four Allium spp. (A. tuberosum, A. senescens, A. thunbergia, and A. sacculiferum) were measured immediately after cultivation ( Figure 1 and Table 2). The total length was similar among the Allium spp., at 212-244 mm, except for A. sacculiferum, which was 110.8 mm in length. The stem diameter and root length differed considerably between A. tuberosum and A. sacculiferum, but the number of leaves did not show a significant difference among species.

Antioxidant Activities of Allium Spp.
The antioxidant activity of Allium spp. were evaluated using DPPH and ABTS free radical scavenging methods, which are common and rapid methods for in vitro antioxidant assay [29]. The IC50 values of Allium spp. for DPPH and ABTS + scavenging activity varied from 694.2 to 980.0 and from 44.0 to 396.2 μg/mL, respectively ( Table 3). The extracts of A. tuberosum (694.2 ± 10.0 μg/mL) and A. sacculiferum (723.1 ± 12.3 μg/mL) showed higher scavenging activity for DPPH free radical without significant differences between them. The extract of A. sacculiferum showed the highest ABTS + scavenging (44.0 ± 0.7 μg/mL). The highest yield of Allium spp. was approximately 55% and was consistent with all spp. except for A. tuberosum.

Antioxidant Activities of Allium Spp.
The antioxidant activity of Allium spp. were evaluated using DPPH and ABTS free radical scavenging methods, which are common and rapid methods for in vitro antioxidant assay [29]. The IC 50 values of Allium spp. for DPPH and ABTS + scavenging activity varied from 694.2 to 980.0 and from 44.0 to 396.2 µg/mL, respectively ( Table 3). The extracts of A. tuberosum (694.2 ± 10.0 µg/mL) and A. sacculiferum (723.1 ± 12.3 µg/mL) showed higher scavenging activity for DPPH free radical without significant differences between them. The extract of A. sacculiferum showed the highest ABTS + scavenging (44.0 ± 0.7 µg/mL). The highest yield of Allium spp. was approximately 55% and was consistent with all spp. except for A. tuberosum.

Cell Viability of Allium Spp.
The viability of 3T3-L1 cells, when treated with the Allium spp. Extract, was investigated using the MTS assay, which is commonly used for cell viability analyses by measuring the activity of mitochondrial dehydrogenase (Figure 2) [30]. There was no difference observed in the viability of 3T3-L1 cells when treated with 25-200 µg/mL (≥100%) of Allium spp. extract. It has been reported that cells are considered highly viable if the viability is more than 90% [31]. Thus, extracts of Allium spp. of concentrations up to 100 µg/mL (≥90%) were chosen for subsequent experiments.
The viability of 3T3-L1 cells, when treated with the Allium spp. Extract, was investigated using the MTS assay, which is commonly used for cell viability analyses by measuring the activity of mitochondrial dehydrogenase (Figure 2) [30]. There was no difference observed in the viability of 3T3-L1 cells when treated with 25-200 μg/mL (≥100%) of Allium spp. extract. It has been reported that cells are considered highly viable if the viability is more than 90% [31]. Thus, extracts of Allium spp. of concentrations up to 100 μg/mL (≥90%) were chosen for subsequent experiments.

Effect of The Differentiation Inhibition of Allium Spp. by ORO Staining
Adipogenesis has been reported to contribute to the differentiation of pre-adipocytes into adipocytes, lipid accumulation, and growth arrest [32]. Lipid droplet accumulation in adipocytes is known to be an indicator of adipogenesis [33]. Therefore, the anti-adipogenic effect of Allium spp. extracts, with a media of differentiation (MDI), were evaluated using ORO in 3T3-L1 cells ( Figure 3A). Microscopy revealed that the MDI positive control had more stained lipid droplets than in the undifferentiated negative controls. The amount was also significantly increased in the differentiated controls, to 2.01-fold higher than the undifferentiated control. Cells treated with extracts of A. tuberosum (50 and 100 μg/mL; 0.82 and 0.74-fold vs. MDI control, p < 0.05) and A. sacculiferum (50 and 100 μg/mL; 0.69 and 0.57-fold vs. MDI control, p < 0.05) had markedly decreased lipid droplets, as shown in Figure 3B.

Effect of The Differentiation Inhibition of Allium Spp. by ORO Staining
Adipogenesis has been reported to contribute to the differentiation of pre-adipocytes into adipocytes, lipid accumulation, and growth arrest [32]. Lipid droplet accumulation in adipocytes is known to be an indicator of adipogenesis [33]. Therefore, the anti-adipogenic effect of Allium spp. extracts, with a media of differentiation (MDI), were evaluated using ORO in 3T3-L1 cells ( Figure 3A). Microscopy revealed that the MDI positive control had more stained lipid droplets than in the undifferentiated negative controls. The amount was also significantly increased in the differentiated controls, to 2.01-fold higher than the undifferentiated control. Cells treated with extracts of A. tuberosum (50 and 100 µg/mL; 0.82 and 0.74-fold vs. MDI control, p < 0.05) and A. sacculiferum (50 and 100 µg/mL; 0.69 and 0.57-fold vs. MDI control, p < 0.05) had markedly decreased lipid droplets, as shown in Figure 3B.

Profiling of Phenolics in Allium Spp.
Chromatographic techniques are necessary for identifying bioactive compounds [34]. Thus, we performed a high performance liquid chromatography (HPLC) analysis to determine the phenolics present in Allium spp. Homogenetic acids were detected in all Allium spp. Additionally, caffeic acid and ferulic acid were detected in A. tuberosum and A. sacculiferum. Veratric acid and quercetin were detected in A. senescens, and ferulic acid, veratric acid, and quercetin were detected in A. thunbergii (Figure 4).

Profiling of Phenolics in Allium Spp.
Chromatographic techniques are necessary for identifying bioactive compounds [34]. Thus, we performed a high performance liquid chromatography (HPLC) analysis to determine the phenolics present in Allium spp. Homogenetic acids were detected in all Allium spp. Additionally, caffeic acid and ferulic acid were detected in A. tuberosum and A. sacculiferum. Veratric acid and quercetin were detected in A. senescens, and ferulic acid, veratric acid, and quercetin were detected in A. thunbergii (Figure 4).

Discussion
A. tuberosum is widely consumed as a traditional ingredient in Korea and has been used as an herbal medicine for a long time [12]. However, the biological activity of leeks grown in Korea is only known for only few of the Allium spp. There have been many studies analyzing the functional activity of Allium spp. methanol extracts in preventing obesity [16,17,19]. However, methanol extracts cannot be used as food. We selected three types of Allium spp. with anti-obesity effects, and A. thunbergii, which is often used interchangeably with A. sacculiferum although it was found to be an independent spp. In a recent study [35]. In this study, we investigated the growth characteristics, secondary metabolites, and beneficial effects of Allium spp. In Korea for use as food, and the correlation between them.
The antioxidant effect of plants is known to be beneficial not only for plants but also for humans. For example, various phenolic substances have been reported to have an excellent free radical scavenging ability. Free radicals in vivo affect aging and disease in living tissue. Plant phenolic compounds are known to exhibit physiologically active functions by binding phenolic hydroxyl groups to macromolecules, such as enzymes [36]. TP is known to have a high antioxidant activity, which is because the phenolic hydroxyl group (OH) of the compound binds to proteins and polymers. In general, it has been reported that as the content of TP increases, physiological activities such as antioxidant activity increase [37]. DPPH and ABTS radicals are relatively stable free radicals and are reduced by sulfur-containing amino acids such as cysteine, GSH, ascorbate, and butylated hydroxyanisole. Accordingly, DPPH and ABTS are widely used to analyze the in vitro antioxidant activity of various extracts [38]. On comparing the antioxidant effect of all Allium spp., A. sacculiferum was identified to have the highest radical scavenging effect and TP content.
Adipogenesis is similar to the development of obesity in humans, and the excessive differentiation of 3T3-L1 cells into pre-adipocytes can induce obesity; therefore, these cells have been widely used in studies that assess the characteristics of adipogenesis and obesity [39]. A correlation between antioxidants and adipogenesis has been reported to promote the conversion of pre-adipocytes to mature adipocytes [24]. Thus, to investigate the inhibitory effect of A. tuberosum and A. sacculiferum extracts, which were found to have high antioxidant activity, ORO staining was performed. They had an inhibitory effect on the formation of lipid droplets. Therefore, A. tuberosum and A. sacculiferum extracts, which have been confirmed to have antioxidant activity, can be utilized for their anti-adipogenic effects.
According to a number of recent studies, plant-derived phenolic compounds inhibit adipogenesis [40]. Phenolic profiling was performed utilizing an HPLC analysis to determine the component responsible for the inhibitory action of Allium spp. In our study, caffeic acid was detected in A. tuberosum and A. sacculiferum. Caffeic acid has been reported to be an anti-obesity agent [41]. Using our data, an analysis was conducted to determine the correlation between phenolics and the biological activities of Allium spp. (Table 5). The anti-adipogenic and antioxidant effects of A. tuberosum and A. sacculiferum may be due to its caffeic acid content.
Collectively, these results suggest that Allium spp. have potential antioxidant and antiadipogenic effects, which derive from the presence of phenolic compounds, particularly caffeic acid.

Conclusions
Among Allium spp., A. tuberosum and A. sacculiferum have the antioxidant potential of scavenging free radicals and also inhibit adipogenesis in 3T3-L1 cells by decreasing lipid accumulation. The properties of these species were found to be correlated with their phenolic content, specifically caffeic acid. Consequently, various metabolites and phenolic compounds were found to differ according to Allium spp., thus showing differences among the Allium spp., with respect to their beneficial biological effects.