Antioxidant and Antiadipogenic Activities of Galkeun-Tang, a Traditional Korean Herbal Formula

Galkeun-tang (GKT; Galgen-tang in Chinese and Kakkon-to in Japanese), a traditional herbal formula, has been used for treatment of the common cold. Here, we report in vitro antioxidant and antiadipogenic effects of GKT. GKT increased the activities of scavenging 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. GKT also significantly reduced the malondialdehyde (MDA) generation during low-density lipoprotein (LDL) oxidation and the electrophoretic mobility of oxidized LDL, indicating inhibitory effects of GKT on Cu2+-mediated oxidation of LDL. Regarding antiadipogenic activity, GKT treatment significantly suppressed lipid accumulation, triglyceride production, and glycerol-3-phosphate dehydrogenase (GPDH) activity in differentiated 3T3-L1 adipocytes. Consistent with this, GKT significantly reduced the secretion of leptin, a major adipokine, in differentiated 3T3-L1 adipocytes. Overall, our findings suggest that GKT has the potential for antioxidative and antiadipogenic properties.


Introduction
Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and antioxidative defenses [1]. Disruption in normal redox signaling can mediate toxic effects and, thus, is closely associated with various human diseases [2]. Obesity is a metabolic disorder caused by excess fat accumulation in adipose tissue [3]. Recent papers have confirmed a critical role for oxidative stress in the pathogenesis of obesity or its associated diseases [4][5][6]. A change in protection against antioxidative mechanisms was observed in an obese rodent model and in obese human males [7,8]. Thus, the development of antioxidants could be a valuable approach for treating and preventing obesity or its related diseases. Antioxidative activities of synthetic or natural products have been reported in obesity models. Natural products are considered particularly attractive antiobesity drug candidates because of their higher efficacies and fewer side effects.
Many traditional herbal medicines have attracted increasing attention for their complementary therapeutic effects with few or no side effects compared with Western medicines [9,10]. Galkeun-tang (GKT; Galgen-tang in Chinese and Kakkon-to in Japanese), a traditional herbal formula, has been used widely for treatment of the common cold, flu, and fever. In recent research papers, several pharmacological activities of GKT have been reported including antiviral, anti-inflammatory, and immune-regulating activities [11][12][13][14][15]. However, the effect of GKT on obesity or obesity-related diseases has not been reported. The pathogenesis of obesity and its associated diseases is closely related to an inflammatory reaction caused by nutrient excess and imbalance that has been termed "metainflammation" [16]. Thus, we hypothesized that the previously reported anti-inflammatory activity of GKT may influence obesity or its related diseases.
We, therefore, investigated the antioxidant and antiadipogenic effects of GKT. We analyzed its scavenging activities on 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals in in vitro systems. The effect on low-density lipoprotein (LDL) oxidation was assessed by measuring production of malondialdehyde (MDA). In addition, its inhibitory effects on adipogenesis, the process by which preadipocytes become differentiated adipocytes [17], were determined by Oil Red O staining and assays for triglyceride content,  For HPLC analysis, lyophilized GKT extract was weighed (200 mg) into a 20 mL flask and distilled water was added to the volumetric mark, and then the mixture was passed through a 0.2 m syringe filter before injection into the HPLC system. 2.17. Statistical Analysis. All data were presented as mean ± standard error of the mean (S.E.M.). Group differences were assessed by one-way ANOVA and post hoc Tukey's multiple comparison test using Graphpad InStat ver.3.10 (Graphpad Software, Inc., San Diego, CA). Significance of differences from the normal control was taken as < 0.05.

HPLC Analysis of GKT.
All calibration curves were obtained by assessment of peak areas from standard   ( 2 ), limit of detection (LOD), and limit of quantification (LOQ) of the six marker compounds are summarized in Table 2. Using optimized chromatography conditions, a three-dimensional chromatogram was obtained using the HPLC-PDA detector ( Figure 1). The concentrations of the six marker compounds were 2.01-12.17 mg/g and are summarized in Table 3.

Antioxidant Activity of GKT.
To evaluate the antioxidant activity of GKT, we tested its scavenging activities on ABTS and DPPH radicals. The ABTS radical scavenging activity of GKT is presented in Table 4. The extracts of GKT showed a dose-dependent radical scavenging activity. The concentration of GKT required for 50% inhibition (IC 50 ) of ABTS radicals was 51.16 g/mL, while the IC 50 value of ascorbic acid, as a positive control, was 3.22 g/mL. The antioxidant activities obtained for GKT by the DPPH method are shown in Table 5. Similar to the ABTS assay, GKT reduced DPPH radical formation in a concentration-dependent manner. The IC 50 of GKT against DPPH radicals was 242.96 g/mL, while the IC 50 value of ascorbic acid was 10.43 g/mL.

Effect of GKT on Cu 2+ -Mediated Oxidation of LDL.
The generation of MDA equivalents during LDL oxidation was estimated by the TBARS assay. As shown in Figure 2(a), when LDL was incubated with CuSO 4 for 6 h, a significant     increase in TBARS was detected. In contrast, GKT significantly reduced the amount of TBARS formed in a dosedependent manner (IC 50 : 53.23 g/mL). The alteration of mobility in agarose gel electrophoresis reflects the increase in the negative charge of LDL particles which occurs during oxidation [20]. When the oxidation was carried out in the presence of GKT, the increase in electrophoretic mobility of oxLDL was significantly reduced (Figure 2(b)). These data suggest that GKT has an inhibitory effect on LDL oxidation.

Cytotoxic Effects of GKT in 3T3-L1 Cells.
To evaluate the possible cytotoxicity of GKT against 3T3-L1 preadipocytes, the cells were treated with various concentrations of GKT for 24 h. As shown in Figure 3(a), GKT had no cytotoxicity against 3T3-L1 preadipocytes. Additionally, the cytotoxicity of GKT was assessed in the differentiated adipocytes. During differentiation for 8 days, the cells were exposed to various concentrations of GKT. No significant cytotoxic effect was observed in GKT-treated adipocytes (Figure 3(b)).

The Inhibitory Effects of GKT on Adipogenesis of 3T3-L1
Adipocytes. Triglyceride production is one of the important events which occurs during adipogenesis [21]. Oil Red O staining was carried out to examine lipid accumulation in the differentiated 3T3-L1 adipocytes. The number of lipid droplets detectable with Oil Red O staining was obviously increased in adipocytes compared with preadipocytes ( Figure 4(a)). However, GKT treatment significantly reduced lipid accumulation compared with the untreated differentiated cells. In addition, the intracellular triglyceride content was measured in 3T3-L1 adipocytes treated with GKT. Consistent with the results of Oil Red O staining, triglyceride production was significantly increased in the differentiated adipocytes compared with preadipocytes, but GKT significantly inhibited triglyceride production compared with untreated differentiated cells (Figure 4(b)).
GPDH is an enzyme that generates glycerol-3-phosphate from dihydroxyacetone phosphate for lipid biosynthesis in adipocytes [22]. As shown in Figure 5(a), treatment with GKT at the concentration of 25∼400 g/mL significantly inhibited the activity of GPDH compared with untreated differentiated adipocytes. Consistent with this, GKT also had a suppressive effect on secretion of leptin, a key adipokine [23], compared with the untreated differentiated cells ( Figure 5(b)). Similarly, treatment with GW9662, the positive control, dramatically inhibited adipogenesis in 3T3-L1 cells.

Discussion
In the present study, we demonstrate that a traditional herbal medicine GKT has antioxidant and antiadipogenesis properties. For quality control of the GKT extract, we performed a quantitative determination of the six main components in GKT using HPLC coupled with a PDA. The investigated components were as follows: puerarin form Puerariae Radix, cinnamaldehyde and cinnamic acid from Cinnamomi Ramulus, paeoniflorin from Paeoniae Radix, and liquiritin and glycyrrhizin from Glycyrrhizae Radix et Rhizoma. The optimized HPLC-PDA method was applied for simultaneous quantitation of the six components in GKT (Figure 1). Among these components, puerarin and glycyrrhizin, which are marker components of Puerariae Radix and Glycyrrhizae Radix et Rhizoma, were detected at 10.29 mg/g and 12.17 mg/g, respectively, as the major components of GKT (Table 3). The establishment of this HPLC-PDA method will be helpful in improving quality control of GKT.
In in vitro assay systems, GKT showed significant scavenging effects against ABTS and DPPH radicals (Table 4)   Evidence-Based Complementary and Alternative Medicine triglyceride production, GPDH activity, and leptin production in adipocytes without cytotoxic effects (Figures 3-5).
Oxidative stress plays an important role in the pathogenesis of various diseases including obesity. Thus, the "oxidative stress paradigm" is an appealing concept for developing novel therapeutics [24]. Excess oxidative stress in obese patients coincides with fat accumulation in adipocytes [4]. We induced the cellular differentiation of 3T3-L1 mouse preadipocytes into adipocytes. This cell line is useful for studying adipogenesis [25]. After adipocyte differentiation, a significant increase of intracellular lipid accumulation compared with untreated preadipocytes was observed by Oil Red O staining. In contrast, GKT significantly inhibited the amount of lipid accumulation compared with untreated differentiated adipocytes (Figure 4(a)). Microscopy further confirmed the inhibitory effect of GKT on adipogenesis. GKT treatment markedly reduced the increase in the number and size of adipocytes, a hallmark of adipocyte endocrine function [26]. Lipid droplets consist of a core of lipid esters and a surface lined with a phospholipid monolayer, and, in adipocytes, the lipid ester core contains triglycerides [27][28][29]. Consistent with the results of Oil Red O staining, GKT significantly decreased the content of triglyceride in adipocytes (Figure 4(b)). Furthermore, we evaluated the GPDH activity in differentiated 3T3-L1 adipocytes. GPDH is activated strongly in mature adipocytes and plays a role in the triglyceride biosynthesis pathway [30,31]. GPDH enzyme activity was significantly decreased in GKT-treated adipocytes ( Figure 5(a)). The level of another key factor in adipogenesis, leptin, was measured in adipocytes differentiated in the absence or presence of GKT. Leptin is an adipokine exclusively produced by adipocytes in proportion to triglyceride accumulation [32]. As expected, GKT significantly inhibited the level of secretion of leptin in differentiated 3T3-L1 adipocytes ( Figure 5(b)). Similar to our study, several groups have reported dual effects of natural products, including purple sweet potato [33], safflower seed [34], and buckwheat sprout [35], on oxidative stress and adipogenesis. Together, these results suggest the potential of natural products, including herbal medicines, as adipogenesis-preventing substances with antioxidant properties.
Interestingly, several herbal components of GKT have been reported to have antioxidant properties or antiobesity effects. Pueraria lobata extract ameliorated impaired glucose and lipid metabolism in obese mice [36]. Pueraria lobata extract also inhibited tert-butyl hydroperoxideinduced ROS generation [37]. An extract of Cinnamomum cassia twigs inhibited adipocyte differentiation via activation of the insulin signaling pathway in 3T3-L1 preadipocytes [38]. Ephedra sinica extract exerted antioxidant effects by accelerating free radical scavenging activity and oxidant reducing power [39]. These data could support antiadipogenic/antioxidant activities of GKT. Additional studies will be required to confirm the antioxidant/antiobesity effects of GKT using a high fat diet-fed obese animal model.