## 4 Discussion  
Tilianin, a bioactive compound of _Agastache rugosa_, has been reported to have various functions as mentioned in introduction. However, the anti-obesity effect of tilianin remains unclear. _A. rugosa_, which has antifungal, antibacterial and antipyretic effects [25], also showed anti-adipogenic properties in this study. We evaluated the anti-adipogenic effects of ARE and tilianin using 3T3-L1 cells. Tilianin is plant-derived flavonoids abundant in _A. rugosa_. Flavonoids exhibit antioxidant activities and play various biological and pharmaceutical roles as plant secondary metabolites. Moreover, some research suggest that plant-derived flavonoids are endowed with obesity prevention or treatment [26; 27]. The effects of flavonoids on obesity that recently researched are: diminution of food intake and regulation of appetite; less intestinal fat absorption; control of adipocyte differentiation, adipogenesis and lipolysis [28]. Here, ARE and its flavonoid compound tilianin were clearly shown to have anti-obesity effects on 3T3-L1 adipocytes. More prominently, tilianin dose-dependently suppressed lipid accumulation in differentiated adipose cells. Lipid accumulation (%) is calculated by relative oil-red-o absorbance (485 nm) that eluted by treating 2-propanol on colored cells. Oil-red-O dyes lipid droplets which is known to be composed of neutral lipids, triglyceride and sterol esters. Most of the lipid droplets in white adipose tissues  
Figure 5: Anti-lipogenic effect of ARE and tilianin on mRNA and protein level and lypolytic effect of tilianin on mRNA level of 3T3-L1 cells. (**A**). mRNA expression of ARE-treated group (n = 3). (**B**,**C**). mRNA expression of tilianin-treated group (n = 4). (**D**). SREBP1 protein expression of ARE-treated group (n = 3). (**E**). SREBP1 protein expression of tilianin-treated group (n = 3). Different letters (a–e) indicate significantly different values in samples (\(p<0.05\), ANOVA Duncan’s test). consists of triglyceride [29]. Differentiated 3T3-L1 adipocytes which behaves similar to white adipose tissue form lipid droplets full of triglyceride. Therefore, we can conclude that lipid accumulation decreases, triglyceride content would decrease as well. Especially tilianin showed remarkable effects than ARE in lipid accumulation and mRNA expression levels. It can be assumed that single compounds may have more clear effects because it is a highly refined matter. The possibility cannot be ruled out that ARE is consisted of various compounds so other components may have offset the anti-obesity effects in ARE. Inhibition of lipid droplet accumulation in adipocytes can have a variety of causes. [PERSON] et al. demonstrated that the key adipogenic transcription factors, PPAR\(\gamma\) and C/EBP\(a\), cooperate with the activation of adipocyte gene programs to result in adipogenic differentiation [30]. According to the related studies to date, it can be hypothesized that ARE and tilianin inhibit the differentiation of preadipocytes into adipocytes. In order to investigate the inhibitory activity of ARE and tilianin on the expression of PPAR-\(\gamma\) and C/EBP\(\alpha\), the expression of these factors was observed at the gene and protein level, and it was confirmed that the expression was reduced. Therefore, the hypothesis can be demonstrated that the inhibition of fat accumulation by ARE and tilianin is due to the inhibition of the adipogenic differentiation by these bioactive substances. Our results showed that tilianin treatments clearly appeared significant diminution of the mRNA levels of the adipogenesis-related genes PPAR-\(\gamma\), C/EBP\(\alpha\) and FABP4 and the lipogenesis-related genes SREBPP1, ACC and FAS. Additionally, the levels of CPT1, a lipolysis factor, increased following tilianin treatment. ARE exhibited anti-obesity effects, with ARE treatment causing decreases in lipid droplets by 20% compared with untreated cells and causing decreases in mRNA and protein levels of adipogenesis- and lipogenesis-related genes.