Eupatilin Alleviates Hyperlipidemia in Mice by Inhibiting HMG-CoA Reductase

Artemisia princeps (family Asteraceae) is a natural product broadly used as an antioxidative, hepatoprotective, antibacterial, and anti-inflammatory agent in East Asia. In the present study, eupatilin, the main constituent of Artemisia princeps, was investigated as an antihyperlipidemic agent. Eupatilin inhibited 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HCR), an enzyme that is a therapeutic target for hyperlipidemia, in an ex vivo assay using rat liver. In addition, oral administration of eupatilin significantly lowered the serum levels of total cholesterol (TC) and triglycerides (TG) in corn oil-induced and Triton WR-1339-induced hyperlipidemic mice. These results suggest that eupatilin can alleviate hyperlipidemia by inhibiting HCR.


Introduction
Artemisia princeps (AP) (family Asteraceae) is a natural product that is broadly used as an antioxidative, hepatoprotective, antibacterial, and anti-infammatory agent in China, Japan, and Korea [1]. Eupatilin, the main component of AP, ameliorates gastric ulcers [2], motivates apoptosis of human gastric cancer cells [3], and suppresses the infammatory response induced by carrageenan through the control of nuclear factor-kappa B (NF-κB) [1].
Lipid metabolism homeostasis is maintained by a balance between lipid synthesis and degradation, and disruption in this balance can lead to hyperlipidemia [4]. Te enzyme 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HCR), a rate-limiting enzyme for the biosynthesis of cholesterol from acetate, is a therapeutic target for hyperlipidemia, and several HCR inhibitors have been developed [5]. Statins are HCR inhibitors widely used as a treatment for hyperlipidemia. Statins mediate their anti-infammatory efects by inhibiting NF-κB [6][7][8].
In addition, statins are efective in terms diabetic neuropathic pain through their anti-infammatory efects. Tese efects of statins may be like those of N-acetylcysteine [9,10]. However, although the anti-infammatory efects of eupatilin have been reported [1], its hypolipidemic efect has not been thoroughly studied.
Te present study aimed to investigate eupatilin as a natural antihyperlipidemic agent. We evaluated the HCRinhibiting activity of eupatilin and investigated its antihyperlipidemic efects. Tis study suggests eupatilin as an HCR inhibitor.

Partial Purifcation and Activity of HCR.
Te HCRinhibitory efect of eupatilin was analyzed according to the previously described method [5,11,12]. Te rate of NADPH oxidation was determined using HCR isolated from the liver of three 8-week-old male Sprague-Dawley (SD) rats (weight: 250-300 g) stimulated with 5% cholestyramine. To confrm the oxidation rate, a reaction mixture at pH 6.8 containing 0. 16

Induction of Hyperlipidemia in Mice.
To confrm the antihyperlipidemic efect of eupatilin, hyperlipidemia was induced in mice using corn oil [13]. Each group consisted of six mice. Corn oil (1 g/kg) was administered orally 2 hours after a single oral treatment of eupatilin (25 and 50 mg/kg) or lovastatin (10 mg/kg). Blood samples were collected by cardiac puncture under anesthesia 2 hours after corn oil administration.
In addition, hyperlipidemia was induced in mice using Triton WR-1339 [5]. Six mice were used for each group. Eupatilin (25 and 50 mg/kg) or lovastatin (10 mg/kg) was orally treated once daily for three days, and Triton WR-1339 was administered through the tail vein 16 hours after the last treatment. Fasting was maintained for 16 hours until administration with Triton WR-1339. Blood samples were collected by cardiac puncture under anesthesia 18 hours after administration of Triton WR-1339.

Determination of Serum TC, TG, and HDL Cholesterol
Levels. Serum TC, TG, and HDL cholesterol levels were measured using commercially available assay kits and previously published protocols [14][15][16]. Serum TC was quantifed using an assay kit for measuring TC to which the enzyme method of Allain et al. [14] was applied. 3.0 ml of enzyme solution was added to 0.02 ml of serum, mixed, and reacted at 37°C for 5 minutes. Te absorbance of the reactants was measured at a wavelength of 500 nm using a spectrophotometer (BioPhotometer, Eppendorf ), and the TC content was quantifed by substituting it into a calibration curve. Serum TG was measured using an assay kit for measuring TG according to the principle of the color development method using the enzymatic method of Sardesai and Manning [15]. 3.0 mL of enzyme solution was added to 0.02 mL of serum, mixed, and reacted at 37°C for 10 minutes. TG content was quantifed by measuring the absorbance of the reactant at a spectrophotometer (BioPhotometer, Eppendorf ) wavelength of 550 nm and substituting it into a calibration curve.
In addition, serum HDL-cholesterol was measured using an assay kit for measuring HDL-cholesterol [16]. 0.2 mL of separation solution was added to 0.2 mL of serum, mixed, and left at room temperature for 10 minutes. 0.1 mL of supernatant obtained by centrifugation at 3,000 rpm for 10 minutes was taken, mixed well with 3.0 mL of enzyme reagent, and reacted at 37°C for 5 minutes. Te absorbance was measured at a wavelength of 500 nm with a spectrophotometer (BioPhotometer, Eppendorf ), and HDL cholesterol content was quantifed by substituting it into a calibration curve.

Statistical Analysis.
All the data are expressed as a mean ± standard deviation, and statistical signifcance was analyzed using one-way ANOVA followed by the Student's t-test and the Student-Newman-Kelus test. Statistical signifcance was set at p < 0.05.

Results
Te HCR-inhibiting efect of eupatilin, the main constituent of AP, was evaluated to investigate the antihyperlipidemic efects of this natural herb. Eupatilin potently inhibited HCR activity with an IC 50 value of 34.2 μM (Figure 2).
In addition, because HCR inhibitors, including lovastatin, are used to treat hyperlipidemia, the antihyperlipidemic efects of eupatilin were investigated in corn oil-and Triton WR-1339induced hyperlipidemic mice. Orally treated eupatilin signifcantly reduced TC and TG levels in the serum of corn-oilinduced hyperlipidemic mice in a dose-dependent efect compared to those in the control group. Moreover, HDL cholesterol levels improved; however, the result was not signifcant ( Figure 3 and Table 1).
Eupatilin also signifcantly lowered TC and TG levels by 48.1% and 16.6%, respectively, in Triton WR-1339-induced hyperlipidemic mice when administered orally at a concentration of 50 mg/kg. In contrast to corn oil-induced hyperlipidemia in mice, orally administered eupatilin signifcantly increased HDL cholesterol levels in Triton WR-1339-induced hyperlipidemic mice. Notably, these antihyperlipidemic efects of eupatilin were dose-dependent ( Figure 4 and Table 2).

Discussion
Homeostasis is important for lipid metabolism, and hyperlipidemia develops when homeostasis is disrupted. Te importance of HCR as a therapeutic target for hyperlipidemia has been demonstrated in several studies. HCR, which acts as a rate-limiting enzyme in the process of cholesterol biosynthesis from acetate, is typically inhibited by statins, which have been long used as a treatment for hyperlipidemia [4,5]. Statins are generally safe drugs; however, they are related with several side efects such as muscle pain [17] and the risk of diabetes [18]. In addition, the therapeutic efect of statin may be limited in certain age groups [19]. Herbal medicines have gained increasing attention for their antihyperlipidemic efects as safe and highly efective drugs to replace statins. We have previously analyzed the antihyperlipidemic efect of natural ingredients with NF-kB inhibitory efects by reversely interpreting the results of studies showing that statins exhibit anti-infammatory efects by inhibiting NF-κB inhibition [6][7][8].
Eupatilin, the main component of AP, is widely used to treat gastric infammation in East Asia. Eupatilin exhibits anti-infammatory efects by inhibiting NF-κB [1]. Here, we evaluated the HCR-inhibiting and antihyperlipidemic efects  Te serum levels are expressed as the mean ± S.D, (n � 6). a,b,c,d Items with the same letter in each column were not signifcantly diferent (p < 0.05).
Biochemistry Research International 3 of eupatilin. We revealed that eupatilin efectively inhibited HCR and signifcantly reduced serum TC and TG levels in hyperlipidemic mouse models, demonstrating its capability as a curative agent for hyperlipidemia. However, eupatilin showed a diferential HDL cholesterol-enhancing efect in Triton WR-1339-and corn oil-induced hyperlipidemic mice. Triton WR-1339 induces oxidative reactions and prevents the catabolism of triacylglycerol-rich lipoproteins by lipoprotein lipase (LPL) to induce hyperlipidemia [20]. In contrast, corn oil acts as a fat source when administered to animals [21], and the transient increase in this fat is thought to induce hyperlipidemia. In addition to NF-kB inhibition, eupatilin exhibits anti-infammatory efects through antioxidant effects [22]; therefore, it exhibits a superior efect in recovering HDL cholesterol levels in Triton WR-1339-induced hyperlipidemia mice that exhibit oxidative action. Tis study demonstrates that eupatilin can reduce hyperlipidemia by inhibiting HCR activity. However, longterm administration experiments, toxicity tests, and clinical trials are needed to confrm the therapeutic potential of eupatilin in treating hyperlipidemia.

Conclusion
To discover a natural component that suppresses hyperlipidemia, the efect of eupatilin, the main component of AP widely used in infammatory diseases in East Asia, was confrmed. Eupatilin, which inhibits HCR, lowered TC and TG in mice induced with hyperlipidemia using corn oil or Triton WR-1339. In addition, HDL cholesterol, lowered by Triton WR-1339, was increased by eupatilin. In conclusion, eupatilin exhibits antihyperlipidemic efects through HCR inhibition.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest. Serum level (mg/dl) Figure 4: Efects of eupatilin on serum TG, TC and HDL cholesterol levels in Triton WR-1339-induced hyperlipidemic mice. Triton WR-1339 was administered intravenously in the control, Eu25, Eu50, and Lova10 groups. Te test agents (control, saline alone; Eu25, 25 mg/kg of eupatilin; Eu50, 50 mg/kg of eupatilin; Lova10; 10 mg/kg of lovastatin) were administered orally once a day for three days. Te last oral administration was conducted 16 h before Triton WR-1339 treatment. All values are represented as mean ± SD (n � 6). #signifcantly diferent compared with the normal group (p < 0.05) * signifcantly diferent compared with the control group (p < 0.05). Te serum levels are expressed as the mean ± S.D, (n � 6). a, b, c ,d, e Items with the same letter in each column were not signifcantly diferent (p < 0.05).