A Novel Herbal Medicine for Dyslipidemia: Assessments in Experimental Models

Dyslipidemia substantially contributes to the risk of cardiovascular diseases. The polyherbal formulation has been a traditional therapeutic strategy used to treat dyslipidemia. This study was designed to evaluate the effects of a novel herbal medicine called “GANMO” on an experimental animal model with endogenous dyslipidemia and exogenous dyslipidemia. In the endogenous hyperlipidemia model, rats were previously treated with GANMO tablets and intraperitoneally injected with poloxamer 407 to induce hyperlipidemia. In the exogenous hyperlipidemia model, rats were given oral administration of oil-cholesterol mixture and GANMO for 4 consecutive weeks. Serum lipid profiles were assessed at all experimental animals. In both models, GANMO at both doses significantly decreased the serum total cholesterol (TC) level and non-high-density lipoprotein (HDL) cholesterol level as compared with the model group. HDL cholesterol levels increased in rats with high doses of GANMO compared to those with low doses. GANMO at both doses substantially reduced TG level in the endogenous hyperlipidemia model. In conclusion, GANMO tablets posed a positive effect on serum lipid modulations in dyslipidemia models.


Introduction
Dyslipidemia refers to the excess status of fatty substances including cholesterol, triglyceride, and decreased highdensity lipoprotein cholesterol (HDL-C) in the bloodstream [1]. Besides, dyslipidemia is a major contribution to the onset of cardiovascular diseases (CVD) such as atherosclerosis, myocardial infarction, and cerebral vascular accidents, which are the main causes of the global burden of diseases [2]. Currently, although statins (e.g., simvastatin, atorvastatin, and rosuvastatin) have been widely used to reduce plasma lipids, their usage may be limited due to their side effects such as hepatotoxicity, rhabdomyolysis, or skeletal muscle injury [3]. us, natural alternatives have been proposed to control lipid metabolism.
Using multiple herbs (i.e., polyherbal) has been used in the traditional medicine approach given that it can improve the effects of therapy in curing a specific disease [4]. Several previous studies in Asian countries such as Indonesia [5] or China [6] have attempted to produce polyherbal medicine to treat dyslipidemia. In Vietnam, we have produced a novel polyherbal medicine called "GANMO" in improving serum lipid profiles. Each drug tablet contains Pericarpium Citri Reticulatae, Atractylodes macrocephala Koidz., Reynoutria japonica Houtt., Folium Nelumbinis, Sargassum pallidum (Tum), Cassia tora L., Alisma plantago aquatica L., Fallopia multiflora ( unb.) Haraldson, Crataegus cuneara Sied. et Zucc., and Rheum palmatum L. Several previous studies have shown the benefits of these herbal medicines in reducing serum lipid levels [7][8][9]. is study aimed to assess the effects of GANMO tablets on serum lipid profiles of dyslipidemia experimental animals.

Animal Preparation.
Wistar rats (males and females, weighing 180-220 g) were provided by the Center of Experimental Animals, DanPhuong, Hanoi, Vietnam. Healthy Swiss albino mice (of both sexes, weighing 21-25 g) were originated from the National Institute of Hygiene and Epidemiology (NIHE), Vietnam. Animals were housed under the standard environment (temperature: 25°C ± 2°C and relative humidity: 80% ± 10%), 12-hour dark/light time (lights on at 6: 00 AM). We fed the mice with standard animal feed and allowed free access to water. After randomizing the rats into different intervention groups as well as before implementing the experiment, mice were transferred to the laboratory conditions for 7 days at the laboratory of Pharmacology Department, Hanoi Medical University. All protocols used in this study were approved by the Scientific Board Committee of Hanoi Medical University (ref. number: IRB00003121).

2.4.
Procedure. Exogenous dyslipidemia model: dyslipidemia was induced in rats by oral administration of cholesterol mixture (cholesterol 10%, cholic acid 1%, propylthiouracil (PTU) 0.5%, and peanut oil added to precisely 1 mL) for four weeks [10]. Wistar Albino rats (180-220 g) were divided into 5 groups with 10 rats per group. Rats were given oral medication twice per day, at least two hours apart: Rat's body weight was recorded at baseline, after 2 weeks, and after 4 weeks. On day 15 and day 29, rats fasted overnight. Blood was collected to measure serum TC, TG, and HDLconcentrations. LDL-C concentration was calculated using the Friedewald formula: LDL-C � TC−(HDL-C)−(TG/2.2) (mmol/L) [11].
Endogenous dyslipidemia model: poloxamer 407-(P-407-) induced dyslipidemia model was described by Millar et al. [12]. Our mice were randomly separated into five groups with 10 rats per group.
Group 1 (control group): mice were given oral distilled water 1 mL/100 g b.w/day and then received intraperitoneal (IP) injection 0.9% NaCl 10 mL/kg b.w on day 7. Group 2 (model group): mice were given oral distilled water 1 mL/100 g b.w/day and then received IP injection 2% P-407 at the dose of 200 mg/kg b.w on day 7 Group 3 (atorvastatin): mice were given oral atorvastatin at the dose of 100 mg/kg b.w/day and then received IP injection 2% P-407 at the dose of 200 mg/kg b.w on day 7 Group 4 (GANMO-low dose): mice were given oral GANMO at the dose of 30.2 g/kg b.w/day (equivalent to clinical dose); then received IP injection 2% P-407 at the dose of 200 mg/kg b.w on day 7. Group 5 (GANMO-high dose): mice were given per oral GANMO at the dose of 90.6 g/kg b.w/day (3 times as high as clinical dose) and then injected i.p. 2% P-407at the dose of 200 mg/kg b.w on day 7.
Blood was collected at 24 h after IP injection of P-407 and analyzed for serum lipids including TG, TC, and HDL-C. Non-HDL-cholesterol (non-HDL-C) was estimated: non-HDL-C � TC−(HDL-C).

Statistical
Analysis. Data were analyzed using SPSS software version 20.0. T-test and ANOVA tests were performed to examine the differences in serum lipid profiles in different groups. A p value <0.05 was considered to be statistically significant.

Effects of GANMO Tablets on Lipid Levels in Exogenous
Dyslipidemia Model. Table 1 shows that, for all time points, no significant difference in body weight was found among groups (p > 0.05). Table 2 shows that atorvastatin tended to decreased levels of TG, TC, and LDL-C, and GANMO at the dose of 45.3 g/kg had a tendency to reduce TG level as compared with the model group after 2 weeks of treatment, but no significant change was observed (p > 0.05). Figure 1 and Table 3 show that, after 4 weeks of treatment, in the groups treated with atorvastatin and GANMO, there was a considerable decrease in levels of LDL-C and TC as compared with the model group. In terms of the HDL-C level, the group treated with GANMO at the dose of 45.3 g/ kg tended to increase the HDL-C level, but no significant difference was observed (p > 0.05). No differential change was observed between groups treated with GANMO at the dose of 15.1 g/kg and the model group (p > 0.05). Table 4 illustrated that there was a dramatic development in all lipid levels in the model group as compared with the control group (p < 0.001). Table 5 shows that there was a substantial reduction in the TC level and non-HDL-C level and a significant increase in HDL-C level in the group treated atorvastatin as compared with the model group. GANMO at both doses of 90.6 g/kg b.w and 30.2 g/kg b.w significantly decreases TG level, TC level, and non-HDL-C level developing the HDL-C level as compared with the model group.

Discussion
In literature, the model of exogenous dyslipidemia induced by oil-cholesterol mixture (cholesterol with bile acid and hypothyroidism-inducing agents) is widely used to screen natural or synthetic drugs [13]. In this study, we evaluated the effects of GANMO tablets on the changes in serum lipid levels. e findings of this study suggested that there was no significant difference in body weight among intervention and control groups at all time points after four weeks of the high-fat diet. However, the model of hyperlipidemia was induced successfully through the significant change of serum biochemical indexes. In particular, rats in the model group given the cholesterol-rich diet exhibited high plasma lipid levels, including TC, TG, and LDL-C levels. ere was a significant improvement in serum lipid indexes in the group treated with GANMO tablets as compared with the control group after 4 weeks. e lipid-lowering effect of GANMO was a dose-dependent effect, and GANMO at a high dose posed a greater effect than the low dose.
A model of endogenous hyperlipidemia was developed by IP injection of P-407 200 mg/kg b.w. P-407, a polyetherbased nonionic surface-active-agent (surfactant), providing an attractive means of inducing hyperlipidemia because of its rapid onset and seeming lack of over toxicity as compared with Triton WR-1339. P-407 has been known to cause significant dose-dependent hypercholesterolemia and hypertriglyceridemia in rats via several mechanisms (e.g., inhibition of lipoprotein lipase, indirect stimulation of HMG-CoA (3-hydroxy-3-methylglutarylCo-A) reductase, and promotion of concentration of hepatic cholesterol content) [14]. Based on its mechanism and shreds of evidence of efficiency, the statin is chosen as the drug reference standard. It inhibits HMG-CoA reductase, which counters the effect of P-407, thus decreases the serum TC. In addition, it also lowers the level of LDL by lowering the level of its precursor (VLDL and IDL), which further enhances its lipidlowering effect. Currently, seven different statins are available generically, but only atorvastatin and rosuvastatin are used in high-intensity therapy [3].
Because of its safety, various P-407-induced hyperlipidemia models in rats and mice have been often conducted with the standard dose of 500 mg/kg. In this study, the dose of 200 mg/kg was chosen to evaluate and compare the effects of the regimens. Within 24 h of its IP injection, a profound hyperlipidemia state was achieved. Our finding showed that TG concentration increased substantially by 6.8-fold, and TC levels and non-HDL-C concentrations increased by 2.9fold and 4.1-fold, respectively. Based on the success of the P-407-induced hyperlipidemia model, the effects of the GANMO tablet could be evaluated precisely. e data indicated that there was a substantial decrease in the levels of TG, TC, and non-HDL-C and a significant increase in the HDL-C level in dyslipidemia mice treated with GANMO at both doses of 30.2 g/kg b.w and 90.6 g/kg b.w. Mechanisms of compounds in GANMO tablets in the treatment are very complex.

Conclusion
In conclusion, our results demonstrated that oral administration of GANMO at doses of 30.2 g/kg and 90.6 g/kg for consecutive 7 days significantly reduced levels of TC, TG, non-HDL-C and increased the HDL-C level in P-407-induced hyperlipidemic mice. e exogenous dyslipidemia model induced by oil-cholesterol mixture and oral administration of GANMO tablets for 4 weeks at both doses of 15.1 g/kg and 45.3 g/kg reduced TC level and non-HDL-C level in rats. Moreover, GANMO tablets at the dose of 45.3 g/ kg tended to increase HDL-C level as compared with the model group.
Data Availability e data used to support the findings of this study are available from the corresponding author upon request.