Hyperlipidemia presents as a principal factor of accelerating the progression of Cardiovascular disease (CVD) which results in morbidity and mortality in developed countries [21]. Treatments of lipid lowering is well-recognized as a notable strategy by all the health care organizations around the world for prevention of CVD [22]. A large number of natural plant products found in traditional Chinese medicine (TCM) have been proved to possess lipid-lowing activities with fewer side effects, which draw great attention worldwide [23]. These findings have enhanced the searching for the alternative drugs with fewer adverse effects to prevent the risk of CVD. The combination of berberine and evodiamine is from a two-herb Chinese medicinal formula composed of Coptis chinensis and Evodia rutaecarpa, which has been widely used to treat multiple diseases in terms of different ratios [24]. Berberine is the active ingredient of the traditional herbal Coptidis Rhizoma which has been investigated in many directions, including anti-inflammatory, anti-cancer,anti-microbial, as well as cardiovascular fields [7,8,9,10]. Evodiamine is a naturally indoloquinazoline alkaloid isolated from Evodia rutaecarpa, attracting close attentions with its anti-cancer, anti-inflammation, and anti-obesity effects [13,14,15,16,17]. Many researches have indicated that both of berberine and evodiamine exerted on regulation of lipid metabolism [18, 25], but whether they have a synergistic effect on preventing hyperlipidemia when combined together remains completely unclear.
In the present study, the effect of the berberine combined with evodiamine was compared to the effects of a single herb on the parameters of hyperlipidemia in HFD-fed rats. The current study demonstrated that berberine, evodiamine, and their combinations could obviously reduce the serum lipid hyperlipidemic rats induced by HFD. Moreover, the combination of berberine and evodiamine notably decreased the content of TC in serum and liver, which showed best lipid-lowing effect compared with the either drug alone. The results appeared to be show a synergistic action of berberine and evodiamine as the effect on serum TC reduction. The underlying mechanism was exerted by the characterization of key proteins involved in the process of cholesterol absorption.
Diet habits play a significant role in the development of cholesterol metabolism. It is believed that the consumption of high fat diet which contains of saturated fats is the major risk factor for development of obesity and metabolic syndrome [26]. In this study, the hyperlipidemic animal model was successfully established in rats by the induction of high-fat, high-cholesterol diet, as evidenced by increased lipid parameters in serum and liver. In our study, a dramatic increase in serum lipid levels of TC, TG, LDL-C, and hepatic TC levels was observed in hyperlipidemic rats induced with HFD in 4 weeks (Fig. 2). After administration with berberine, evodiamine, and their combination, the elevated serum levels of TC, TG, and LDL-C were significantly decreased at eight week (Fig. 3). Meanwhile, cholesterol content in liver was obviously decreased after the supplementation of BB + EV. Notably, treatment with BB + EV showed the best effect on normalizing the serum TC, TG, and hepatic TC concentrations in 8 weeks, which appeared superior to either herb alone. However, BB and BB + EV did not develop a significant effect on body weight compared with HFD group (Fig. 1a, b), which agreed with the study of Yanwen Wang et al. [12], who found that berberine did not show a significant effect on body weight. Berberine was conformed to possess obvious cholesterol-lowering effect on serum TC, LDL-C, or non HDL cholesterol (non HDL-C) in plenty of researches [12, 27,28,29]. Evodiamine was reported to have effect on lipid metabolism by regulation the key genes in fat synthesis [18]. The reductions of liver cholesterol might be a result of decreased cholesterol input arise from the inhibition of intestinal cholesterol absorption [30]. Our research showed that the combination of berberine and evodiamine had the best lipid-lowing effect on lipid profile in serum and liver, compared to either drug alone. Therefore, we speculate that the berberine combined with evodiamine had a synergistic effect on reducing cholesterol in the plasma and liver by inhibiting intestinal cholesterol absorption.
Large numbers of studies have demonstrated the hypolipiemic properties of berberine and evodiamine by animal and clinical researches. Kong et al. [31] has been reported that 1000 mg/day berberine for three months could significantly lower total cholesterol and LDL-cholesterol by 29% and 25%. Another study has showed that evodiamine as a supplement comprising 0.02, 0.04, and 0.06% of the diet fed to mice for 4 weeks could influence lipid metabolism in time and dose-dependent manners [18]. However, minimal information on the adverse drug reactions of berberine and evodiamine supplementation is available. Some studies have demonstrated that the maternal toxicity lowest observed adverse effect levels of berberine were 531 mg/kg/day and 841 mg/kg/day in rats and mice [32], and berberine could present certain side effects in hypervagotonic people [33]. The incidence of toxic adverse effect is connected with the doses of berberine, which showed that the dose of berberine increase lead to the rising risk of toxic side effect [34]. In our current study, the doses of berberine and evodiamine were 72.6 mg/kg and 16.6 mg/kg, which were equal to 5 g/day of the human dose, much lower than that used in previous studies. Whether the supplementation of berberine combined with evodiamine has any side effects for a long time as a treatment on hyperlipidemia needs a further study in the future.
Hyperlipidmia is characterized as the increased serum lipid index especially serum total cholesterol and LDL cholesterol levels, which results in coronary morbidity and mortality. The previous researches suggested that the ratios of serum cholestanol and plant sterols to cholesterol were the markers of cholesterol absorption, which reliably depicted the cholesterol absorption efficiency. Non-cholesterol sterols which include cholestanol and plant sterols, are regarded to be significant markers of cholesterol absorption [35]. Thongtang et al. [36] found that the ratio of ß-sitosterol to total cholesterol was positively related to absorption activity of cholesterol. In this study, we found that supplementation with HFD increased the level of ß-sitosterol and ß-sitosterol/cholesterol ratio, which were attenuated by berberine, evodiamine, and their combination, suggesting that berberine, evodiamine, and their combination could successfully decrease the markers of cholesterol absorption, ultimately inhibiting absorption of cholesterol. Notably, treatment with the combination of berberine and evodiamine showed remarkable reduction in ß-sitosterol by 48.2% compared with the HFD group. Although berberine alone also inhibited the cholesterol absorption marker, this effect was intensified when it was combined with evodiamine. Therefore, we hypothesized that the hyperlipidemic ability of co-administration of berberine and evodiamine may be associated with their effects on cholesterol absorption.
Absorption of cholesterol is a significant physiological process involved in maintaining lipid homeostasis [4]. Cholesterol absorption occurs mainly in the small intestine and proximal jejunum,which is regulated by a number of proteins. Niemann-Pick C1 like1 (NPC1L1) protein and Acyl-CoA cholesterol acyltransferase 2 (ACAT2) are essential factors on regulating the intestinal absorption of cholesterol. Cholesterol absorption starts with cholesterol uptake by enterocyte. Apart from the passive penetration, free cholesterol which is hydrolyzed from dietary cholesterol esters and biliary sources are transported actively through NPC1L1 from the lumen into enterocytes [37]. Subsequently, free cholesterol is converted to cholesteryl ester (CE), packed into chylomicrons (CM) and then transferring into the lymphatics. This process is catalyzed by ACAT2, which is a key cholesterol esterifying enzyme for chylomicrons biosynthesis from cholesteryl esters [38]. After the general circulation, cholesterol is ultimately delivered to the liver as the form of CM. Our study showed that the co-administration of berberine and evodiamine favorably inhibit the expressions of NPC1L1 and ACAT2 accompany by the reductions of serum cholesterol levels, which suggested that the hyperlipidemic effect of the combination might be attributed to the down-regulation of NPC1L1 and ACAT2 in HFD-fed rats. Elimination of excessive cholesterol contained mainly two ways, which included firstly, combining the intracellular cholesterol into cholesterol esters in a way activation of ACAT2. Second, excess cholesterol was converted to bile acids in the liver, finally eliminated into feces [39]. The second possible mechanism for the cholesterol-lowering activity of berberine and evodiamine supplementation was mediated by increasing the bile acid synthesis. We plan to investigate the effect of combination of berberine and evodiamine on bile acid profile in the future study.
Apolipoprotein B is an important plasma particle which is the formation of chylomicrons and the other cholesterol transport molecules as low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) for cholesterol transportation between liver and peripheral tissues [40]. ApoB48 is the transport molecules essential for CM, responsible for packing the dietary fats to form CMs for transportation [41]. In fact, ApoB48 was accepted as a vital element involved in lipid metabolism, even a risk factor for CVD [42]. In line with previous study, the current study found that the expression of ApoB48 in intestine was importantly elevated in HFD group by comparison with the CON group. Interestingly, berberine combined with evodiamine significantly decreased intestinal ApoB48 expression in HFD-fed rats. These results indicated that the combination of berberine and evodiamine exhibited the lipid-lowing effect partly on inhibiting the intestinal absorption of dietary fat by reducing ApoB48 overproduction.