Potential antihyperlipidemic effects of myrcenol and curzerene in high-fat fed rats

Abstract

The study evaluated the anti-hyperlipidemic effects of myrcenol and curzerene on a high fat diet induced hyperlipidemia rat model. Thirty male albino rats were fed on a high-fat diet for four months. The HFD-induced hyperperlipidemia rats were treated with rosuvastatin (10 mg/kg), curzerene (130 mg/kg) and myrcenol (100 mg/kg) for four weeks. Blood samples were collected for further analysis. Aorta and heart were harvested for histopathological evaluation. Hepatic lipase and HMG-CoA reductase were determined by ELISA. FST and Y-maze tests were performed to assess the stress level in hyperlipidemia rats. The phytochemical compounds (Curzerene and Myrcenol) and the standard drug (Rosuvastatin) resulted in decreased body weight as well as reduced levels of LDL, TG, TC, AST and ALT as compared to the diseased group. Additionally, the treated groups displayed improved HDL levels and less depressed behavior. The ELISA results revealed that the Curzerene and myrcenol had significantly increased the protein concentration of hepatic lipase than the diseased group whereas both compounds significantly lowered the HMG-CoA reductase concentrations compared to the diseased group. The findings suggested that myrcenol and curzerene had the potential to be therapeutic agents for managing hyperlipidemia and reducing the risk of heart-related conditions associated with high lipid levels.

Introduction

Hyperlipidemia is a complex pathological condition characterized by various acquired or genetic disorders that result in high plasma lipid levels. The intake of saturated fat and cholesterol had a significant impact on how obesity manifested in individuals with hyperlipidemia [1]. Lipids, including cholesterol, lipoproteins, chylomicrons, VLDL lipoprotein, LDL, Apo lipoproteins and HDL are mainly stored in adipose tissues for energy. Cholesterol is a fatty substance that is transported in blood along with proteins in the form of lipoproteins and its excess can precipitate on blood vessel walls forming plaque, reducing arterial diameter and resulting in numerous cardiovascular diseases [2].

LDL is responsible for carrying cholesterol particles from the liver to body tissues, merging them into cell membranes, and forming fatty buildup in artery walls. Many trials and studies have repeatedly proven that high LDL cholesterol levels enhance a person’s risk of developing atherosclerotic plaques and consequent vascular disease [3]. VLDL is synthesized in the liver from triglycerides and total cholesterol esters and undergoes lysis by the lipoprotein lipase (LPL) enzyme to generate intermediate-density lipoproteins IDL or VLDL remnants. LDL is transformed into bile acids and discharged into the intestines, while in non-hepatic tissues, it is used in hormone assembly and cell membrane fusion. Macrophages and other cells can consume it, or it may accumulate to form foam cells [4]. Atherosclerosis remains unnoticed, until the plaque stenosis occurred. The underlying endothelial damage that leads to atherosclerosis appears to be caused by the endothelium’s loss of nitric oxide. Increased inflammation is caused by this process immediately surrounding the area of malfunction, which allows lipids to build up in the innermost layer of the endothelium wall [5].

In the fasting state, VLDL is used as a substitute chylomicron carrier by the liver and controls lipoprotein metabolism. Cholesterol and triglyceride produced by the liver and released into the plasma as a component of VLDL are degraded by lipolysis to low-density lipoprotein (LDL) via intermediate density lipoprotein (IDL). Directly taken up VLDLs are converted into IDL and then IDL converted to LDLs by hepatic lipase [6].

Hyperlipidemia rarely has signs and symptoms, but it can lead to problems like heart stroke, arteriosclerosis, coronary artery diseases, vascular disease, diabetes, and myocardial infarction. Treatment options include medication, fibrates, omega-3 fatty acid supplements and statins [7].

The enzyme 3-hydroxy-methylglutaryl CoA reductase (HMG-CoA) plays a crucial role in regulating the synthesis of cholesterol within the body. Its rate-limiting step is the primary determinant of cholesterol levels in the body [8]. The lipolysis enzyme (hepatic lipase) that catabolized the hydrolysis of TG and phospholipids in an endogenous pathway. Maintained the IDL and LDL levels in blood circulation by metabolizing and removing TG from VLDL [9]. Researchers have been investigating the possible impacts of medicinal and herbal plants because of their anti-hyperlipidemia, hypoglycemic, and anti-depressed properties due to the side effects of current anti-hyperlipidemia drug (statins) such as rhabdomyolysis, myalgia and myositis [10].

The phytochemicals compounds curzerene and myrcenol used as anti-hyperlipidemic agents. Curcuma zedoaria, Eugenia uniflora plants that contained large amounts of curzerene and lemongrass, hop, bay and thyme avidently have Myrcenol [11, 12]. In previous studies, curzerene and myrcenol improved coronary artery disease and arteriosclerosis, reduced blood sugar levels and maintained lipid profiles [13].

This study is aimed to assess the anti-hyperlipidemia effects of Curzerene and Myrcenol in HFD rats focusing on biochemical and hematological analysis including AST, ALT, triglycerides, high density lipoprotein, cholesterol and very low-level density lipoprotein. Histopathological study of heart and aorta were also conducted to determine the morphological changes. HMG-CoA reductase and hepatic lipase protein concentration were evaluated by ELISA.

Materials and methods

Chemicals

PBS and RIPA buffer were purchased from Sigma-Aldrich (Life Science, Germany). Cholesterol and cholic acid were purchased from Sigma-Aldrich (Germany). Rosuvastatin calcium was gifted by Medpak Pharmaceuticals (Lahore, Pakistan). Treatment compounds Curzerene was purchased from Biosynth Carbosynth (UK) and myrcenol from Sigma Life Science Aldrich (Germany). Hepatic lipase and HMG-CoA Elisa kits were purchased from ZOKEYO (Wuhan, China). All the other chemicals used were of analytical grade.

Animals

Thirty male albino rats weighing around140-180 g were kept in the animal house of The University of Lahore under standard conditions. The animals were acclimatized to laboratory conditions, and experiments were performed by OECD (organization for economic co-operation and development) guidelines.

High -fat -diet induced hyperlipidemia

Hyperlipidemia was induced for 4-month by HFD (60%) homogeneous mixture of (1%w/w) cholic acid, (2%w/w) cholesterol, (9% w/w) egg yolk, sucrose (50% w/w) along with standard diet [14]. Serum TC and TG were measured on the last day of 4th month of HFD. Rats with triglyceride levels higher than threshold range i.e. 126–145 mg/dL were chosen for additional studies [15]. Hyperlipidemia rats were selected and treated with a standard drug (Rosuvastatin 10 mg/kg), curzerene (130 mg/kg) and Myrcenol (100 mg/kg) for 4 weeks by oral gavage. After 4 weeks, they were sacrificed and blood samples were collected for biochemical and hematological assays.

Thirty male albino rats were divided into following five groups (n = 6).

Group 1	Normal group	600 g/kg NF+NS
Group 2	Diseased group	HFD
Group 3	Standard group (Rosuvastatin)	10 mg/kg + HFD
Group 4	Curzerene treated	130 mg/kg + HFD
Group 5	Myrcenol treated	100 mg/kg + HFD

Measurement of rats body weight

Body weight of all rats was measured on weekly basis during the whole experiments.

Behavioral tests

Forced swimming test (FST) is a preclinical animal model to evaluate the depressive-like behavior in rats by observing hyperactivity, paddling, climbing, jumping and late immobilized time for 6 min [16, 17]. Y MAZE behavioral test was conducted to assess stress levels in rats. This test involved rats navigating each arm of a Y-shaped maze at 120º angle, moving from arm A to arms B and C for a duration of 5 to 8 min [18].

Sample collection

After being anesthetized with intraperitoneal injection of 100 mg/kg Ketamine and 20 mg/kg Xylazine, animals were subjected to blood and tissue analysis. Noteworthy, tissue samples of the heart, aorta and were obtained and preserved in 10% formalin for further use. Adipose tissue samples were placed in Eppendorf tubes containing 1 ml of Trizole.

Hematological assay

Serum TC, TG, HDL, LDL and VLDL were measured by using automated analyzer (COBASC311) according to manufacturer’s instruction.

Biochemical analysis

Alanine Transaminase (ALT) and Aspartate Aminotransferase (AST) levels were measured using automated analyzer (COBAS C311). Through biochemical analysis, the impact of the treatment on the liver and the progression of hyperlipidemia was measured [19].

Histopathology test

Histopathological examinations were conducted on the heart and aorta to assess morphological changes. Tissues were fixed in 10% formalin and then cut into 2–3 cm sections. The tissue sample were stained with hematoxylin and eosin and Sudan red oil to observe the fat accumulations and atherosclerosis lesions under the light microscope [20].

Protein assay test

Adipose tissues were carefully washed with pre-cooled PBS to remove any residual blood and internal debris. Then, the sample was treated with 1 ml of Trizole and the precise amount of RIBA lysis buffer (1 ml) was added. The tissue was homogenized, followed by a 30-minute lysis on ice. After a 1-minute sonication, the samples were centrifuged at 12,000 rpm for 10 min at 4^oC. The supernatant was extracted and stored at -80 °C for HMG-CoA Reductase and hepatic lipase ELISA [21].

Measurement of HMG-CoA reductase and hepatic lipase by ELISA

The levels of HMG-CoA Reductase and hepatic lipase were measured using an ELISA kit’s guidelines provided by manufacturer. Standard concentration used for hepatic lipase were 200,100, 50, 25, 12.5, 0 ng/ml and for HMG-CoA standard concentration were 20, 10, 5, 2.5, 1.25, 0 ng/ml. Samples and standards were arranged and added to microplate strips. Diluent (1:20) and HPR-conjugate reagent were added to each well, covered and incubated for 60 min at 37 °C. The wells were washed and the process was repeated four times. Optical density (OD) was measured at 450 nm wavelength in ELISA.

Statistical analysis

Results were presented as the mean ± standard deviation (SD) and data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad prism 8.0 version. P value was considered as statistically significant using the following abbreviations. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 when compared to diseased group whereas #= p˂0.05, ##= p˂0.01 and ### = p˂0.001 were considered statistically significant when compared from control group.

Results

Curzerene and Myrcenol reduced body weight in high-fat diet rats

The research findings revealed that rats fed on high-fat diet experienced a considerable increased in body weight. Among the groups, the diseased rats experienced the most significant weight gain. However, when compared to the standard group, it was observed that curzerene and myrcenol exhibited a notable reduction in weight gain in albino rats. Furthermore, the curzerene treated group displayed a decrease in weight that was similar to the normal group (Fig. 1).

Fig. 1

Body weight of rats. ** indicates p < 0.01 significance decrease in body weight in the myrcenol and curzerene groups compared to the diseased groups. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 significance and ### = p < 0.001 significance compared to two control groups i.e., normal group vs. diseased group

Curzerene and myrcenol alleviated depressive behavior induced by HFD

Hyperlipidemia rats showed less locomotor activity and were immobilized for 4 min out of 6 min FS, depicting their depressive behavior. Normal rats were immobilized only for 1–2 min as compared to depressed rats. Myrcenol and curzerene significantly reduced the duration of immobility. In Y-Maze test, Phytochemical’s compound group rats showed more entries than the diseased group indicating less stressed behavior (Table 1, Fig. 2).

Table 1 Curzerene and Myrcenol effect in depressive behavior induced by HFD

Fig. 2

FST (forced swimming test) and Y-Maze test. Curzerene and Myrcenol auspiciously reduced the stress level in HFD-hyperlipidemia. Behavioral test was performed one day before dissection. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 significant compared to diseased group whereas ### = p ˂ 0.001 significant when compared from control group

Curzerene and Myrcenol regulated lipid Profile levels in HFD-induced hyperlipidemia rats

Curzerene and myrcenol effectively restored normal lipid profile levels in hyperlipidemia rats. The HDL levels were increased in all groups except in the diseased group and normal group. When compared to the diseased group, curzerene and myrcenol significantly lowered the levels of LDL. The myrcenol compound displayed a maximum significant reduction in the TG value than the diseased group. Phytochemical compounds showed a lower TC level in rats than in the diseased group. The myrcenol graph exhibited more significant values than the standard group (Table 2, Figs 3–4).

Table 2 Effects of curzerene and Myrcenol on HDL, LDL, TG, TC levels in HFD-induced hyperlipidemic rats

Fig. 3

HDL (High-density lipoprotein), LDL (Low-density lipoprotein), TG (Triglycerides), TC (Total cholesterol). Curzerene and myrcenol regulated the lipid profile level after 4 weeks of treatment. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 significant compared to diseased group whereas ### = p ˂ 0.001 significant when compared from control group

Fig. 4

AST (Aspartate transaminase), ALT (Alanine transaminase). Biochemical analysis was performed on the serum samples after the blood samples were collected in a gel tube after dissection. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 significant compared to diseased group whereas ### = p˂0.001 significant when compared from control group

Curzerene and Myrcenol mitigated AST and ALT levels in HFD induced hyperlipidemic rats

AST and ALT levels elevates with the progression in hyperlipidemia. Curzerene and myrcenol AST and ALT mean ± SD were markedly reduced in HFD rats when compared to the diseased group. However, the AST and ALT levels were high compared to the standard group (Table 3).

Table 3 Effects of Curzerene and Myrcenol in AST and ALT levels in HFD-induced

Curzerene and myrcenol maintained HFD-induced hyperlipidemia by upregulating hepatic lipase and downregulating HMG-CoA reductase

ELISA testing revealed that curzerene and myrcenol had impact on hepatic lipase levels and HMG-CoA reductase levels by upregulating hepatic lipase and downregulating HMG-CoA. Analysis of the protein concentration graph indicated that HL levels were higher than in the treated group compared to the diseased group. The HMG-CoA protein level were significantly reduced in the curzerene and myrcenol group as compared to the diseased group (Table 4, Fig. 5).

Table 4 Effects of curzerene and myrcenol in hepatic lipase and HMG-CoA concentration

Fig. 5

Curzerene and Myrcenol regulated the HL and HMG-CoA protein concentrations that were measured by the ELISA kit. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 significant compared to diseased group whereas #= p˂0.05, ##= p˂0.01 and ### = p˂0.001 significant when compared from control group

Curzerene and myrcenol improved histopathology in HFD-induced hyperlipidemia rats

Histopathology of normal group heart revealed no discernible morphological changes. Myrcenol and Curzerene significantly improved heart’s pathology compared to the control group. The heart histology of the diseased group revealed excessive fat accumulation in many places. The diseased group’s cardiac adiposity is linked to atherosclerosis influenced by HFD induction. Sudan red oil staining made the fat plaques visible (Fig. 6).

Fig. 6

Heart longitudinally viewed images stained with H&E and Sudan red oil were observed under a 40x magnification microscope. SG (standard group), CG (curzerene group), MG (Myrcenol group), DG (diseased group), NG (normal group). The diseased heart histology viewed showed excessive fat accumulation at various spots

Discussion

This study investigates the anti-hyperlipidemia activity of curzerene and myrcenol in an HFD-induced model. Hepatic lipase and HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase) were the main targeted biomarkers in this study. The HFD was composed of 8 egg yolks, 1% colic acid, 2% cholesterol, and 50% sucrose. The HFD-induced hypercholesterolemia, increased lipid profile level and increased body weight also showed evidence in the elevation of the stress level and influenced the development of health conditions like hyperlipidemia that lead toward heart-associated conditions such as arteriosclerosis [22]. In previous studies, Curzerene and Myrcenol showed evidence of lowering lipid levels, fast metabolism, fat reduction and decreased arteriosclerosis development. Myrcenol was reported to lower LDL levels, increase HDL levels and lower stress levels [23]. Shokoohi reported that the herbal combo of C mukul, T chebula and C myrrh (curzerene) has a favorable effect on the lipid profile in hyperlipidemia type 2 diabetic patients and showed evidence of a suggestive reduction in blood glucose levels, cholesterol levels and LDL levels as compared to the placebo group [7]. In diabetic rats, thyme extract (Myrcenol) decreased serum LDL and triglycerides and augmented serum HDL levels after 28 days [24].The outcome exhibited the bay leaf (Myrcenol) lowering the TC, TG, LDL and VLDL. Hence, bay leaf is a convenient agent for dropping hyperlipidemia [25].

Curzerene was orally administered at 130 mg/kg for anti-hyperlipidemia activity in HFD rats for 4 weeks. Myrcenol was 100 mg/kg orally administered for 4 weeks. Current study demonstrated significant effects of myrcenol and curzerene on lowering lipid profile levels in the compound-treated group as compared to the diseased group. Significant decrease in LDL level and triglycerides and a prominent elevation in HDL serum level. Among all the statins, only Rosuvastatin have a high HDL level (8–10%), but fortunately, our Myrcenol compound showed almost the same results as the standard drug in HDL level. However, both compounds’ values were higher than those of the diseased group. Higher HDL levels in the body lowered the LDL levels. Our compound groups demonstrated a remarkable reduction in LDL levels as compared to the diseased group. This indicates that our compounds have the potential to significantly improve the LDL level. Curzerene showed the same result as in the normal group. Both compounds had better results in TG than the diseased group (hypertriglyceridemia). Myrcenol was more effective in reducing TG levels compared to Rosuvastatin. in the TC graph, the diseased group showed the highest level influenced by HFD induction. Both compounds showed lower TC results than the diseased group. Myrcenol compounds showed the lowest values of TC rather than the other groups, including the standard group. Curzerene and standard groups had the same results as the normal control group. Obesity is the underlying cause of elevated triglycerides and low HDL levels. Moreover, higher levels of triglycerides and cholesterol can trigger the development of heart conditions such as hyperlipidemia and atherosclerosis [26].

That excessive weight gain and fat accumulation can lead to various metabolic disorders associated with obesity, such as hyperlipidemia. Previous study revealed that weight gain caused by a high-fat diet results in significant changes in blood lipids. These changes include increased serum triglycerides, LDL cholesterol, and total cholesterol levels, as well as a decreased serum HDL/TC ratio. This confirms that the weight management is crucial to preventing the onset of obesity-related metabolic complications [27]. After HFD, rat’s body weight also increased noticeably as compared to the normal control group, which continued on normal feed during that period. Obesity is also a risk factor for hypercholesterolemia. Excessive use of sucrose causes belly fat and later on insulin resistance, which directly relates to hyperlipidemia [28]. In many studies, high sucrose diets developed NAFLD, and evidently, adipose tissues showed those morphological changes [29]. Our study was demonstrated through lipid profiles, liver analysis and ELISA by comparing treated compounds vs. normal and diseased control groups. The body weight of rats was double than the normal group, NG were fed only on normal feed. The weight reduction after 4 weeks of curzerene and myrcenol treatment suggested that phytochemicals have positive impact on control body weight.

Assessed the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) because there is evidence that a high-fat diet can affect liver function and lead to fatty liver disease [14]. Biochemical analysis through AST and ALT results revealed low levels in the treated compound group than the diseased group. According to established studies the elevated level of these enzyme may be related to the hepatic cell injury [30].Myrcenol significantly decreased the AST level. After induction of HFD for 4 months. Stress can cause chronic health conditions like hyperlipidemia, hyperglycemia, and hypertension. Managing stress is crucial in preventing them [31]. The results of the behavioral test confirmed that the stress level was noticeable in the diseased group as compared to the normal control group. After being treated with myrcenol and curzerene, rats experienced lower levels of stress and increased activity, all while displaying normal behavior. The treatment administered to the rats had a remarkable impact on their behavior and overall well-being. The rats that were treated showed a significant reduction in immobility time during the FST, indicating a reduction in depressive symptoms. Furthermore, the treatment also improved their behavior in the Y-maze test, which is indicative of better memory function. In contrast, the diseased rats continued to exhibit the same negative behavior as before, remaining immobile and hiding in arm A. These findings clearly demonstrate the positive impact of the treatment on the rats and its potential to improve these similar conditions. The treatment also resulted in a decrease in immobility time during the FST and improved behavior in the Y-maze test as compared to the diseased group.

Hyperlipidemia can trigger various risk factors that may lead to the early development of atherosclerosis and injuries to blood vessels [27]. Histopathology results distinctly revealed pathological differences among the normal control group, the diseased group and the compound-treated group. Diseased group heart and aorta morphological changes, like excessive fat storage, fatty streak development and plaque formation in layers of the aorta, were totally absent in the compounds-treated group. The standard group had minute amounts of lipids, such as fat droplets, as compared to the normal control group. Sudan red oil (fat absorbent) does not show any red-brown color in the normal control group.

Hepatic lipase breaks down lipids like phospholipids and triglycerides. It can reduce hyperlipidemia by breaking down TG but converts LDL to sdLDL, which contains small amounts of TG and TC. Low HL protein activity can increase the risk of coronary artery disease, atherosclerosis and hypercholesteremia. Changes in HL activity can affect plasma levels and make it easier to develop hyperlipidemia from a high-fat diet [32]. HL can also convert VLDL into IDL and then into sdLDL. Additionally, hepatic lipase functions as a ligand that promotes lipoprotein absorption by proteoglycans and cell surface receptors, thus impacting the distribution of lipids within cells [6].The rate-limiting enzyme is HMG-CoA reductase. HMG-CoA reductase produces mevalonate, a significant intermediate in cholesterol production. HMG-CoA reductase inhibitors, or statins, prevent cholesterol synthesis as competitive inhibitors [28].

The ELISA results of the hepatic lipase and HMG-CoA reductase exhibited protein concentrations present in the sample at 450 nm absorbance. In the hepatic lipase ELISA, both compounds (Myrcenol and Curzerene) had a higher protein concentration than the diseased group. Myrcenol test compound showed results near those of the normal control group. Curzerene had a higher protein content than the standard group. HMG-CoA reductase ELISA results showed the highest protein concentration in the DG group. Myrcenol and curzerene both evidently had equal protein concentrations but were significantly lower than the standard group. Our compounds significantly showed better results in the HMG-CoA. The standard drug showed less protein content than the diseased group but higher than our two compounds. All these results confirmed that curzerene and myrcenol are potential drugs to control hyperlipidemia.

Conclusions

Myrcenol and curzerene have been found to have anti-hyperlipidemia effects by upregulating hepatic lipase and downregulating HMG-CoA reductase. Furthermore, the two phytochemical compounds were discovered to considerably decrease stress level and regulating the lipid profile values. These discoveries could be valuable in upcoming research on atherosclerosis and in clinical trials.