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Abstract

In the present study, coumarin and some coumarin derivatives (esculetin, scoparone, and 4-methylumbelliferone) were investigated for their lipid-lowering effect in rats. Male Sprague–Dawley rats (150–200 g) were divided into six groups and each group comprised of five rats. Hepatic injury-dependent hyperlipidemia was induced by carbon tetrachloride (CCl₄, 1.25 ml/kg). Coumarin and coumarin derivatives esculetin (35 mg/kg), scoparone (35 mg/kg), 4-methylumbelliferone (35 mg/kg), or coumarin (30 mg/kg) were administered to experimental groups at 12-h intervals. Animals received the derivatives esculetin, scoparone or 4-methylumbelliferone prior to the administration of a single toxic dose of CCl_4. Serum total cholesterol (TC), triglyceride (TG), very low-density lipoprotein cholesterol (VLDL-C), and low-density lipoprotein cholesterol (LDL-C) levels significantly increased in CCl₄-treated group (p < 0.05, p < 0.01, p < 0.01, and p < 0.05, respectively), while levels of serum high-density lipoprotein cholesterol (HDL-C) decreased (p < 0.01). 4-Methylumbelliferone had no recovery effects on serum TC levels, however, significantly prevented CCl₄-induced hyperlipidemia by reducing TG and VLDL-C levels (p < 0.05 and p < 0.05, respectively). In addition, coumarin had no recovery effect on any of the serum lipid parameters against CCl₄-induced hyperlipidemia. Among the coumarin derivatives only esculetin and scoparone significantly prevented serum HDL-C in CCl₄-induced dyslipidemia. The results from this study indicate that the chemical structure of coumarins plays an important role on the regulation of serum lipid profiles.

Keywords

Coumarins lipid rat

Introduction

Liver disease is one of the serious health problems throughout the world. Liver injury can be caused by toxic chemicals, drugs, or infection.¹ Carbon tetrachloride (CCl₄) has been widely used to investigate chemical toxin-induced liver damage.^2,3 This toxic agent mainly damages the liver due to the breakdown of membrane structures caused by lipid peroxide or covalent binding of CCl₄ metabolites with macromolecules in several tissues.^4,5 The liver is particularly susceptible to oxidative stress due to the release of CCl₄ metabolites, which induces an inflammatory response. High levels of reactive oxygen species lead to various human pathologies, including liver cirrhosis and fibrosis.⁶ It is suggested that cirrhosis interferes with disturbance of lipid metabolism,⁷ which is mostly characterized by elevated serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C), and decreased high-density lipoprotein cholesterol (HLDL-C) levels.⁸ According to Wang et al.,⁹ serum TC, TG, LDL-C, and VLDL-C levels were increased in CCl₄ applied rats.

Herbal products with antioxidant and lipid-lowering capacity have been used for centuries in every culture. There is a growing interest in elucidation of the biological roles of phenolic compounds throughout the world. Coumarins comprise a group of natural phenolic compounds and are found in human dietary fruits and vegetables. More than 1300 coumarins have been isolated from natural sources, and some have been synthesized in several laboratories.^10
–12

So far some coumarins (e.g. warfarin and carbochromen) have been approved for the therapeutic implications as antithrombotic agents in clinics.^13,14 Their popularity has increased because of the variety of pharmacological properties demonstrated in antimicrobial, anti-inflammatory, and antioxidant activities.^15

–19 Dyslipidemia is among the most significant risk factors for the development of cardiovascular disease.²⁰

So far it has also been well established that coumarin and certain coumarin derivatives have the ability to bring about a lipid-lowering effect in hyperlipidemia associated with various cases such as diabetes,^21,22 alcoholism,^23,24 and feeding with a high-fat diet.^25–26 However, to date, the antihyperlipidemic potential of the coumarins with various chemical structures against CCl₄-induced hyperlipidemia has not been investigated. Therefore, in the current study, the possible lipid-lowering effects of coumarin and some coumarin derivatives in rats exposed to CCl₄ were evaluated for the first time.

According to our previous study,¹⁷ and other previous studies,^19,27,28 it has been demonstrated that esculetine and scoparone have a potent antioxidant activity, whereas 4-methylumbelliferone and simple coumarin have a lesser extent than esculetine and scoparone. We can hypothesize that there might be a correlation between the antioxidant potent and antihyperlipidemic effect of coumarin and coumarin derivatives by considering literature knowledge.

Materials and methods

Animals

Male Sprague–Dawley rats (150–200 g) were used in this study. The rats were kept at a temperature of 21 ± 2°C in 12/12-h light–dark cycle with free access to a standard pellet diet and with ad libitum access to water. Ethical approval for the research was obtained from the committee for animal experiments at the Dicle University Medical Research Centre (Diyarbakır, Turkey; project number: DÜAPK 03TF42).

Chemicals

CCl₄, coumarin (1,2-benzopyrone), and 4-methylumbelliferone (7-hydoxy-4-methyl coumarin) were obtained from Sigma Chemicals Company. Scoparone (6,7-dimethoxycoumarin) and esculetin (6,7-dihydroxycoumarin) were purchased from Aldrich Chemicals Company. Corn oil was purchased from the local market.

Animal groups

The rats (150–200 g) were divided into six groups and each group comprised of five rats. The control and experimental groups of animals were weight matched (150–200 g). Hepatic injury-dependent hyperlipidemia was induced by CCl₄ (1.25 ml/kg). The CCl₄ was suspended in corn oil (20% v/v) and administered orally with an intragastric cannula. The control animals received four doses of saline (3 ml/kg) orally at 12-h intervals up to 48 h, and corn oil was applied orally 1 h after the last dose. Similarly, the CCl₄ group received four doses of saline at 12-h intervals, and CCl₄ was administered 1 h after the last saline treatment. Coumarin and coumarin derivatives treated groups were treated similarly as the CCl₄ group, except that esculetin (35 mg/kg), scoparone (35 mg/kg), 4-methylumbelliferone (35 mg/kg), or coumarin (30 mg/kg) were administered instead of saline at 12-h intervals. The doses for coumarin and various derivatives were designed according to the previous studies.^29,30

Animals were anesthetized with ketamine 24 h after the last treatment and blood was collected by cardiac puncture. Serum TC, TG, LDL-C, VLDL-C, and HDL-C were determined by standard automated techniques in Biochemistry Department of Dicle University Faculty of Medicine.

Statistical analysis

Statistical analysis was performed using the SPSS for Windows statistical package, version 20.0 (SPSS Inc. Chicago, illinois, USA). Each value represents the mean ± standard deviation of the replicates. The effects of drug treatments were evaluated statistically using Kruskal–Wallis test followed by Mann–Whitney U test to determine the difference between any two groups of rats. Statistical significance was assumed at p < 0.05.

Results

The estimated serum TC value in the control group was found to be 57.4 ± 6 mg/dl. TC level was significantly elevated to 77 ± 8 mg/dl in the CCl₄-treated group (p < 0.05). Pretreatment of animals with esculetin and scoparone significantly decreased TC values to 60.1 ± 7 mg/dl and 62.2 ± 6 mg/dl, respectively (p < 0.05, p < 0.05; Figure 1(a)). As observed in other serum lipid parameters, coumarin and 4-methylumbelliferone had no recovery effects on serum TC levels in rats.

Figure 1.

Effect of pretreatment with esculetin, scoparone, 4-methylumbelliferone, and coumarin on CCl₄-induced lipid profile changes: TC (a), TG (b), VLDL-C (c), LDL-C (d), HDL-C (e), and each value represent mean ± SD (n = 5). a′, as compared with control group; b′, as compared with CCl₄ group. *p < 0.05; **p < 0.01. TC: total cholesterol; TG: triglyceride; VLDL-C: very low-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; CCl₄: carbon tetrachloride.

The effect of coumarins on serum TG levels is shown in Figure 1(b). TG level of the CCl₄ group (54.6 ± 12 mg/dl) was significantly higher than that of control group (21.2 ± 8 mg/dl, p < 0.01). Pretreatment with esculetin, scoparone, and 4-methylumbelliferone significantly prevented CCl₄-induced hyperlipidemia as TG levels were found to be 28.0 ± 5 mg/dl, 30.8 ± 4 mg/ml, and 33.2 ± 5 mg/dl, respectively, and therefore close to the control level (p < 0.05, p < 0.05, p < 0.05, respectively). Coumarin treatment slightly decreased TG levels (42.0 ± 12.9 mg/dl) and this was found to be insignificant when compared with the CCl₄ group (54.6 ± 12 mg/dl).

Serum VLDL-C levels are shown in Figure 1(c). Animals treated with CCl₄ showed a marked increase in VLDL-C level as 10.5 ± 1.5 mg/dl when compared to the control level of 4.26 ± 1 mg/dl (p < 0. 01). Esculetin, scoparone, and 4-methylumbelliferone showed a significant reduction in VLDL-C levels as 5.2 ± 1.2, 5.4 ± 2, and 6.6 ± 1mg/dl, respectively, compared to that of CCl₄-treated group (p < 0.05). Administration of coumarin did not attenuate CCl₄-induced increase in VLDL-C.

Serum LDL-C levels are shown in Figure 1(d). LDL-C level of the CCl₄ group (45.3 ± 10 mg/dl) was higher than that of control group (23.51 ± 8 mg/dl, p < 0.05). In this study, pretreatment of animals with esculetin and scoparone maintained normal LDL-C levels against CCl₄-induced hyperlipidemia. LDL-C levels in esculetin and scoparone groups were found to be 25.2 ± 6 and 27.5 ± 4 mg/dl, respectively, which were significantly lower than the values of the CCl₄-treated group and close to control group values (p < 0.05). The other two chemicals 4-methylumbelliferone and coumarin had no recovery effects against CCl₄-induced increase in serum LDL-C level.

It was also observed that the administration of CCl₄ decreased HDL-C levels significantly (21.0 ± 2 mg/dl) below the control levels (29.6 ± 4 mg/dl, p < 0.01; Figure 1(e)). In the groups of animals that were pretreated with esculetin and scoparone, this parameter was returned to normal levels of 29.6 ± 4 and 29.2 ± 4 mg/dl, respectively (p < 0.01). Administration of 4-methylumbelliferone and coumarin did not show any recovery effect on serum HDL-C levels against the decreasing effect of CCl₄.

Discussion

In this preliminary study, coumarin and coumarin derivatives (4-methylumbelliferone, scoparone, and esculetin) were studied according to their molecular structures for determining whether they had any lipid-lowering effects against acute hepatic injury induced by CCl₄ in rats. The liver is the major site for the synthesis and metabolism of lipids.³¹ Marked alterations in lipid metabolism have been reported in CCl₄-induced hepatotoxicity in rats.^2,3 Numerous studies have indicated that the administration of CCl₄ increased TC, TG, LDL-C, and VLDL-C levels while decreasing HDL-C levels.³² The possible reasons for CCl₄-induced lipid metabolism changes in rats have been explained by various mechanisms. Hepatotoxic substances such as CCl₄ interfere with hepatic phospholipid synthesis and cause an elevation of lipid profile components as well as a decreased release of hepatic lipoproteins.⁹ ^, ³³ The high concentration of serum lipids in CCl₄-treated rats is mainly due to an increase in the mobilization of free fatty acids from fat deposits.³⁴ The increased plasma LDL-C concentration in CCl₄-induced rats might also be due to the defect in LDL receptor production or its function.³⁵ According to our results, serum TG, TC, VLDL-C, and LDL-C levels were increased while HDL-C levels were decreased in rats exposed to CCl₄. Our findings related to the change in serum lipid profiles of CCl₄-treated rats are in agreement with the results of previous studies.^9,3

Natural and synthetic coumarins have drawn much attention due to their potential beneficial effects on human health.^13,14,16 Coumarin and its derivatives were shown to have lipid-lowering potential in alcoholic fatty liver,²⁴ hyperlipidemia due to a high-fat diet, and strepzotocin-induced diabetes in rat.^{21,22,25
–27,36} According to Satheesh et al.,²⁵ coumarin has hypolipidemic activity in rats fed with high-fat diet.²⁵ This lipid-lowering effect may be due to the inhibition of hepatic cholesterogenesis or due to the increase in excretion of fecal sterol.³⁷

A recent study indicated that the simple compound coumarin has antihyperlipidemic effect in experimental type 2 diabetic rats by alleviating the lipid profiles and lipid metabolizing enzyme.²²

Umbelliferone, a coumarin derivative, was shown to have an antihyperlipidemic effect in alcoholic fatty liver. The results of Kim et al.²⁴ indicated that umbelliferone application significantly reduced the plasma trigliceride, free fatty acid, and cholesterol levels compared to the alcohol control group. Although umbelliferone did not affect the HDL-C levels, it attenuated the decrease in the HDL-C/TC ratio that was decreased by alcohol. The lipid-lowering effect of umbelliferone was also shown by Ramesh et al.²¹ in streptozotocin-induced diabetic rats. They indicated that the antihyperlipidemic effect of 4-methylumbelliferone in diabetic rats could be due to an increase in insulin secretion which, in turn, inhibits hormone-sensitive lipase and increases the utilization of glucose, thereby decreasing the mobilization of free fatty acids.

Scoparone is also one of the coumarin derivatives which significantly reduced the elevated levels of TC, TG, VLDL-C, LDL-C, and HDL-C in hyperlipidemic diabetic rabbits. These protective effects of scoparone were related partly to its free radical scavenging property that may affect the lipoprotein levels.³⁶ The antihyperlipidemic effect of scoparone at CCl₄-induced liver injury was also studied by Choi et al.²⁷ They have indicated that scoparone exerted this protective effect by attenuating hepatic lipid depots and reducing oxidative stress.

In the present study, pretreatment with 4-methylumbelliferone, scoparone, and esculetin prevented CCl₄-induced increase in serum TC, TG, VLDL-C, and LDL-C levels while decreasing HDL-C levels. On the other hand, coumarin had no effect on CCl₄-induced changes in serum lipid profiles. These results demonstrate that coumarin analogs can provide an antihyperlipidemic activity against CCl₄-induced hyperlipidemia, whereas coumarin itself has no protective effect. So far, possible antihyperlipidemic potential of the coumarin and its derivatives against CCl₄-induced hyperlipidemia has not been investigated, although many coumarins and their analogs underwent extensive investigation aimed to assess antihyperlipidemic effects in experimental diabetes,^21,22 alcoholic fatty liver,^23,24 and hyperlipidemia due to high-fat diet.^25,26 Therefore, we cannot compare our results with the earlier studies.

The coumarins are extremely variable in structure, due to the various types of substitutions in their basic structure, which in turn can influence their biological activity. Among coumarin derivatives, some compounds have the ability to scavenge reactive oxygen species and others have potent antihyperlipidemic activity.^18,38 The results of various studies^28,39 demonstrated that the chemical structure of esculetin is an effective radical scavenger. In our previous study,¹⁷ esculetin and scoparone were the most effective radical scavengers among the compounds tested. Among the four compounds tested, esculetin showed the most potent antihyperlipidemic activity against CCl₄-induced hepatotoxicity. It significantly lowered TC, TG, VLDL-C, and LDL-C levels by 23%, 49%, 51%, and 45%, respectively, and increased HDL-C by 29%. Based on our findings, scoparone significantly lowered TC, TG, VLDL-C, and LDL-C levels by 9%, 44%, 49%, and 39%, respectively, and increased HDL-C level by 28%, which was to a lesser extent than esculetin. The other coumarin derivative, 4-methylumbelliferone had interestingly antihyperlipidemic effects on TG and VLDL-C levels. It decreased TG and VLDL-C levels by 39% and 37%, respectively, which was to a lesser extent than esculetin and scoparone.

In the present study, coumarin itself did not show antihyperlipidemic activity. In our previous study, coumarin did not show antioxidant activity against CCl₄-induced hepatotoxicity in rats.¹⁷ According to our results, it can be inferred that coumarin has neither a role in the process related to lipid metabolism nor in the hepatoprotective effect against CCl₄-induced injury. Nevertheless, Leelavinothan et al.²² demonstrated that coumarin has antihyperlipidemic effect in experimental type 2 diabetic rats and Satheesh et al.²⁵ also showed lipid-lowering effect of coumarin in rats fed with high-fat diet.

In light of our results and the published literature, it can be stated that certain coumarin derivativees have showed antihyperlipidemic effects in the various hyperlipidemic status.^{18,21,27,36,38} Antihyperlipidemic effects of coumarins might be stated that by inhibition of hepatic cholesterologenesis, inhibition of free fatty acid release from fat depots, stimulation of lipoprotein lipase activity, or modulation of the number of lipoprotein receptors in tissues.

In conclusion, these certain coumarins, (esculetin, scoparone, and 4-methylumbelliferone) might be useful therapeutic agents against various hyperlipidemias and to prevent developing atherosclerotic complications. Further studies are required to confirm the efficiency of coumarins, esculetin, scoparone, and 4-methylumbelliferone as nutritional supplements for the prevention of hyperlipidemia.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Influence of coumarin and some coumarin derivatives on serum lipid profiles in carbontetrachloride-exposed rats

Abstract

Keywords

Introduction

Materials and methods

Animals

Chemicals

Animal groups

Statistical analysis

Results

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

References