Protective Effect of Laurus nobilis Extract against Hypercholesterolemia Damage in Male Rats

Amr N. El-Shahat1*, A.M. Abdul Azeem1 and Mohamed H.M. Abd el Megid2

1Food Irradiation Research Department, Egyptian Atomic Energy Authority, 3 Ahmed El-Zomor St., El-Zohour District, 8th District, Nasr City, Cairo

2Natural Products Department, Egyptian Atomic Energy Authority, 3 Ahmed El-Zomor St., El-Zohour District, 8th District, Nasr City, Cairo

ABSTRACT

Laurus nobilis (Bay leaf) has been shown to possess various biological activities such as antioxidant activity, anti-inflammatory activity and prevention of cardiovascular diseases. The present study aims to investigate the effectiveness of bay leaf extract (BLE) in protection against high cholesterol diet (HCD) induced hypercholesterolemia. About thirteen compounds of essential oils were identified by GC/MS analysis including 1.8-cineole, Linalool and sabinene which represent the main volatile oils of bay leaf. Supplementation of BLE with HCD for 10 weeks resulted in reduction in the levels of some lipid contents (total cholesterol, triglycerides, Low-density lipoprotein-cholesterol and very Low-density lipoprotein-cholesterol), tumor necrotic factor-alpha and interleukin-6 as well as reduced activity of hepatic and cardiac enzymes. Also, feeding rats on HCD with BLE enhance the antioxidant status in liver and heart tissues with indicated inhibition of lipid peroxidation by reducing the level of malondialdehyde compared to HCD-fed rats. The results concluded that BLE may have an effective role in reducing high cholesterol levels.

Article Information

Received 28 August 2021

Revised 05 April 2022

Accepted 21 April 2022

Available online 10 June 2022

(early access)

Published 05 June 2023

Authors’ Contribution

ANE performed animal experiments. ANAA and MHMAM performed the biological study and collected blood samples. ANE, ANAA and MHMAM wrote the manuscript

Key words

Laurus nobilis, High cholesterol diet, Tumor necrotic factor-alpha, Interleukin-6, Antioxidant activity

DOI: https://dx.doi.org/10.17582/journal.pjz/20210828210818

* Corresponding author: amrshahat22@yahoo.com

0030-9923/2023/0004-1613 $ 9.00/0

Copyright 2023 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Hypercholesterolemia is understood as elevation of total cholesterol levels within the blood which may have occurred as a result of inherited diseases, obesity, unbalanced diet or different diseases like diabetes (Makkos et al., 2020). It is one among metabolic disorders that plays an important role within the occurrence of atherosclerosis and may be a risk factor for coronary heart diseases (Pluijmert et al., 2019). Hypercholesterolemia can impair left ventricular (LV) function by decreasing coronary blood flow reserve and capillary density with induction of apoptosis (Yao et al., 2020). Additionally, hypercholesterolemia induces oxidative and nitrative stress that plays a role in several cardiac disfunction (Pluijmert et al., 2019). Therefore, modulation of hypercholesterolemia appears to be mandatory approach to avoid hypercholesterolemic myocardium complications (Csonka et al., 2016) but there are some of unexpected side effects could be occurred due to using of anti-cholesterol chemical drugs in a long-term (Hartanti et al., 2019). Thus, there is an obvious need for more efficacious and alternative treatment options such as using of herbal plants that contain different components characterised by their pharmacological effectiveness without any complications.

Laurus nobilis (Bay leaf) is an aromatic herb that belonging to the Lauraceae family and widely used as a condiment and spice (Mohammed et al., 2021). The tea of bay leaves is used traditionally as a therapy against diarrhea, for rheumatic pains and treatment of asthma, and cardiac diseases (Mohammed et al., 2021). This herbal plant possesses several types of metabolites that characterized by their antioxidant properties, anti-inflammatory actions, inhibition of oxidative enzymes (Hartanti et al., 2019).Also, the bioactive components in bay leaves such as saponin, terpenoid, flavonoid, polyphenol, alkaloid, and essential oil have been shown to have effects on insulin sensitivity and can lower cholesterol levels by inhibiting the action of HMG-CoA Reductase (Hartanti et al., 2019).Therefore, this study aims to investigate the possible hypocholesterolaemic effect of Laurus nobilis leave extract in male rats fed a high-cholesterol diet (HCD) to avoid the side effects induced by anti-cholesterol chemical drugs.

MaterialS and Methods

All experiments were carried out during 2021 at the Egyptian atomic energy authority, food irradiation department. Chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Essential oil components of Laurus nobilis

Fresh Bay leaf leaves (Laurus nobilis) were purchased from local market (Cairo, Egypt). Separation and identification of essential oil components were performed by using Gas chromatography instrument, Model Hewlett-Packard- MS (5970) series II Condition analysis as follow: Column: 30m hp Methyl silicon 0.1mm; Temperature: Initial 60 ºC; Rate: 3 ºC/ min up to 240 ºC; Carrier gas: Helium 1.0 ml/min; Injection port; Temperature: 250 ºC; Detector temperature: 270ºC; Integration: By using HP software Data; Injection volume: 0.3ml. The isolated peaks were identified by matching with data from the library of mass spectra and compared to those of authentic compounds and published data (Adams, 1995). Quantitative determination was carried out based on peak area integration.

Preparation of Bay leaves extract (BLE)

Bay leaves (BL) were cleaned, washed and dried at room temperature. Then, leaves were grounded for 2 min by electrical grinder.20 g of the leaves were soaked in 200 mL distilled water and was heated at 70 °C in 10 min. by a heater-stirrer (500 r/min). Finally, all the plant residues were removed by filtration and centrifugation of the extract.

Experimental animal

Male albino rats Sprague Dawley (170 to 200g body weight (B. wt.)) were purchased from the Egyptian Holding Company for Biological Products and Vaccines (Cairo, Egypt) and used for the different investigations carried out in the present study. Rats were acclimated to controlled laboratory conditions for two weeks. Rats were maintained on rodent diet and tap water ad libitum. All animals’ procedures were carried out in accordance with the Ethics Committee of the National Research Centre conformed to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH Publication No. 85-23, 1996). The high-cholesterol diet (HCD) contained 100 g cholesterol, 30 g propyl thio-uracil, and 100 g cholic acid in 1 L of peanut oil (Inoue et al., 1990).

Experimental design

The study was performed on the adult male rats divided in four groups, each of 7 rats: Group 1 (Control group): was rats fed with normal pellet diet for 10 weeks, group 2 (HCD group): was fed with HCD for 10 weeks, group 3 (BLE group): was given 200 mg kg−1 of BLE (Mohammed et al.,2021) that administered every day orally using intragastric tube for 10th weeks during the examination and group 4 (HCD and BLE): was fed with HCD plus BLE (200 mg/kg B.wt./day/10 weeks) by using intragastric tube.

At the end of 10th week, rats were fasted for 24 h and anaesthetized with diethyl ether. Blood samples were collected through heart puncture and allowed to coagulate and centrifuged for to obtain serum for biochemical analysis. Also, liver and heart tissues were removed for biochemical investigation.

Biochemical analysis

Total cholesterol (TC), triglycerides (TG) and high-density lipoprotein-cholesterol (HDL-C) were determined according to procedure described by Allain et al. (1974), Fossati and Prencipe (1982) and Demacker et al. (1980), respectively. Low-density lipoprotein-cholesterol and very low-density lipoprotein-cholesterol were evaluated according to Friedwald’s formula (Friedwald et al., 1972) by the following equations: LDL-C (mg/dl) = TC-(TG/5+HDL-C), vLDL (mg/dl) = TG/5. The levels of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) were determined by the method of King (1965). Creatinine kinase-MB (CK-MB) and cardiac troponin I (cTnI) were performed by ELISA technique (BioSource International, Camarillo, CA, USA) according to the manufacturer’s instructions. The activity of serum aspartate transaminase (AST) and alanine transaminase (ALT) was estimated according to Reitman and Frankel (1957), serum γ-glutamyl transferase (GGT) was assessed according to Rosalki (1975) and serum alkaline phosphatase activity (ALP) was assessed according to Kind and King (1954). Detection of serum tumour necrotic factor-alpha (TNF-α) and interleukin-6 (IL-6) was performed by ELISA technique (BioSource International, Camarillo, CA, USA) according to the manufacturer’s instructions.

Hepatic and cardiac tissues (100 mg tissue/ml buffer) were homogenized in 50 mM phosphate buffer (pH 7.2; St. Louis, MO, USA); the homogenates were then centrifuged at 1,200 x g for 15 min and the supernatant was used for determination of the concentration of malondialdehyde (MDA)was according to Yoshioka et al. (1979), GSH content by Beutler et al. (1963), superoxide dismutase activity (SOD) by the method of Minami and Yoshikawa (1979) and catalase activity (CAT) by Johansson and Borg (1988).

Statistical analysis

Results were presented as mean ± SE (n= 6). Experimental data were analysed using one way analysis of variance (ANOVA). Duncan’s multiple range test was used to determine significant differences between means. Data were statistically analysed by the aid of Statistical Package of the Social Sciences, SPSS version 25 (copyrighted by IBM SPSS software, USA). Differences between means were considered significant at P < 0.05.

Results

Table I shows the essential oil contents extracted from bay leaves. 1.8-cineole represents the main volatile oil with value 36.42g/100 g, followed by Linalool 15.17 g/100 g and sabinene 9.39 g/100 g.

As shown in the results under the influence of HCD a significant increase was observed in the level of lipid profile contents (TC, TG, LDL-C, vLDL-C), inflammatory factors (TNF-α and IL-6), liver and cardiac markers (ALP, γGT, ALT, AST, LDH, CK-MB and cTnI) and the level of hepatic and cardiac MDA accompanied by reduction in the level of HDL-C, GSH and the activity of SOD and CAT compared to control group (Tables II, III).

Supplementation of HCD-rats with BLE caused significant reduction in the levels of TC, TG, LDL-C, vLDL-C, TNF-α, IL-6, ALP, γGT, ALT, AST, LDH, CK-MB, cTnI and MDA associated with significant elevation in the level of HDL-C, GSH and the activity of antioxidant enzymes (SOD and CAT) compared to rats fed with HCD.

 

Table I. Essential oil contents extracted from Laurus nobilis.

S. No	Compound	Rt. (min)	g/100 g
1	α-Thujene	15.38	0.37
2	α-Pinene	15.48	3.11
3	Sabinene	16.17	9.39
4	β -Pinene	16.20	2.86
5	β -Myrcene	16.50	1.24
6	1.8-Cineole	17.13	36.42
7	γ-Terpinene	17.60	0.61
8	α -Terpinolene	18.10	0.26
9	β -Terpineol	18.32	0.32
10	Linalool	18.37	15.17
11	Terpinen-4-ol	19.60	1.03
12	α - Terpinyl acetate	22.48	10.05
13	α -Farnesene	24.98	2.62
	Total	-	83.45
	Total unknown	-	16.55

 

Table II. Effect of bay leaves extract (BLE) on lipid profile, liver function, heart function, TNF-α and interleukin-6 in high cholesterol diet (HCD) induced hypercholesteraemic rats.

Parameters	C	BLE	HCD	HCD & BLE
TC (mg/dl)	146.18±6.92c	140.73±6.43c	285.46±7.62a	192.27±6.37b
TG (mg/dl)	109.45±5.83c	102.54±5.12c	207.32±5.71a	146.34±4.92b
HDL-C (mg/dl)	41.58±2.64a	43.92±2.45a	30.68 ±1.29c	37.83±1.46b
LDL-C (mg/dl)	82.71±4.16c	76.30±4.24c	213.32±5.68a	125.18±6.13b
vLDL-C(mg/dl)	21.89±1.48c	20.50±1.56c	41.46±1.85a	29.26±1.44b
AST (U/ml)	31.52±1.57c	30.42±1.38c	54.24±2.31a	42.58±1.73b
ALT (U/ml)	28.65±1.42c	26.47±0.92c	49.52±1.76a	37.65±0.93b
ALP(U/100ml)	6.58±0.64c	5.87±0.71c	21.23±0.81a	15.24±0.67b
γGT (U/ml)	5.12±0.48c	4.86±0.42c	13.25±0.63a	8.29±0.61b
LDH (U/ml)	229.31±16.21c	223.61±18.65c	539.43±19.27a	345.31±17.12b
CPK (U/L)	259.65±7.59c	256.71±9.82c	464.29±10.10a	308.34±9.95b
CK-MB(ng/mL)	3.15 ±0.69c	3.11±0.76c	7.29±1.22a	4.97 ±0.97b
CTnI (ng/mL)	24.63±1.57c	23.59±1.34c	62.74±2.86a	36.17±1.48b
TNF--α (pg/mL)	621.34± 24.51c	609.37 ± 21.72c	908.23±37.15a	702.43 ± 32.11b
IL-6 (pg/mL)	321.25 ± 22.41c	315.37± 21.13c	492.38±26.97a	371.21± 23.16b

 

TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein-cholesterol; LDL-C, Low-density lipoprotein-cholesterol; vLDL-C, very Low-density lipoprotein-cholesterol. ALT, alanine transaminase; AST, aspartate transaminase; ALP, alkaline phosphatase; γGT, γ-glutamyl transferase; LDH, lactate dehydrogenase; CPK, creatine phosphokinase; CK-MB, creatinine kinase-MB; cTnI, cardiac troponin I; TNF-α, tumour necrotic factor-alpha; IL-6; interleukin-6. Values are expressed as means ± S.E. (n=7). Values in the same row with different superscript are significantly different at P<0.05.

 

Table III. Effect of BLE on hepatic and cardiac lipid peroxidation and antioxidant status in high cholesterol diet (HCD) induced hypercholesteraemic rats.

Parameters		C	BLE	HCD	HCD and BLE
MDA (n mol/g tissue)	Liver	223.12±4.17c	216.42±5.20c	386.12±7.14a	259.36±6.12b
	Heart	136.25±4.31c	137.42±4.51c	241.63±5.36a	172.40±4.22b
GSH (mg/g tissue)	Liver	34.32±0.61a	36.56±0.92a	16.92±0.57c	28.91±0.92b
	Heart	5.39 ± 0.26a	5.89 ± 0.25a	3.05 ± 0.21c	4.81 ± 0.24b
SOD (U/mg protein)	Liver	45.71±1.23a	47.35±1.17a	26.35±1.30c	39.21±1.19b
	Heart	28.57±1.34a	29.32±1.28a	17.24±1.16c	21.96±1.19b
CAT (U/mg protein)	Liver	49.83±1.61a	50.91±1.72a	31.76±1.52c	44.15±1.35b
	Heart	42.95±1.35a	44.18±1.30a	21.62±1.23c	35.29±1.27b

 

MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; CAT, Catalase. Values are expressed as means ± S.E. (n=7). Values in the same row with different superscript are significantly different at P<0.05.

 

Discussion

Feeding high-cholesterol diet (HCD) on a long term is one of risk factors that can cause arteriosclerosis, thrombosis and infarction (Csonka et al., 2016). Bay leaves represent a good source of bioactive components that help to elevate the total antioxidant status and protect against lipid peroxidation induced by oxidative stress (Casamassima et al., 2017).

The results of essential oil composition revealed that bay leaf possesses a lot of effective volatile oils that characterized by their antioxidant properties such as 1.8-cineole, Linalool and sabinene. These results agree with the results of Da Silveira et al. (2014) and Flamini et al. (2007). The composition of the essential oil is highly affected by genotype of the plant species, seasonality, and geographic and weather conditions (Da Silveira et al., 2014).

The obtained results revealed that there is a significant increase in lipid profile in HCD- group compared to control group. Li et al. (2011) concluded that long-lasting high-fat diet leads to lipid metabolism disturbance and hyper-lipidemia by reducing lipid metabolic enzymes such as hepatic lipase and lipoprotein lipase. The study of Fungwe et al. (1994) demonstrated that severe hypercholesterolemia resulted in inhibition of rate-limiting enzyme in cholesterol catabolism cholesterol 7a-hydroxylase, enhancement of hepatic TG synthesis and the reduction of fatty acid beta-oxidation. The increased serum levels of LDL-C and vLDL-C in this study indicate that more cholesterol and triglyceride are being transported from the liver to the extra-hepatic tissues to be taken up by those tissues (Adekunle et al., 2013). Additionally, feeding HCD found to increase the level of LDL-C which could be attributed to the ability of cholesterol and saturated fatty acids included in the diet to induce down regulation in LDL receptors (Mustad et al., 1997). The elevation of LDL-C, total cholesterol, triglycerides, and reduction of HDL can lead to the development and progression of atherosclerosis (Adams, 2005).

Also, the results showed that HCD induced over production of free fatty acids into blood stream resulted in excessive release of pro-inflammatory adipocytokines such as IL-6 and TNFα (Soto-Vaca et al., 2013) with marked elevation in the level of hepatic and cardiac MDA and reduction of GSH content and the activity of antioxidant enzymes (SOD and CAT) when compared with control group. The inflammation and oxidation induced by feeding HCD can cause cellular damage, loss of functional integrity, and/ or permeability of cell membrane that can induce the leakage of LDH, CPK, AST, ALT (Dikshit et al., 1995) and CK-MB into the plasma (Mitani et al., 2003). The release of these enzymes from the damaged hepatic or myocardial membranes indicates hypercholesterolemia-induced hepatic and myocardial necrosis (Fouad, 2020).

A significant reduction in the group of rats integrated with HCD and BLE has been observed in the level of lipid contents, enzymatic parameters of liver and heart function, Ro-inflammatory factors (IL-6 and TNF-α) and MDA. While a marked elevation in the same group has been observed in the level of HDL-C, GSH content and the activity of antioxidant enzymes (SOD and CAT) when compared with the hypercholesterolemic group. The results agreed with Casamassima et al. (2016) who showed the bay leaf recovering lipid profile in hyperlipidaemic rabbits. Asadi-Samani et al. (2014) reported that medicinal plants such as bay leaf may reduce hyperlipidaemia, supress atherosclerosis and vascular endothelium damage. The effect of BLE could be attributed to its content of essential oil, flavonoids and phenolic compounds that induced improvements in insulin sensitivity which would lead to improvements in the level of glucose and blood lipids (Al-Samarrai et al., 2017). Aljamal (2010) suggested that there is a positive effect of bay leaves consumption in preventing atherosclerosis by increasing the level of HDL that can prevent the accumulation of lipid peroxides on LD. Gasparyan et al. (2015) found that injection of ethanol extract of dried bay leaves in carbon tetrachloride-mice improved liver function by reducing the level of ALT, AST, ALP and gamma-GT. The antioxidant effect of BLE could be related to Bay leaves’ scavenger activity of essential oil and its main components that can reduce superoxide and hydroxyl radicals (Basak and Candan, 2013). In addition, Casamassima et al. (2016) indicated that the antioxidant activity of bay leaves could be attributed to the ability of its phenol compounds to act as donors of hydrogen, metal chelators and radical scavenger of peroxides and superoxides.

Conclusion

The results of present research underline that the treatment of hypercholesterolemia with bay leaf leaves extract is highly effective in lowering hyperlipidaemia. Also, the results obtain that BLE has high potential effect in improving liver function and preventing atherosclerosis and controlling the oxidative status. The effective role of BLE could be attributed to the presence of several type of essential oil (1.8-cineole, Linalool and sabinene) that have been identified in this study by using Gas chromatography instrument.

Statement of conflict of interest

The authors have declared no conflict of interest.

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