Antibacterial effect, histological impact and oxidative stress studies from laurus nobilis extract  

LAURUS NOBILIS EXTRACT JAZILA EL MALTI

and HAMID AMAROUCH

Laboratoire de Microbiologie Biotechnologie et Environnement Université Hassan II – Aïn Chock

Faculté des Sciences km 8 route d’El Jadida BP. 5366

Casablanca, Morocco

Received for Publication July 6, 2007 Accepted for Publication March 9, 2008

ABSTRACT

Laurus nobilis is one of the most broadly used spices in Moroccan gastronomy. Its antimicrobial activity against both gram-positive and gram- negative bacterial species was demonstrated. Likewise, its toxicity was inves- tigated on Swiss albinos’ mice. Daily, mice were treated orally with 0.003 and 0.3 mg during 7 days. Plasmatic markers and enzymatic systems were assessed and histological alterations were evaluated. A significant increase in creatine phosphokinase level was observed. The microscopic evaluation showed that L. nobilis induces morphological perturbation in mice’s liver.

The results also showed an inhibitory effect of glyceraldehydes 3-phosphate dehydrogenase and an important increase in the level of thiobarbituric acid reactive substances and succinate dehydrogenase and no change in catalase activities. The results showed that L. nobilis induces toxicity at 0.3 mg/g mouse and affects energy metabolism and oxidative stress.

PRACTICAL APPLICATIONS

The aim of the present study is to determine the antimicrobial effect of Laurus nobilis on pathogens for potential application as herbal medicine in infusion or oral preparation. The effect of the spice extract was investigated on the metabolic markers, stress biomarkers and clinical parameters. This study was completed with histological coupes. To our knowledge, this is the first

1

Corresponding author. TEL: +(212) 22-23-06-80/84; FAX: +(212) 22-23-06-74; EMAIL: eljazila@

yahoo.fr

Journal of Food Quality 32 (2009) 190–208.

DOI: 10.1111/j.1745-4557.2009.00245.x 190

© 2009 The Author(s)

Journal compilation © 2009 Wiley Periodicals, Inc.

report to demonstrate the laurel effect on energy metabolism and oxidative stress.

INTRODUCTION

Laurus nobilis, commonly known as Bay, Sweet Bay, True Laurel or Roman Laurel, is well known as a culinary spice. It is widely distributed in the Mediterranean area and Europe. Bay leaf has been used traditionally as herbal medicine to treat rheumatism, earaches, indigestion and sprains and to promote perspiration. The methanolic extract of L. nobilis leaves potently inhibited blood ethanol evaluation in ethanol-loaded rats and hydrochloric acid–ethanol-induced gastric lesions in rats (Matsuda et al. 2000a,b). In vivo gastroprotective and anti-ulcerogenic activities of the sesquiterpenes from the leaves have been studied (Yoshikawa 2001). Using rat epididymal adipocyte assay, Broadhurst et al. (2000) reported insulin-like biological activity for the aqueous leaf extract of L. nobilis. The potential use of laurel in skin diseases and wound healing is already found in ethnobotanical surveys (Ali-Shtayeh et al. 2000; Shivananda et al. 2006).

The leaves of this plant have been used to treat epilepsy, neuralgia and Parkinsonism (Aqili Khorasani 1992). Pharmacological studies have demon- strated the anesthetic, hypothermic, muscle relaxant and anticonvulsant activity of eugenol and methyleugenol (Dallmeier and Carlini 1981) as well as the antistress effect of eugenol (Sen et al. 1992).

Herbal plants and spices are an important part of the culture and traditions of Moroccan people. Bay leaf has been used as herbal medicine and has pharmacological activity. Renewed interest in traditional pharmacopoeias means that researchers are concerned about determining the scientific rationale for the plant’s usage. Instead of relying on trial and error, as in random screening procedures, scientists can use traditional knowledge to target plants that may be medicinally useful (Cox and Balick 1994). Recent scientific research has shown that many plants used as food or in traditional medicine are potentially toxic, mutagenic and carcinogenic (Higashimoto and Purintrapiban 1993; De Sã Ferrira and Ferrão Vargas 1999). Recently, our group participated in a research project aimed at investigating the potential hazards associated with the long-term effect of herbal and spices plants commonly used in Morocco.

The aim of the present study was to determine the antimicrobial effect of

L. nobilis on pathogens, for potential application as an antimicrobial in foods,

and the impact of the spice extract on metabolic markers, stress biomarkers,

clinical parameters and histological injury on Swiss albinos’ mice. In addition,

to the best of our knowledge, this is the first report showing the introduction of

toxicity and perturbation of enzymatic system by L. nobilis.

MATERIALS AND METHODS Chemicals

NAD+ (free acid) was purchased from Boehringer (Mannheim, Germany) and all other chemicals were of analytical grade.

Antibacterial Effect

Spice Material. Bay laurel leaves were collected in the region of Houcima, Morocco, during the summer of 2005. They were air-dried in a shady, well-ventilated room at 25–32C for 10 days and then packed in paper bags and stored for 1 month before their use in the laboratory.

Preparation of Spice Extracts. The leaves of laurel were finely ground (100 g) and macerated at room temperature in 100% ethanol during 5 days.

The extract was subsequently filtered and concentrated to dryness in vacuo.

The residue was dissolved in distilled water to produce a concentration of 150 mg/mL of stock solution.

Microorganisms. The isolated strains were obtained from American Type Collection Culture, Service d’Hygiène de Rabat (Maroc), Institut Pasteur du Maroc (Casablanca); Laboratoire Microbiologie Biotechnologie et Envi- ronnement (Faculté des sciences Aïn Chock, Casablanca, Maroc), Laboratoire Microbiologie Appliquée et Biotechnologie (Faculté des sciences, El jadida, Maroc). Antibacterial activity was determined against gram-positive bacteria (Lactobacillus delbrueckii, Bacillus cereus 11778, Listeria monocytogenes 4d, Staphylococcus aureus 25923) and gram-negative bacteria (Shigella sonnei, Escherichia coli, Pseudomona aeruginosa, Salmonella enteridis, Yersinia enterolitica, Proteus vulgaricus, Proteus mettegeri, Proteus penneri, Kleb- siella pneumoniae, Enterobacter cloacea, Morganella morganii, Citrobacter freundii). The stock cultures were maintained on Luria broth agar medium at 4C. All cultures were revived from stocks stored at -20C by two successive growth cycles at 37C for 24 h in Trypticase Soy Broth (Oxoid, Basingstoke, U.K.). They were subsequently checked for purity by growth at 37C for 18 h on Muller Hinton (MH) agar (Difco, Detroit, MI) plates for all strains except L. monocytogenes on Palcam agar (Merck, Darmstadt, Germany) at 37C for 24 h and L. delbrueckii on De Man, Rogosa and Sharpe (MRS, Oxoid) agar at 30C for 18 h (Table 1). For the determination of antibacterial activity, all strains were grown in 10 mL Muller Hinton (Difco) broth for 24 h at 37C.

These bacteria were selected because they are frequently reported in human

infection and are resistant to several antibiotics.

MIC Agar Dilution Assay. The MIC values of the isolates were studied based on the agar dilution method (Gulluce et al. 2003). Serial twofold dilu- tions were made for laurel extract in 10-mL sterile test tubes containing nutrient broth. One milliliter of the extract was added aseptically to 19 mL of sterile MH agar (Difco) to produce the concentration range of 1–150 mg/mL.

The resulting MH agar solutions were immediately poured into petri plates after vortexing. The plates were inoculated with 5 mL of overnight culture bacteria (10

cfu/mL). Each microorganism was inoculated in brain heart infusion (BHI) (Difco) broth and incubated overnight at 37C, and diluted to approximately 10

cfu/mL. The inoculated plates were incubated at corre- sponding temperatures for each isolates for 24 h. At the end of incubation period, the plates were evaluated for the presence or the absence of growth.

MIC values were defined as the lowest concentration of extract that completely inhibited bacterial growth.

Inhibitory Effect by the Agar-well Diffusion Method. Simple suscep- tibility screening test using agar-well diffusion method as adapted earlier was used (Bagamboula et al. 2004). Each microorganism was suspended in BHI

TABLE 1.

BACTERIA STRAINS AND GROWTH CONDITIONS USED FOR THIS STUDY

Strains Source Growth temperature (C) Growth broth

1. Gram-positive bacteria

Lactobacillus delbrueckii LNM 30 MRS

Bacillus cereus 11778 ATCC 30 Muller Hinton

Listeria monocytogenes 4d LMAB 37 Palcam agar

Staphylococcus aureus 25923 ATCC 37 Muller Hinton

2. Gram-negative bacteria

Enterobacter cloacea LNM 37 Muller Hinton

Citrobacter freundii LNM 37 Muller Hinton

Yersinia enterolitica LNM 37 Muller Hinton

Proteus penneri LNM 37 Muller Hinton

Proteus mettegeri LNM 37 Muller Hinton

Escherichia coli IPM 30 Muller Hinton

Shigella sonnei LNM 37 Muller Hinton

Klebsiella pneumoniae LMBE 37 Muller Hinton

Salmonella enteridis LNM 37 Muller Hinton

Morganella morganii LNM 37 Muller Hinton

Proteus vulgarices LNM 37 Muller Hinton

Pseudomona aeruginosa ATCC 37 Muller Hinton

LNM, Service d’Hygiène de Rabat (Maroc); ATCC, American Type Collection Culture; IPM, Institut

Pasteur du Maroc (Casablanca); LMBE, Laboratoire Microbiologie Biotechnologie et Environnement

(Faculté des sciences Aïn Chock, Casablanca, Maroc); LMAB, Laboratoire Microbiologie Appliquée

et Biotechnologie (Faculté des sciences, El jadida, Maroc).

(Difco) broth and diluted with peptone water to provide initial cell counts of about 10

to 10

colony forming unit (cfu/mL). Each strain of bacteria was inoculated on duplicate plates on agar. The plates were allowed to dry at 4C for 1 h. The wells (5-mm diameter) were formed and filled with 30 mL from spice extract (150 mg/mL). The disks of antibiotics (Sigma, St. Louis, MO) were then placed aseptically over the bacterial cultures on plates by the disk diffu- sion method (Collins et al. 1989). The plates were incubated for 24 h at the appropriate temperature. The diameter of the inhibition zone was measured with calipers.

Animal and Administration of L. nobilis

Swiss albinos’ mice were housed under controlled temperature (22C) and 14 h light and 10 h dark cycle, and were given free access to regular laboratory chow diet and water.

A total of 45 animals were randomized into five groups of nine mice each, and the laurel extract was administered daily by oral injection during 7 days.

L. nobilis extract was given to mice at 0.003, 0.03, 0.3 and 3 mg/g of mice’s weight, while sterile water was given to corresponding groups as control.

Blood Analysis

The determination of the CPK, AST and ALT, creatinine and urea were carried out by Laboratoire des analyses médicales du Centre National de la Sécurité Sociale, Casablanca.

Crude Extract Preparation

All procedures were carried out at 4C. Samples of liver were quickly weighed and then homogenized 1/3 (w/v) in 50 mM potassium phosphate buffer pH 7.4 containing 1 mM EDTA, 1 mM dithiothreitol (DTT). The homo- genates were then filtered on eight layers of gas and stored at -20C until used.

Biochemical Assays

All assays were conducted at 25C using Jenway 6405 UV/Visible spectrophotometer (Jenway, Essex, UK).

Catalase. The consumption of 7.5 mM H

O

in 50 mM potassium phosphate buffer (pH 7) was monitored at 240 nm (Aebi 1984).

Thiobarbituric Acid Reactive Substances (TBARS). The assessment

of the extent of hepatic lipid peroxidation relied on the determinations of MDA

content in the crude liver homogenates. Duplicate determinations from each

liver were made and the average of the two measurements was used in the subsequent statistical analysis of the data. Lipid peroxidation was estimated by the formation of TBARS and quantified in terms of MDA equivalents (Samokyszyn and Marnett 1990).

One mL of samples is added to 1 mL solution (0.375 % thiobarbituric acid and 15% trichloracetic acid in 0.25 M hydrochloric acid). The tubes are heated at 100C for 15 min. Then, they are cooled in ice to stop the reaction.

Centrifugation was then carried out at 1,000 ¥ g for 10 min. The reading of supernatant is made at 535 nm. A standard curve of MDA (8–50 nmol) was freshly prepared. Results are expressed as TBARS (nmol/mg protein) using e = 1.56 ¥ 10

/M/cm.

GAPDH. GAPDH activity in the oxidative phosphorylation was deter- mined by monitoring nicotinamide adenine dinucleotide (NADH) generation at 340 nm (Serrano et al. 1991). The reaction mixture of 1 mL contained 50 mM Tricine-NaOH buffer pH 8.5, 10 mM sodium arsenate, 1 mM NAD+

and 2 mM D-G3P.

Succinate Dehydrogenase. The enzyme was assayed according to King (1967) with assay conditions: 100 mM potassium phosphate buffer (pH 7.4), 0.3 mM EDTA, 0.053 mM DCIP and 100 mg of protein. The mixture was preincubated 10 min at 37C before being added to 50 mL of potassium cyanide (KCN) succinate (containing 3.25 mg/mL of KCN in 0.5 M succinate). The absorbance reflecting the activity was read at 625 nm.

Protein Quantification. Protein content in liver was measured according to the Bradford procedure, using bovine serum albumin as standard (Bradford 1976).

Histopathological Analysis

After 7 days of treatment, the liver, heart, brain, kidneys and bowel 4-mm sections were removed, fixed in Bouin and embedded in paraffin. The organ sections were stained with hematein-eosin, then examinated under light microscopy (Olympus-BH-2, Olympus, Southall, UK).

Statistical Data Analysis

All experiments were conducted in four separate experiments and tests

were duplicated. The experimental data represent the mean ⫾ SD. Means

were compared using the Student’s t-test. Differences were considered

significant at the P < 0.05 level and very significant at the P < 0.01 level

(Sini et al. 2006).

RESULTS AND DISCUSSION Antibacterial Activity

Table 2 shows that the average zone of inhibition of spices against micro- organisms ranged from 7 ⫾ 2 mm (Pseudomonas aeruginosa ATCC 27853) to 20 ⫾ 2 mm (L. monocytogenes). Most disks of antibiotics indicate that they did not possess antibacterial effect on the pathogen. The antibacterial effect of spice extract on the different bacteria strains is shown in Table 3.

Although various strains of bacteria varied in their antimicrobial suscep- tibility, laurel extract was very effective in inhibiting the growth of all the tested strains of a majority of the pathogens, CMI ranged between 4.7 and 9.4 mg/ml with the exception of E. coli, S. enteridis and E. coli ATCC2592 which had a great sensitivity to the spice extract (CMI < 2.34 mg/ml).

Table 3 shows that the laurel extract has a significant role in moderating antimicrobial activities against all microorganisms tested. The composition of the laurel volatile oil has been reported (Kilic et al. 2004). Simic et al. (2004) and Dadalioglu and Evrendilek (2004) reported a strong inhibitory activity against E. coli O157:H7, L. monocytogenes, Salmonella typhimurium and S.

aureus because of the major compounds such cineole, eugenol, sabinene, g-pinene and methyleugenol. Antibacterial activity can be explained by the presence of some compounds of essential oil reported by authors to inhibit peptidoglycan synthesis (Ogunlana et al. 1987), damage microbial membrane structures (Cox et al. 2000), modify bacterial membrane surface hydrophobic- ity (Turi et al. 1997) and modulate quorum sensing (Gao and Teplitski 2003).

It is difficult to perform a detailed comparison with the results obtained by other authors in this type of study. Among the problems worth mentioning are those that arise from the material used, in terms of its nature (spice, extract or essential oil), origin (country of origin, altitude at which it grows, harvest season), production process, level of purity and preservation, all of which help to determine the presence of variable concentrations of antimicrobials in the final product. On the other hand, many tests have been carried out on synthetic growth media, with dilution or diffusion in a solid medium, using disks impregnated with antimicrobial agents (Hsieh et al. 2001).

Toxicity Test

The general state and the mice mortality was followed during the 7 days of treatment. No sign of stress or breathing difficulties in the gavaged mice was observed.

No changes were observed with the amounts of 0.003 and 0.03 mg/g

during 7 days of treatment. On the other hand, a significant decrease in the

T ABLE 2. MEAN CLEARANCE ZONE DIAMETERS FOR LAURUS NOBILIS EXTRACT A T (300 m g/ m L) AND FOR ANTIBIOTICS Microor ganisms LAUREL TE NA V A AM DDA AMX NV C (10 m g) C (30 m g) SST P K CB Lactobacillus delbrueckii –– – – – – – 1 0 ⫾ 3.1 12 ⫾ 2.4 – – – – Bacilus cer eus A TCC 1 1778 Listeria monocytogenes 12 ⫾ 2.1 19 ⫾ 1.0 12 ⫾ 1.3 10 ⫾ 2.4 – – – 1 3 ⫾ 1.5 16 ⫾ 1.2 14 ⫾ 2.6 – – 15 ⫾ 1.4 – Staphylococcus aur eus 20 ⫾ 2.0 20 ⫾ 1.9 – – – – – 1 2 ⫾ 2.2 15 ⫾ 3.1 13 ⫾ 1.9 – – – – Enter obacter clolacea 9 ⫾ 1.7 23 ⫾ 2.0 – 1 6 ⫾ 1.7 – – – 1 4 ⫾ 2.1 7 ⫾ 2.2 20 ⫾ 2.2 – – – – Citr obacter fr eundii 12 ⫾ 1.8 18 ⫾ 2.8 12 ⫾ 1.5 16 ⫾ 2.3 – – – 1 0 ⫾ 1 .4–1 3 ⫾ 2.0 – – – – Y ersinia enter olitica 8 ⫾ 2.0 12 ⫾ 1.5 10 ⫾ 2.0 – – – 1 0 ⫾ 2.0 – – – – – – Pr oteus penneri 8 ⫾ 2.0 – – – – – – 1 2 ⫾ 1.5 9 ⫾ 3.2 12 ⫾ 1.6 – – 6 ⫾ 1.2 – Pr oteus mettegeri 14 ⫾ 1.6 – – 15 ⫾ 2.8 – – – 1 2 ⫾ 1.8 12 ⫾ 1.4 16 ⫾ 1.4 – – – – Escherichia coli 8 ⫾ 1.6 17 ⫾ 1.9 – 1 2 ⫾ 1.1 – – – 1 5 ⫾ 1.4 12 ⫾ 1.2 1 1 ⫾ 2.2 – – – – Shigella sonnei 13 ⫾ 1 .9 ––– – – –– – – – – 1 4 ⫾ 1.9 – Klebsiella pneumoniae 7 ⫾ 1.8 – – 16 ⫾ 1.6 – – – – – – – – – – Salmonella enteridis 9 ⫾ 2.4 12 ⫾ 2.0 – – – – – – 14 ⫾ 2.2 15 ⫾ 1.6 – – – – Mor ganella mor ganii 12 ⫾ 2 .1 ––– – – –– – – – – – – Pr oteus vulgaricus 7 ⫾ 1 .6 ––– – – –– – – – – – – Pseudomonas aeruginosa A TCC 27853 8 ⫾ 1.8 – – – – – – 1 2 ⫾ 1.4 10 ⫾ 1.8 13 ⫾ 1.5 – – – – V alues (mm) are expressed as mean ⫾ SD of four experiments. –, no inhibition zone; n.d., not determined. T e, tetrac ycline 30 m g; NA, nalidixic acid; A, v ancomycine 30 m g; AM, ampicillin 10 m g; DA, clindamycin 2 mg; AMX, amoxycillin 25 m g; NV , n o v obiocine 5 m g; C, chloramphenicol 10 m g/30 m g; SST , sulf athiazol 0.25 mg; P, penicillin 10 m g; K, kanamycine 30 UI; CB, carbenicillin 100 m g.

weight was observed (21%) and (38 %) at 0.3 and 3 mg/g, respectively. No mortality was observed in the mice during the 7 days of treatment (Fig. 1).

The body weight of the mice was altered by the extract L. nobilis at 0.3 and 3 mg/g. These results agree with those of enzymatic markers and also suggest that L. nobilis ethanol extract is toxic at doses > 0.3 mg/g.

Effect of L. nobilis Extract on Plasmatic Parameters

The blood analysis indicates an important increase in CPK (¥1.59) and ALT (¥1.26) level at 0.3 mg/g. No changes were observed in AST (Table 4).

No changes were observed with the amounts of 0.003 mg/g on plasmatic parameters. There appeared to be no renal toxicity in the mice because we found no change in creatinine and urea at 0.3 and 0.003 mg/g.

In vivo Effect of L. nobilis Extract on Stress and Metabolic Biomarkers For stress biomarkers, the treatment of the mice showed that laurel induced a significant increase in the level of TBARS (¥3.5) at 0.3 mg/g compared with the control. This increase in activities shows the beginning of oxidative stress in liver. Effectively, oxidative stress occurs as a consequence of imbalance between the formation of oxygen free radicals and inactivation of these species by the antioxidant defense system. Lipid peroxidation has been implicated in the pathogenesis of increased membrane rigidity, osmotic fragility, reduced erythrocyte survival and perturbations in lipid fluidity.

TABLE 3.

MIC (mg/mL) OF SPICE EXTRACTS OF LAUREL

Bacteria MIC (mg/mL)

Lactobacillus delbrueckii 4.7

Bacillus cereus ATCC11778 9.4

Staphylococcus aureus 4.7

Listeria momocytogenes < 2.34

Enterobacter cloacea < 2.34

Citrobacter freundii 4.7

Yersinia enterolitica 4.7

Proteus penneri < 2.34

Proteus mettegeri 9.4

Escherichia coli < 2.34

Shigella sonnei 9.4

Klebsiella pneumoniae 9.4

Salmonella enteridis < 2.34

Morganella morganii 4.7

Proteus vulgaricus 4.7

Pseudomonas aeruginosa ATCC27853 4.7

Free radical-induced oxidative stress has been implicated in the pathogen- esis of a wide variety of clinical disorders, resulting usually from deficiency of natural antioxidant defenses. Potential antioxidant therapy should therefore include either natural free radical scavenging antioxidant enzymes or agents that are capable of augmenting the activity of these enzymes, which include superoxide dismutase, catalase and glutathione peroxidase.

FIG. 1. WEIGHT VARIATION OF THE SWISS ALBINOS’ MICE BEFORE AND AFTER GAVAGES (7 DAYS) WITH 0.003 mg/g MOUSE, 0.03 mg/g MOUSE, 0.3 mg/g MOUSE,

3 mg/g MOUSE OF LAUREL EXTRACT

Values are expressed as mean ⫾ SD. *P < 0.05 (Student’s t-test), n = 9% indicates the percentage of decrease or increase in the values compared with the control.

TABLE 4.

PLASMATIC PARAMETERS FROM CONTROL AND LAUREL TREATED MOUSE AT 0.003 AND 0.3 mg

Control Laurel

0.003 mg

Laurel 0.3 mg

GOT (U/l) 285 ⫾ 56 272 ⫾ 28 276.5 ⫾ 35

GPT (U/l) 45.6 ⫾ 7 39 ⫾ 2.8 57.5 ⫾ 0.70* (¥1.26)

CPK (U/l) 2,915 ⫾ 134 2,328 ⫾ 694 4,645 ⫾ 77.78* (¥1.59)

Urea 0.32 ⫾ 0.10 0.35 ⫾ 0.07 0.36 ⫾ 0.06

Creatinine 8.66 ⫾ 1.52 9 ⫾ 1.41 8.5 ⫾ 0.70

Values (mm) are expressed as mean ⫾ SD (n = 9). Numbers in parentheses indicate how many times the values have increased compared with the control values.

* P < 0.05 (Student’s t-test).

GOT, glutamic oxalic transaminase; GPT, glutamic pyruvic transaminase.

For the metabolic markers, GAPDH was very sensitive to L. nobilis extract at 0.3 mg/g; it significantly decreased (52%). In contrast, the SDH activity increased significantly (¥4.94) at 0.3 mg/g. This increased SDH activity suggests that the recovery process increased the production of energy in liver, and that metabolism energetics in heart and liver are perturbed, which caused serious problems such as cirrhoses of liver and myocardial infarcts over a long time.

Living tissues are endowed with innate antioxidant defense mecha- nisms, such as the presence of the enzyme catalase (CAT). A reduction in the activities of these enzymes is associated with the accumulation of highly reactive free radicals, leading to deleterious effects such as loss of integrity and function of cell membranes (Sheela and Angusti 1995).

This probably explains the unchanged activities of CAT at 0.3 mg/g (Table 5).

No changes were observed with the amounts of 0.003 mg/g on stress and metabolic biomarkers.

Other plants such as Elettaria cardamomum have the same effects as laurel against free radical damage induced during stress (El Malti et al. 2007).

Effectively, cardamomum induces toxicity at 0.3 mg/g mouse and affects energy metabolism, which induces inflammation in the brain, oxidative stress and cell necrosis in the heart.

The development of synthetic compounds capable of scavenging reac- tive oxygen species has been a great success; however, it has been suggested that these substances may be inappropriate for chronic human consumption

TABLE 5.

EFFECT IN VIVO OF LAUREL AT 0.003 AND 0.3 mg ON RESPONSE OF OXIDATIVE STRESS AND METABOLIC BIOMARKERS

Control Laurel

0.003 mg

Laurel 0.3 mg Catalase

(mmol/min/mg of protein)

3.51 ⫾ 0.58 3.17 ⫾ 0.48 2.28 ⫾ 0.89 Thiobarbituric acid reactive substances

(nmol/mg of protein)

0.42 ⫾ 0.16 0.32 ⫾ 0.08 1.47 ⫾ 0.15* (¥3.5)†

Glyceraldehyde-3-phosphate dehydrogenase (mmol/min/mg of protein)

0.50 ⫾ 0.15 0.40 ⫾ 0.19 0.24 ⫾ 0.04* (-52%)‡

Succinate dehydrogenase (Absorbance/min/mg of protein)

1.48 ⫾ 0.76 1.98 ⫾ 0.12 7.32 ⫾ 1.32* (¥4.94)†

Values (mm) are expressed as mean ⫾ SD (n = 9).

* P < 0.05 (Student’s t-test).

† Indicate how many times the values have increased compared with the control values.

‡ Indicate the percentage of decrease in the values compared with the control values.

(Madhavi and Salunkhe 1995). Antioxidants are compounds that inhibit or delay oxidation of other molecules by terminating the initiation or propaga- tion of oxidizing chain reactions. The potential value of antioxidants has prompted researchers to look for natural antioxidants with low cytotoxicity.

A great number of spices and aromatic herbs contain chemical compounds exhibiting antioxidant properties (Madsen and Bertelsen 1995). These prop- erties are attributed to a variety of active phytochemicals, including vitamins, carotenoids, terpenoids, alkaloids, flavonoids, lignans, simple phenols and phenolic acids (Liu and Ng 2000). Antimicrobial and antioxidant properties of spices make them useful as preservative agents.

In previous studies, the hexane and ethyl acetate extracts of Bay leaf exhibited potent biological activity in inducing apoptosis (Fang et al. 2005).

Reynosin, santamarine, spirafolide and costunolide were found in Bay leaf in previous studies. Reynosin and santamarine have been previously reported to exhibit cytotoxic activity against the human lung carcinoma cell Line GLC4 and the colorectal cancer cell line COLO 320 (IC50 = 124 mM) (Goren et al.

1996). They have also been found to have inhibitory effects on alcohol absorption and nitric oxide production (Matsuda et al. 2000a). Spirafolide has been reported as having an anti-inflammatory effect (Matsuda et al.

2000a) and the ability to inhibit alcohol absorption (Yoshikawa et al.

2000). Costunolide is the most studied sesquiterpene lactone; it is found in numerous species belonging to many genera of the composite, and a known source is costus root oil. This latter compound has been reported to enhance liver glutathione S-transferase activity (Wada et al. 1997). A recent study found that costunolide also induces apoptosis by decreasing the anti- apoptotic protein Bcl-2 (Babish et al. 2002). Reynosin, santamarine, spi- rafolide and costunolide have been reported to possess antimicrobial (Dadalioglu and Evrendilek 2004) and antioxidant properties (Ferreira et al.

2006).

These properties are also strongly needed by the food industry in order to find possible alternatives to synthetic preservatives. In this context, as laurel extract gave interesting results, it can be considered one of the promising extracts in terms of antimicrobial activity and the ability to neutralize free radicals and to prevent unsaturated fatty acid oxidation. This confirms the potential use of herbs and spices in the food industry to increase the shelf life of foodstuffs.

Immunological Study

In order to explore the impact of laurel extract on immunological cells, the number of lymphocytes, monocytes and polynuclears were calculated.

Figure 2 shows an important increase in lymphocyte level (100%) and a

significant decrease in polynuclear level (59%) at 0.3 mg/g compared with the control. No changes were observed for monocytes.

Lymphocyte proliferation is a very sensitive test and is used as a potential biomarker for toxic exposures. In the present study, Fig. 2 showed an impor- tant increase in lymphocyte level. Further, the liver and heart treated with 0.3 mg/g show cell necrosis and inflammation (Figs. 3 and 4). The necrosis of the cells causes the importation of the immunizing cells and the development of the inflammatory reactions.

Morphological and Histopathological Study

The macroscopic analysis of the removed organs during the dissection did not reveal any changes compared with the control.

Optical microscopy of the liver of the of mice treated with 0.3 mg/g showed a manifold centrilobular necrotic areas filled with detritus and con- nective tissue, hyperplasia of hepatocytes, as well as cytoplasmic dystrophy characterized by small vacuoles tightly packed in the cytoplasm. Additionally, sharply dilated central veins were noted (Fig. 3).

In the heart of the mice treated with 0.3 mg/g, a necrosis and dissolu- tion of the nucleus and nuclear membrane (karyolysis) was observed (Fig. 4).

FIG. 2. LEUKOCYTE NUMBERS AFTER 7 DAYS OF GAVAGES IN COMPARISON WITH CONTROL

Values are expressed as mean ⫾ SD. *P < 0.05 (Student’s t-test), n = 9% indicates the percentage of

decrease or increase in the values compared with the control.

Inflammatory focus (A)

(B)

(C)

FIG. 3. HISTOLOGICAL SECTIONS OF LIVER FROM CONTROL (A) AND GAVAGE WITH LAUREL 0.003 mg (B) AND 0.3 mg (C) AFTER 7 DAYS OF TREATMENTS

Sections are stained with hematosin–eosin. Magnification: 400¥.

A

B

C

FIG. 4. HISTOLOGICAL SECTIONS OF HEART FROM CONTROL (A) AND GAVAGE WITH LAUREL 0.03 mg (B) AND 0.3 mg (C) AFTER 7 DAYS OF TREATMENTS

Sections are stained with hematein-eosin. Magnification: 400¥.

CONCLUSION

The present study confirmed the antibacterial effect of L. nobilis extract and the toxicity of laurel at 0.3 mg/g, which induced inflammation in liver, oxidative stress and cell necrosis in the heart. The use of L. nobilis as a spice should not exceed 0.003 mg/g because at this amount no negative effects were observed.

NOMENCLATURE

ALT glutamic pyruvic transaminase

AST glutamic oxalic transaminase

CPK creatine phosphokinase

DCIP dichloroindophenol

EDTA ethylenediamine tetraacetic acid

GAPDH glyceraldehyde 3-phosphate dehydrogenase

KCN potassium cyanide

MDA malondialdehyde

MIC minimal inhibitory concentrations

NAD nicotinamide adenine dinucleotide oxidized form

SDH succinate dehydrogenase

REFERENCES

AEBI, H. 1984. Catalase in vivo. Methods Enzymol. 105, 121–126.

ALI-SHTAYEH, M.S., YANIV, Z. and JAMAL, M. 2000. Ethnobotanical survey in the Palestinian area: A classification of the healing potential of medicinal plants. J. Ethnopharmacol. 73, 221–232.

AQILI KHORASANI, M.S. 1992. Collection of Drugs (Materia Media), pp. 624–630, Enqelab-e-Eslami Publishing and Educational Organization, Tehran, Iran.

BABISH, J.G., HOWELL, T.M. and PACIORETTY, L.M. 2002. Combina- tions of sesquiterpene lactones and diterpene triepoxide lactones for synergistic inhibition of cyclooxygenase-2. Patent Corporation Treaty (PCT) International Applications. 67.

BAGAMBOULA, C.F., UYTTENDAELE, M. and DEBEVERE, J. 2004.

Inhibitory effect of thyme and basil essential oils, carvacrol, thymol, estragol, linalool and p-cymene towards Shigella sonnei and S. flexneri.

Food Microbiol. 21, 33–42.

BRADFORD, M. 1976. A rapid and sensitive method for the quantization of

microgram quantities of protein utilizing the principle of protein dye

binding. Anal. Biochem. 72, 248–254.

BROADHURST, C.L., POLANSKY, M.M. and ANDERSON, R. 2000.

Insulin-like activity of culinary and medicinal plant aqueous extracts in vitro. J. Agric. Food Chem. 48, 849–852.

COLLINS, C.H., LYNE, P.M. and GRANGE, J.M. 1989. Microbiological Methods, 6th Ed., Butterworths, London, U.K.

COX, P.A. and BALICK, M.J. 1994. The ethnobotanical approach to drug discovery. Sci. Am. 270, 60–65.

COX, S.D., MANN, J.L., MARKHAM, H.C., BELL, J.E., GUSTAFSON, J.R.

and WARMINGTON, S.G. 2000. The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). J. Appl. Microbiol.

88, 170–175.

DADALIOGLU, I. and EVRENDILEK, G.A. 2004. Chemical compositions and antibacterial effects of essential oils of Turkish oregano (Origanum minutiflorum), bay laurel (Laurus nobilis), Spanish lavender (Lavandula stoechas L.), and fennel (Foeniculum vulgare) on common foodborne pathogens. J. Agric. Food Chem. 52, 8255–8260.

DALLMEIER, K. and CARLINI, E.A. 1981. Anesthetic, hypothermic, myo- relaxant and anticonvulsant effects of synthetic eugenol derivatives and natural analogues. Pharmacology 22, 113–127.

DE SÃ FERRIRA, I.C.F. and FERRÃO VARGAS, V.M. 1999. Mutagenicity of medicinal plant extracts in Salmonella/microsome assay. Phytother.

Res. 13, 397–400.

EL MALTI, J., MOUNTASSIF, D. and AMAROUCH, H. 2007. Antimicrobial activity of Elettaria cardamomum: Toxicity, biochemical and histological studies. Food Chem. 104, 1560–1568.

FANG, F., SANG, S., CHEN, K.Y., GOSSLAU, A., HO, C.-T. and ROSEN, R.T. 2005. Isolation and identification of cytotoxic compounds from Bay leaf (Laurus nobilis). Food Chem. 93, 497–501.

FERREIRA, A., PROENC, C., SERRALHEIRO, M.L.M. and ARÀUJO, M.E.M. 2006. The in vitro screening for acetylcholinesterase inhibition and antioxidant activity of medicinal plants from Portugal. J. Ethnophar- macol. 108, 31–37.

GAO, M. and TEPLITSKI, J.B. 2003. Production of substances by Medicago truncatula that affect bacterial quorum sensing. Mol. Plant-Microbe In.

16, 827–834.

GOREN, N., WOERDENBAG, H.J. and BOZOK-JOHANSSON, C. 1996.

Cytotoxic and antibacterial activities of sesquiterpene lactones isolated from Tanacetum praeteritum. Planta Med. 62, 419–422.

GULLUCE, M., SOKMEN, M., DAFERERA, D., AGAR, G., OZKAN, H.,

KARTAL, N., POLISSIOU, M., SOKMEN, A. and SAHIN, F. 2003. In

vitro antibacterial, antifungal, and antioxidant activities of the essential

oil and methanol extracts of herbal parts and callus cultures of Satureja hortensis L. J. Agric. Food Chem. 51, 3958–3965.

HIGASHIMOTO, M., PURINTRAPIBAN, J., KATAOKA, K., KINOUCHI, T., VIMITKETKUMNUEN, U., AKIMOTO, S., MATSUMOTO, H., and OHNISHI, Y. 1993. Mutagenicity and antimutagenicity of extracts of three spices and a medicinal plant in Thailand. Mutation Research 303, 135–142.

HSIEH, P.C., MAU, J.L. and HUANG, S.H. 2001. Antimicrobial effect of various combinations of plant extracts. Food Microbiol. 18, 35–43.

KILIC, A., HAFIZOGLU, H., KOLLMANNSBERGER, H. and NITZ, S.

2004. Volatile constituents and key odorants in leaves, buds, flowers and fruits of Laurus nobilis L. J. Agric. Food Chem. 52, 1601–1606.

KING, T.E. 1967. Preparation of succinate cytochrome c reductase and cyto-chrome b-cl particles and reconstitution of succinate-cytochrome c reductase. In Methods in Enzymology Vol. 10 (R. Estabrook, ed.) pp. 216–225, Academic Press, New York, NY.

LIU, F. and NG, T.B. 2000. Antioxidative and free radical scavenging activi- ties of selected medicinal herbs. Life Sci. 66, 725–735.

MADHAVI, D.L. and SALUNKHE, D.K. 1995. Toxicological aspects of food antioxidants. In Food Antioxidants: Technological, Toxicological, and Health Perspectivas (D.L. Madhavi, S.S. Deshpande, and D.K. Salunkhe, eds.) pp. 267–359, Marcel Dekker, Inc, New York, NY.

MADSEN, H.L. and BERTELSEN, G. 1995. Spices as antioxidants. Trends Food Sci. Technol. 6, 271–277.

MATSUDA, H., KAGERURA, T., TOGUCHIA, I., UEDA, H., MORIKAWA, T. and YOSHIKAWA, M. 2000a. Inhibitory effects of sesquiterpenes from Bay leaf on nitric oxide production in lipopolysaccharide- activated macrophages: Structure requirement and role of heat shock protein induction. Life Sci. 66, 2151–2157.

MATSUDA, H., UEMURA, T., SHIMODA, H., NINOMIYA, K., YOSHIKAWA, M. and KAWAHARA, Y. 2000b. Anti-alcoholism active constituents from Laurel and Linden. Tennen Yuki Kagobutsu Toronkai Koen Yoshishu. 42, 469–474.

OGUNLANA, E.O., HOEGLUND, S., ONAWUNMI, G. and SKOELD, O.

1987. Effects of lemongrass oil on the morphological characteristics and peptidoglycan synthesis of Escherichia coli cells. Microbios 50, 43–59.

SAMOKYSZYN, V.M. and MARNETT, L.J. 1990. Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid. Free Radical Biol.

Med. 8(5), 491–496.

SEN, P., MAITI, P.C. and PURI, S. 1992. Mechanism of anti-stress activity of

Ocimum sanctum Linn, eugenol and Tinospora malabaria in experimen-

tal animals. Indian J. Exp. Biol. 30, 592–596.

SERRANO, A., MATEOS, M.I. and LOSADA, M. 1991. Differential regula- tion by trophic conditions of phosphorylating and non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenases in Chlorella fusca. Biochem. Bioph. Res. Co. 181, 1077–1083.

SHEELA, C.G. and ANGUSTI, K. 1995. Antiperoxide effects of S-allyl cystein sulphoxide isolated from Allium sativum Linn and gugulipid in chlosterol diet fed rats. Indian J. Exp. Biol. 33, 337–341.

SHIVANANDA, P.N., SANDIFORD, V.B. and ADOGWA, A. 2006. Evalua- tion of wound healing activity of Allamanda cathartica. L. and Laurus nobilis. L. extracts on rats BMC. Compl. Alternative Med. 6, 1–10.

SIMIC, S., RISTIC, S.M., VUKOJEVIC, G.J. and MARIN, P.D. 2004. The chemical composition of some Lauraceae essential oils and their anti- fungal activities. Phytother. Res. 18(9), 713–717.

SINI, S., LATHA, P.G., SASIKUMAR, J.M., RAJASHEKARAN, S., SHYAMAL, S. and SHINE, V.J. 2006. Hepatoprotective studies on Hedyotis corymbosa (L.) Lam. J. Ethnopharmacol. 106, 245–249.

TURI, M., TURI, S.K. and MIKELSAAR, B. 1997. Influence of aqueous extracts of medicinal plants on surface hydrophobicity of Escherichia coli strains of different origin. APMIS 105, 956–962.

WADA, K., UEDA, N., SAWADA, H., AMEMIYA, N. and HAGA, M. 1997.

Nat. Med. 51(3), 283–285.

YOSHIKAWA, M. 2001. Recent research on herbal medicines – characterization of bioactive constituents. Fragrance J. 29, 13–23.

YOSHIKAWA, M., SHIMODA, H., UEMURA, T., MORIKAWA, T., KAWAHARA, Y. and MATSUDA, H. 2000. Bioorganic. Med. Chem.

8, 2071–2077.