沙漠研究 19-1, 117- 120 (2009 )Journal of Arid Land Studies“Desert Technology IX ”
Refereed Paper

Anti-Neuronal Stress Effect of Tunisian Rosmarinus officinalis Extract
Abdelfatteh EL OMRI1, Junkyu HAN1, Ron HASHIZUME2, Manef BEN ABDRABBAH3, Hiroko ISODA*1
Abstract: The present study is aimed to the effect of a Tunisian aromatic plant Rosmarinus
officinalis extract on - a neuronal cell model - PC12 cells and on HSP47. The extract of R.
officinalis and two pure compounds rosmarinic acid and carnosic acid promote markedly theneurite outgrowth of PC12 cells and enhanced the acetylcholinesterase activity same as NGF(neurite outgrowth factor). The investigation of phosphorylation marker illustrates thatRosemary extract treatment showed ERK1/2 phosphorylation same as NGF. Moreover,Rosmarinus extracts and its pure compounds have shown a stress recovery activity on HSP47.
Keywords: Acetylcholine, Neurite outgrowth, Rosmarinus officinalis, Stress recovery

1. Introduction
Stress is a widely prevalent phenomenon that exacts a heavy toll on the quality of human life.
During times of stress, the body responds by releasing hormones that trigger physical changes. Chronic
stress can have negative effects on our well being, leading to many physiological changes. The effects ofstress are supposed to be an outcome of altered activity of different mechanism such as central
neurotransmitters, neurohormonal factors, and free radical generation (Herman et al., 1997). There aresome studies linking stress with cholinergic activity. Moreover, motor neurons have a high threshold for
induction of heat shock proteins (Hsps) in response to stress. Numerous plants have been used to treat
cognitive disorders, over 150 plant species in various preparations and mixtures were cited by (Das et al.,2007) as relevant to dementia, Alzheimer disease and cognitive aging disease. The present study is aimed
to the effect of a Tunisian aromatic plant “R. officinalis” extracts on neuronal stress and its mechanism
using PC12 cells and Heat shock protein HSP47 as a model.
2. Materials and Methods
2.1. Sample preparation and HPLC analysis
Rosemary was collected from Tunisia, from Sammama-Mount kasserine and Zaghouan-Mount during
the period April-May 2007 corresponding to the flowering season of this plant. Fresh leaves were crushed
in a mortar and extracted with different solvent 10% w/v. The extraction with water was carried out inautoclave at 105 ˚C for 15 min. For ethanol (70%) and methanol (70%), the extraction was done in darkin an Erlenmeyer at 25 ˚C, for 1 week, with shaking at least one time a day. HPLC analysis were done byGilson apparatus using a binary acetonitrile/water gradient, Column: Beta max Neutral (150 × 4.6 mm id;
particle size 5 μm), a flow rate of 3 ml/min and detection was recorded using UV detector at 300 and 230
nm.
2.2. Acetylcholinesterase activity
The rat pheochromocytoma PC12 cell line was routinely maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM; Sigma) supplemented with 5% heat inactivated fetal bovine serum, 10% heat inactivatedhorse serum (Gibco®, Invitrogen), and 1% streptomycin (5000 I.U. ml−1)-penicillin (5000 μg ml−1)
solution in tissue culture flasks and incubated at 37 ˚C in a 95% air-5% CO2 incubator. The cells wereplated onto 96-well plates at initial concentrations of 1×104 cells per well in 100 µL of culture medium,incubated and allowed to attach for at least 6 h before the different extract of Rosemary were added toobtain the required final concentrations. After incubating for 24 hr, the medium was carefully removed
and discarded and the cells carefully washed twice with 200 μl of PBS(-). After washing, 20 μl of 5.6
1Alliance for Research on North Africa (ARENA), Graduate School of Life and Environmental Sciences, University ofTsukuba, 1-1-1 Tennodai, Tsukuba City, Ibaraki 305-8572, Japan.
*Corresponding Author: Fax no. +81298535775 E-mail: isoda@sakura.cc.tsukuba.ac.jp
2NOF CORPORATION, 20-3, Ebisu 4, Shibuya, Tokyo 150-6019, Japan
3
Unité de Recherche: Physico-chimie et Moléculaire: Institut Préparatoire aux Etudes Scientifiques et Techniques
IPEST, La Marsa, 2070 Tunis – Tunisia

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mM acetylcholine iodide and 180 μl of lysis solution, pH 7.5 (0.12 M NaCl, 0.2% TritonX-100, 1 mM
EDTA, 50 mM Hepes) were added into each well. After incubating for 2 h in room temperature, 20 μl of
the cell lysates were transferred to a fluorescence reading multiwell plate and incubated for 1 h with 160 μlbuffer solution, pH 5.0 (1 mM EDTA, 0.2% TritonX-100, 50 mM acetate buffer) and 20 μl of 0.4 mM
7-diethylamino-3-(4-maleimidyl-phenyl)- 4-methylcoumarin in acetonitrile. The fluorescence in eachwell was then measured using a multi-detection microplate reader (Powerscan® HT, Dainippon
Pharmaceutical, Japan) at 360 nm/ 460 nm.
2.3. Stress recovery activity
The Chinese hamster ovary (CHO) cells with (HSP(+)) or without (HSP(-)) a heat shock protein
promoter were used for this experiment. Cells were grown as adherent monolayer in 75-cm2 tissue
culture flasks using F12 Medium (Gibco®, Invitrogen, Tokyo, Japan) supplemented with 10% Fetal
Bovine Serum, 200 µg/mL of G418 (Gibco BRL 13075-015) and 0.1 g/L kanamycin solution (Sigma).
HSP47-transformed cells were trypsinized and plated onto 96-well plates at initial concentrations of 1×104
cells per well in 100 µL of culture medium. The cells were allowed to attach for 48h at 37 ˚Csupplemented with 5% CO2, shocked for 90 min at 42 ˚C, 5% CO2 and recovered at 37 ˚C for 2 h in a 5%
CO2 incubator. Then, the medium was removed and 100 µL of samples diluted with medium followed byincubation for 3 h in a 5% CO2 incubator at 37 ˚C. The medium was then carefully removed and the cells
washed twice with PBS(-). 50 µl of lysis buffer (Promega) was then added and the plates incubated for
30 min at room temperature. 20 µl of cell lysates were transferred to a new plate, to which 100 µL ofsubstrate solution (10 mM NaH2PO4•2H2O, 100 mM NaCl, 1% BSA, 0.005% NaN3, 1 mM MgCl2•6H2O,1% 4-methylumbelliferyl-β-D-glactoside (MUG), pH 7) was added in order to trigger the conversion ofMUG into galactose and methylumbelliferyl by galactosidase. After allowing the reaction to occur in thedark for 30 min at Room temperature, 60 µl of reaction stop buffer (1 M glycine-NaOH, pH 10.3) wasadded and the fluorescence at 365 nm excitation/ 450 nm emission was then determined using a
multi-detection microplate reader (Powerscan® HT, Dainippon Pharmaceutical, Japan).
3. Results and Discussion
3.1. Tunisian Rosmarinus officinalis
R. officinalis (Fig. 1) is a woody shrub with a pine needle like leaves. Its trusses of blue flowers lastthrough spring and summer. It will grow to a height of 1.5 m. In Tunisia, R. officinalis is growing wildin bioclimatic zones extending from the sub-humid to the arid, with a rain fall level of 200 to 600 mm/year(Rejeb et al., 2006) on sandy, calcareous or marno-calcareous soils.
Main populations developed in garrigues resulting from the degradation of Pinus halepensis L.,
Juniperus phoenicea L. and Tetraclinis articulata L., (Zaouali and Boussaid, 2007). In Tunisia, this plant
covers an area of about 340000 ha (Rejeb et al., 2006). Leaves of this plant have been used for a longtime in Mediterranean cuisine, not only to improve or modify the flavor of foods, but also to avoid its
deterioration. Also this plant is a symbol of remembrance and has been used since many centuries toimprove memory. The infusion of leaves is used as antispasmodic, antiseptic, diuretic and relaxing.
Dried leaves are used as additives to prevent fat oxidation and as flavoring agent in some meal. In thepresent study, we identified by HPLC analysis from different solvent extract of R. Officinalis leaves about
8 compounds: Ferulic acid, Caffeic acid, p-Coumaric acid, Rosmarinic acid, Apigenin, Luteolin, Carnosoland Carnosic acid. It was reported by (Kosaka and Yokoi, 2003) that Carnosic acid (Fig. 2a), was able topromote markedly the synthesis of NGF (nerve growth factor) in T98 glioblastoma cells. NGF is a factor
vital for the growth and functional maintenance of nerve tissue growth factor. Furthermore, Rosmarinicacid (Fig. 2b) and Caffeic acid may produce antidepressive-like effect in the forced swimming test in mice
(Takeda et al., 2002) i.e. that these compounds have a positive effect on memory and have relaxant effect.
3.2. Acetylcholinesterase activity
AChE is a glycoprotein, responsible of the breakdown of acetylcholine (ACh) (Fig. 3). In fact, Achis cleaved by AChE into choline and acetate. The choline group was quantified at 360 nm excitation and
460 nm emission by adding MDCC (7-diethyl-3(4-maleimidyl-phenyl)-4-methylcoumarin119

CH3CH3HO(a)CH3OHHOCH3HOOOHOHO(b)HOO

Fig. 1. Rosmarinus officinalis

OHHOFig. 2. Chemical structure of carnosic acid (a) androsmarinic acid (b)Fig. 3. Fluorescence detection of AchE activity using PC12 cells

after cell lysis. And AChE activity was reported as percentage of control. R. officinalis solvent extract
enhanced AChE activity same as NGF. Also the microscopic observation of cells treated with Rosemary
extract, has shown that this plant promote markedly neurite outgrowth and cell proliferation. In this study,AChE was increased i.e. that the amount of ACh also was improved. Aloisi et al. (1996) reported that inanimal experimental model, the synthesis and release of ACh in hippocampus is correlated to stress andenvironment changes. Hippocampal cholinergic system is implicated in several functions such as arousaland attention, in certain aspects of learning and memory and in the neuroendocrine and emotional
responsiveness to aversive stimuli. This fact, show clearly that Tunisian R. officinalis has a relaxing
index effect and can be recommended for memory improvement. Such effect can be due to the presenceof some polyphenolic and terpenic compound such as Rosmarinic acid and carnosic acid.
3.3. Molecular mechanism of regulation of AChE
In the present study, mechanism of promoting action of R. officinalis extract of PC12 cells
differentiation and acetylcholinesterase activity seems to be regulated by MAPK: Mitogen-activatedprotein (MAP) kinases ERK1/2 that respond to extracellular stimuli (mitogens) and regulate various
cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis.
3.4. Stress recovery activity of Rosmarinus officinalis
We evaluate this activity using a transformed cell model: transfected Chinese Hamster cell Ovary(CHO) with HSP 47 (Isoda et al., 2003). Heat shock protein HSP 47 is a 47 kDa collagen specificmolecular chaperone localized in the endoplasmic reticulum that plays a critical role during the folding,maturation, and secretion of procollagen (Higuchi et al., 2006).
As HSP47 is a heat shock (stress) protein, its expression is dramatically enhanced after the cells aretreated at high temperature (42-45 ˚C) for short periods (about 30 min). Nuclear run-on assay
demonstrated that a more than ten-fold increase in the transcription rate of HSP47 gene occurred after 2 hof the heat treatment of chick embryo fibroblasts at 45 ˚C, which rapidly returned to the basal level during

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the recovery period at 37 ˚C (Nagata, 1998). In this case heat stress at 42 ˚C lasts 90 min, the recovery for
2 h at 37 ˚C, and the treatment time took 3 h. Then, the expression of the heat shocked protein wasmeasured according to the amount of β-galactosidase that cleave MUG to methylumbelliferyl and galactosethat can be measured by fluorescence at 360 nm emission and 460 nm excitation. Rosemary extracts havean interesting stress recovery activity at some specific concentration.
4. Conclusions
In this study we report that Tunisian R. officinalis extracts have an index of relaxing effect, throughtheir high antioxidant activity, their capacity of enhancing neurite outgrowth and cell differentiation of
PC12 cells and their aptitude of regulation of cholinergic system. The mechanism of PC12 cells
differentiation and AChE activity are regulated by MAPK: ERK1/2. In addition, these extracts have arecovery effect on HSP47. These findings, suggest that this plant has a neuroprotective effect and can beused to prevent from some neurodegenerative disease and stress. The promising future treatment ofneurodegenerative diseases and aging depends on availability of effective brain permeable, ironchelatable/radical scavenger neuroprotective drugs that would prevent the progression of neurodegeneration. Furtherstudies are required to test out its outcome on the protection from neurological diseases of a wide array ofneuronal cell and animal models.
Acknowledgement
This study was supported in part by funds from the Grant-in-Aid for Scientific Research (Grant No. 17255011).
References
Aloisi A.M., Casamenti F., Scali C., Pepeu G., Carli G. (1996): Effects of novelty, pain and stress on hippocampal extracellular
acetylcholine levels in male rats. Brain Research, 748: 219-226.
Das A., Rai D., Dikshit N., Palit G., Nath C. (2004): Nature of stress: Differential effects on brain acetylcholinesterase activity andmemory in rats. Life Sciences, 77: 2299–2311.
Herman J.P., Cullinan W.E. (1997): Neurocircuitary of stress: HPA axis. Trends Neurosci, 20: 78-84.
Higuchi I., Hashiguchi A., Matsuura E., Higashi K., Shiraishi T., Hirata N., Arimura K., Osame M. (2006): Different pattern of HSP47
expression in skeletal muscle of patients with neuromuscular diseases. Neuromuscular disorders, 17: 221-226.
Isoda H., Koyama T., Takashi M., Oka S., Suguiura N., Maekawa T., Inamori Y., Yokota S., Kiktahara M., Nagata K. (2003): Highsensitive detection of environmental pollutants. US Patent and trademark office, 670521.
Kosaka K., Yokoi T. (2003): Carnosic Acid, a component of Rosemary (Rosmarinus officinalis L.), promotes synthesis of Nerve
Growth Factor in T98G human glioblastoma cells. Biol. Pharm. Bull., 26 (11): 1620 -1622.
Nagata H. (1998): Expression and Function of Heat Shock Protein 47 a Collagen-Specific Molecular Chaperone in the EndoplasmicReticulum. Matrix Biology, 16: 379-386.
Rejeb M.N., Khouja M.L., Ghrabi Z., Chemli R., Albouchi A., Khaldi A., Dahman M. (2006): Guide des plantes médicinales etaromatiques, Maghreb Editions. ISBN: 9973-9954-3-0. 129 p.
Takeda H., Tsuji M., Inazu M., Egashira T., Matsumiya T. (2002): Rosmarinic acid and caffeic acid produce antidepressive-like effectin the forced swimming test in mice. European Journal of Pharmacology, 449: 261– 267.
Zaouali Y., Boussaid M. (2007): Isozyme markers and volatiles in Tunisian Rosmarinus officinalis L. (Lamiaceae): A comparative
analysis of population structure: Biochemical Systematics and Ecology, 36 :11-21.