Journal of Applied Sciences Research, 6(9): 1400-1411, 2010© 2010, INSInet Publication

Study on the Effect of Rosemary Extract on Some Neurotransmitters and Their RelatedIons in Different Brain Areas of Adult Male Albino Rats

1Seham, M. Abdel-Kader, 2Mona, Abdel-Rahman and 2Amira, A. Bauomy.

1Radioisotopes Department, Nuclear Research Center, Atomic Energy Authority, Egypt.2Zoology and Entomology Department, Faculty of Science, Helwan University, Egypt.

Abstract: The daily oral administration of Rosmarinus officinalis (Rosemary) extract with a dose of 582.4mg / kg b.wt. (0.5 ml solution/rat) for 4 weeks resulted in a significant reduction in monoamines, Na+ ion,GABA contents and acetylcholine esterase activity in all tested brain areas, almost all over theexperimental period. Also, it caused a significant increase in Cl- ion content. According to the obtainedresults, it could be concluded that the plant extract may decrease monoamines reuptake or induce theirturnover, which eventually stimulate their release. Moreover, since Rosmarinus officinalis wasdemonstrated as an anticonvulsant and has an antinociceptive effect so it blocks persistent sodium currentsin CNS neurons. On the same line, reduction in GABA and increment in Cl- ion contents may be due toanxiolytic and sedative effects of Rosmarinus. The reduction in acetyl cholinesterase activity may be dueto Rosmarinus officinalis has an anti-acetylcholine esterase activity which improving memory.

Key words: Rosmarinus officinalis, dopamine, norepinephrine, serotonin, sodium ion, γ-aminobuteric acid, chloride ion, acetylcholine esterase, rat, brain areas.

INTRODUCTION

Rosmarinus officinalis is a plant belonging to theLabiatae family. It is a symbol of remembrance Bown[1], thus it is well-known as a general tonic andenergizer [2]. It is used for flavoring food, a beveragedrink, as well as a fragrant additive in soaps andcosmetics [3, 4].

Rosmarinus contains a wide variety of volatile andaromatic components such terpenoid, flavonoids [4, 5].

The essential oil has a repellent activity Nerio etal. [6], antimicrobial effects Okoh et al., [7] and antiviralactivities [8]. In addition, Rosmarinus officinalis iswidely used around the world for medicinal purposesOluwatuyi et al. [9], which has an antinociceptiveeffect González-Trujano et al. [4], antioxidant propertiesNieto et al. [10]; Tironi et al. [11] and it is used in thetreatment of memory impairment observed inAlzheimer's disease [12].

MATERIALS AND METHODS

Animals: Male albino rats “Rattus rattus” weighing100-120g, purchased from the breeding unit of HoldingCompany for Biological Products and Vaccines. Theywere allowed to acclimatize to surroundings for fewdays before carrying out the experiments. The animalswere housed under normal environmental conditions oftemperature and humidity.

Materials: Rosmarinus officinalis leaves were obtainedfrom the market. The plant seeds were extractedaccording to [13].

Plant Extract: The animals were daily and orallyadministered plant extract equivalent to 582.4 mg / kgb.wt. (0.5 ml solution/rat) for 4 weeks [14]. Rats weresacrificed after 1, 2, 3 and 4 weeks of the treatment.To study the withdrawal effect of the plant extract,another group of animals were sacrificed after 1 and 2weeks of withdrawal period following the last dose.

Animal Groups: The experimental animals used in thisstudy were divided into two main groups. Each oneconsists of 75 rats. Each group was divided into 6subgroups of 12 rats.

1-First Group: Animals of this group were receiveddistilled water (0.5 ml/100 gm rat) daily and orally for4 weeks and served as a control group.

2- Second Group: Animals of this group werereceived a daily oral administration of Rosmarinusofficinalis extract for 4 weeks at a dose level 582.4mg/kg b.wt. All animals were sacrificed by suddendecapitation after 1, 2, 3, 4 weeks and 1, 2 weeks ofwithdrawal period.

Corresponding Author: Seham, M. Abdel-Kader, Radioisotopes Department, Nuclear Research Center, Atomic EnergyAuthority, Egypt.E-mail:amiraanwar1@gmail.com

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Biochemical Analysis: The brain tissue was rapidlyand carefully removed then divided into 2 halves. Eachhalf was dissected on a dry ice glass plate according tothe method described by [15]. The brain was separatedinto the following regions: cerebellum, striatum,cerebral cortex, hypothalamus, brainstem andhippocampus. Brain tissues were wiped and dried on afilter paper, weighed, wrapped in plastic films and thenin aluminum foil and quickly frozen in – 700C forfurther analysis.

In the present study DA, NE and 5-HT wereextracted and estimated in the brain areas according tothe method of Chang [16] modified by [17]. GABA wasextracted and estimated according to the method of [18].

Metal ions (Na+ and Cl-) were extracted andestimated according to [19]. The acetylcholine esteraseactivity was determined by Ellman method whichmodified by [20]

.Statistical Analysis: Statistical analysis between controland treated animals were performed using student’s t-test [21].

RESULT AND DISCUSSION

Results: Table (1) showed that daily oraladministration of Rosmarinus officinalis extract at adose level 582.4 mg/kg b.wt. resulted in a highlysignificant reduction in DA content in all tested brainareas after 3 and 4 weeks. The maximum effect of DAcontent was observed in striatum on 4th week recording-64.17% as a percentage change. However, DA contentrecorded nearly control level in cerebellum,hypothalamus, brainstem and hippocampus after 2weeks of withdrawal period, DA in striatum andcerebral cortex still showed a highly significantdecrease as compared to its corresponding controlthrough the withdrawal with period.

As noticed in table (2) Rosmarinus officinalisextract gavage caused a highly significant decrease inNE content after 1st week in most brain areas underinvestigation. The maximum decrement was observedon 4th week. Striatum was the most effected arearecording -68.32% as a percentage change. A partialrecover was observed on withdrawal period inbrainstem and hippocampus while NE content incerebellum, striatum, cerebral cortex and hypothalamusshowed more or less near the control level.

The data reported in table (3) showed that theplant extract induced a highly significant reduction in5-HT content in most brain areas under investigationall over experimental period except in cerebral cortexand hippocampus the highly significant reduction wasstarted on 2nd week. After a withdrawal period of 2weeks, 5-HT content approached more or less tonormal levels in all tested brain areas.

Table (4) showed a highly significant decrease inNa+ ion content on 2nd week in all studied brain areasafter Rosmarinus officinalis extract administration.Brainstem and hippocampus are the most effected areason 4th week. A partial recovery was observed on 2nd

withdrawal week in all brain areas under investigationwhile in hippocampus Na+ ion content showed more orless a complete recovery.

As illustrated in table (5), it was appeared that thedaily oral administration of Rosmarinus officinalisextract caused a highly significant reduction in GABAcontent in all studied brain areas on 2nd week except incerebellum the highly significant was started on 3rd

week. The most effected area was hypothalamus on 4th

week (-77.16%). On 2nd withdrawal periodhypothalamus still showed a highly significantreduction while a partial recovery was observed inbrainstem and a complete recover was occurred oncerebellum, striatum, cerebral cortex and hippocampus.

It was clear from data recorded in table (6) thatthe plant extract administration resulted in a highlysignificant increment in Cl- ion content on all studiedbrain areas on 3rd and 4th weeks, the maximum effectwas observed in hypothalamus (409.33%) on 4th week.Although in withdrawal period there was a significantincrease in Cl- ion content in most of brain areas underinvestigation (cerebellum, striatum, hypothalamus andhippocampus), brainstem showed a partial recover andcerebral cortex showed more or less nearly controllevel.

Table (7) demonstrated that acetylcholine esterasewas significantly decreased in all brain areas underinvestigation all over experimental period. The mosteffected area was hippocampus. In spite of being inwithdrawal period (2nd week) a highly significantreduction was observed in all studied brain areasexcept in striatum a partial recover was occurred.

Discussion: From the present results, the daily oralgavage of Rosmarinus officinalis (at a dose level 582.4mg/Kg b.wt.) aqueous extract decreased themonoamines (DA, NE and 5-HT) contents significantlyas well as Na+ ion in all brain areas under investigation(cerebellum, striatum, cerebral cortex, hypothalamus,brainstem and hippocampus) throughout experimentalperiod.

Linalool is a compound commonly found as amajor volatile component of the essential oils inseveral aromatic plant series [22]. Linalool administrationin rats antagonized different pain responses elicited bythe exposure to a chemical nociceptive stimulus. Thedata supposed an involvement of muscarinic, opioidand dopamine transmission [23].

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Table 1: Effect of daily oral administration of Rosmarinus officinalls extract (582.4 mg/kg. b.w.t) on dopamine content in different brain areasof adult male albino rats at different time intervals.

Brain Areas Cerebellum µg/g Striatum µg/g Cerebral Cortex µg/g Hypothalamus µg/g Brainstem µg/g HippocampusTime Intervals µg/g1st week: Control 3.37 ± 0.17 6.90 ± 0.10 1.97 ± 0.02 12.90 ± 0.26 3.99 ± 0.28 12.29 ± 0.60---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 3.25 ± 0.18 6.15 ± 0.17* 1.48 ±0.12** 11.09 ± 0.62* 3.54 ± 0.09 10.73 ±0.03*

% Change -3.56 % -10.87 % -24.87 % -14.03 % -11.28 % -12.69 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week: Control 3.13 ± 0.26 7.49 ± 0.12 1.84 ± 0.03 13.05 ± 0.22 3.27 ± 0.24 10.05 ± 0.16---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 2.57 ± 0.10* 5.53 ± 0.16** 1.29 ± 0.06** 10.33 ± 0.83** 2.78 ± 0.08* 8.75 ± 0.24**

% Change -17.89 % -26.17 % -29.89 % -20.84 % -14.98 % -12.93 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3rd week: Control 3.16 ± 0.24 8.03 ± 0.24 1.57 ± 0.05 12.73 ± 0.60 3.27 ± 0.21 9.01 ± 0.38---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 1.88 ± 0.05** 5.75 ± 0.29** 1.01 ± 0.02** 7.28 ± 0.48** 1.78 ± 0.04** 4.75 ± 0.31**

% Change -40.51 % -28.37 % -35.67 % -42.81 % -45.57 % -47.28 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4th week: Control 2.99 ± 0.15 8.54 ± 0.23 1.57 ± 0.03 12.89 ± 0.60 2.95 ± 0.07 8.53 ± 0.19---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 1.21 ± 0.03** 3.06 ± 0.30** 0.80 ± 0.08** 6.01 ± 0.54** 1.21 ± 0.05** 3.30 ± 0.17**

% Change -59.53 % -64.17 % -49.04 % -53.37 % -58.98 % -61.13 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1st week withdrawal: 2.66 ± 0.13 9.46 ± 0.03 1.46 ± 0.03 12.98 ± 0.18 1.82 ± 0.01 10.31 ± 0.09Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 2.01 ± 0.02** 6.98 ± 0.54** 1.23 ± 0.02** 11.24 ± 0.09** 1.61 ± 0.11* 5.5 ± 0.22**

% Change -24.44 % -26. 26 % -15.75 % -13.41 % -11.54 % -46.65 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week withdrawal: 2.66 ± 0.13 9.46 ± 0.03 1.46 ± 0.03 12.98 ± 0.18 1.82 ± 0.01 10.31 ± 0.09Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 2.51 ± 0.17 8.22 ± 0.22** 1.26 ± 0.06** 12.95 ± 0.64 1.74 ± 0.07 10.14 ± 0.43% Change -5.63 % -13.11 % -13.69 % -0.23 % -4.39 % -1.65 %Data are represented as mean ± S.E. Statistical analyses were carried out between control (6) and treated (6) animals according to student t-test. * Significant at p<0.05 and ** highly significant at p<0.01.

Table 2: Effect of daily oral administration of Rosmarinus officinalls extract (582.4 mg/kg. b.w.t) on norepinephrine content in diffferent brainareas adult male albino rats at different time intervals

Brain Areas Cerebellum µg/g Striatum µg/g Cerebral Cortex µg/g Hypothalamus µg/g Brainstem µg/g HippocampusTime Intervals µg/g1st week: Control 12.64 ± 0.96 26.42 ± 1.33 1.70 ± 0.12 21.24 ± 0.15 11.66 ± 0.91 27.50 ± 0.91---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 10.86 ± 0.21 20.08 ± 1.68** 1.35 ± 0.09* 19.51 ± 0.53** 10.19 ± 0.19 23.60 ± 1.09*

% Change -14.08 % -23.99 % -20.59 % -8.15 % -12.61 % -14.18 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week: Control 13.85 ± 0.75 26.91 ± 1.82 1.25 ± 0.12 26.40 ± 1.39 10.49 ± 0.93 27.99 ± 0.76---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 8.98 ± 0.40** 17.80 ± 0.87** 0.98 ± 0.08* 16.28 ± 1.06** 7.93 ± 0.33* 17.83 ± 1.28**

% Change -35.16 % -33.85 % -21.60 % -38.33 % -24.40 % -36.29 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3rd week: Control 10.71 ± 0.46 13.49 ± 0.96 1.41 ± 0.08 24.86 ± 1.52 12.50 ± 1.08 28.82 ± 0.82---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 6.84 ± 0.10** 8.48 ± 0.82** 1.03 ± 0.03** 13.64 ± 1.09** 6.36 ± 0.38** 14.49 ± 0.57**

% Change -36.13 % -37.14 % -26.95 % -45.13 % -49.12 % -49.72 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4th week: Control 15.00 ± 0.66 15.34 ± 1.10 1.52 ± 0.13 22.50 ± 0.82 9.83 ± 0.63 27.12 ± 0.67---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 5.63 ± 0.16** 4.86 ± 0.46** 0.86 ± 0.08** 11.22 ± 0.60** 4.44 ± 0.14** 8.91 ± 0.77**

% Change -62.47 % -68.32 % -43.42 % -50.13 % -54.83 % -67.15 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1st week withdrawal: 10.03 ± 0.49 18.96 ± 1.28 1.71 ± 0.09 25.89 ± 1.45 7.99 ± 0.52 29.51 ± 0.98Control

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Table 2: Continue.Treated 9.69 ± 0.33 14.25 ± 0.67** 1.58 ± 0.11 21.89 ± 1.30* 6.25 ± 0.15** 23.57 ± 0.97**

% Change -3.39 % -24.84 % -7.60 % -15.45 % -21.78 % -20.13 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week withdrawal: 10.03 ± 0.49 18.96 ± 1.28 1.71 ± 0.09 25.89 ±1.45 7.99 ± 0.52 29.51 ± 0.98 Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 10.30 ± 0.41 18.24 ± 0.46 1.62 ± 0.06 24.75 ± 0.70 7.09 ± 0.15 26.94 ± 0.75*

% Change 2.69 % -3.79 % -5.26 % -4.40 % -11.26 % -8.71 %Data are represented as mean ± S.E. Statistical analyses were carried out between control (6) and treated (6) animals according to student t-test. * Significant at p<0.05 and ** highly significant at p<0.01.

Table 3: Effect of daily oral administration of Rosmarinus officinalis extract (582.4 mg/kg, b.wt.) on serotonin content in different brain areasof adult male albino rats at different time intervals.

Brain Areas Cerebellum µg/g Striatum µg/g Cerebral Cortex µg/g Hypothalamus µg/g Brainstem µg/g HippocampusTime Intervals µg/g1st week: Control 5.39 ± 0.34 20.63 ± 0.67 2.07 ± 0.05 25.39 ± 1.25 4.51 ± 0.04 7.81 ± 0.46---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 3.81 ± 0.21** 14.88 ± 0.56** 1.74 ± 0.14* 20.00 ± 0.87** 4.08 ± 0.10** 6.00 ± 0.48*

% Change -29.31 % -27.87 % -15.94 % -21.23 % -9.53 % -23.18 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week: Control 5.11 ± 0.31 21.39 ± 0.39 2.25 ± 0.08 23.25 ± 0.93 4.74 ± 0.10 9.36 ± 0.39---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 2.84 ± 0.22** 14.62 ± 0.71** 1.53 ± 0.08** 15.81 ± 0.32** 3.54 ± 0.21** 5.48 ± 0.14**

% Change -44.42 % -60.82 % -32.00 % -31.96 % -25.32 % -41.45 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3rd week: Control 5.01 ± 0.17 20.41 ± 0.52 2.88 ± 0.15 20.90 ± 1.92 4.19 ± 0.24 8.47 ± 0.52---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 1.74 ± 0.05** 12.82 ± 0.83** 1.79 ± 0.08** 10.89 ± 0.50** 3.11 ± 0.18** 4.69 ± 0.32**

% Change -65.00 % -37.19 % -37.88 % -47.89 % -25.78 % -44.63 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4th week: Control 5.17 ± 0.11 20.41 ± 0.52 2.88 ± 0.20 20.16 ± 0.69 4.58 ± 0.15 7.08 ± 0.28---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 1.27 ± 0.07** 11.32 ± 0.56** 0.93 ± 0.06** 9.53 ± 0.40** 2.33 ± 0.010** 2.92 ± 0.14**

% Change -75.44 % -44.54 % -67.71 % -52.73 % -49.13 % -58.76 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1st week withdrawal: 4.50 ± 0.13 17.54 ± 0.24 1.79 ± 0.13 23.17 ± 1.02 4.86 ± 0.15 8.12 ± 0.41Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 4.51 ± 0.24 13.50 ± 0.80** 1.53 ± 0.09 19.16 ± 0.60** 3.50 ± 0.13** 4.77 ± 0.36**

% Change 0.22 % -23.03 % -14.53 % -17.31 % -27.98 % -41.26 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week withdrawal: 4.50 ± 0.13 17.54 ± 0.24 1.79 ± 0.13 23.17 ± 1.02 4.86 ± 0.15 8.12 ± 0.41Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 4.48 ± 0.09 17.50 ± 0.19 1.84 ± 0.07 21.23 ± 1.20 4.59 ± 0.10 7.96 ± 0.14% Change -0.44 % -0.23 % 2.79 % -8.37 % -5.56 % -1.97 %Data are represented as mean ± S.E. Statistical analyses were carried out between control (6) and treated (6) animals according to student t-test. * Significant at p<0.05 and ** highly significant at p<0.01.

Table 4: Effect of daily oral administration of Rosmarinus officinalis extract (582.4 mg/kg, b.wt.) on sodium ion content in different brainareas of adult male albino rats at different time intervals.

Brain Areas Cerebellum µg/g Striatum µg/g Cerebral Cortex µg/g Hypothalamus µg/g Brainstem µg/g HippocampusTime Intervals µg/g1st week: Control 281.7 ± 12.1 642.6 ± 29.8 99.6 ± 4.8 686.1 ± 51.4 203.1 ± 11.7 316.6 ± 25.3---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 156.8 ± 5.9** 379.2 ± 15.5** 93.2 ± 3.5 539.9 ± 23.9* 148.7 ± 6.23** 220.6 ± 9.8**

% Change -44.3 % -40.9 % -6.4 % -21.3 % -26.8 % -30.3 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week: Control 281.7 ± 12.1 642.6 ± 29.8 99.6 ± 4.8 686.1 ± 51.4 203.1 ± 11.7 316.6 ± 25.3---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 120.8 ± 6.63** 370.6 ± 12.9** 61.2 ± 4.4** 390.1 ± 15.3** 105.4 ± 8.9** 188.3 ± 8.4**

% Change -57.1 % -42.3 % -38.5 % -43.1 % -48.1 % -40.5 %

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Table 4: Continue3rd week: Control 245.5 ± 11.6 645.0 ± 25.6 94.5 ± 5.9 650.3 ± 25.9 211.6 ± 15.4 340.5 ± 29.2---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 108.6 ± 6.01** 251.8 ± 20.2** 55.2 ± 3.7** 321.9 ± 22.7** 96.7 ± 9.9** 159.6 ± 9.3**

% Change -55.8 % -60.9 % -41.6 % -50.5 % -54.3 % -53.1 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4th week: Control 245.5 ± 11.6 645.0 ± 25.6 94.5 ± 5.9 650.3 ± 25.9 211.6 ± 15.4 340.5 ± 29.2---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 111.3 ± 6.5** 248.6 ± 14.9** 43.3 ± 2.9** 228.8 ± 14.6** 64.2 ± 4.3** 102.7 ± 5.2**

% Change -54.7 % -61.5 % -54.1 % -64.8 % -69.7 % -69.8 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1st week withdrawal:275.4 ± 11.5 671.8 ± 30.6 105.6 ± 9.6 635.5 ± 48.1 229.6 ± 19.5 389.3 ± 24.9Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 118.5 ± 5.7** 251.8 ± 5.9** 62.6 ± 3.4** 337.9 ± 14.3** 113.6 ± 7.5** 228.2 ± 15.6**

% Change -56.9 % -62.5 % -40.8 % -46.8 % -50.5 % -41.4 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week withdrawal:275.4 ± 11.5 671.8 ± 30.6 105.6 ± 6.3 635.5 ± 48.1 229.6 ± 19.5 389.3 ± 24.9Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 172.1 ± 7.9** 545.9 ± 30.7** 84.8 ± 1.9** 394.9 ± 13.8** 132.1 ± 6.5** 375.8 ± 17.1% Change -37.5 % -18.7 % -19.7 % -37.6% -42.5 % -3.5 %Data are represented as mean ± S.E. Statistical analyses were carried out between control (6) and treated (6) animals according to student t-test. * Significant at p<0.05 and ** highly significant at p<0.01.

Table 5: Effect of daily oral administration of Rosmarinus officinalis extract (582.4 mg/kg, b.wt.) on γ-aminobutyric acid content in differentbrain areas of adult male albino rats at different time intervals.

Brain Areas Cerebellum µg/g Striatum µg/g Cerebral Cortex µg/g Hypothalamus µg/g Brainstem µg/g HippocampusTime Intervals µg/g1st week: Control 109.93 ± 10.10 157.80 ± 3.50 33.06 ± 1.96 338.83 ± 11.77 47.80 ± 3.66 152.47 ± 11.18---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 94.70 ± 3.47 140.52 ± 8.71 31.29 ± 0.17 210.20 ± 2.37** 42.12 ± 1.39 128.24 ± 1.34% Change -13. 85 % -10.95 % -5.35 % -37.96 % -11.88 % -15.89 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week: Control 98.30 ± 9.32 164.57 ±6.70 46.83 ± 1.58 339.30 ± 13.67 50.72 ± 4.12 152.00 ± 8.81---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 78.15 ± 3.34 125.12 ± 7.46** 35.24 ± 0.64** 188.92 ± 2.38** 28.16 ± 1.37** 110.21 ± 1.48**

% Change -20.49 % -23.97 % -24.75 % -44.32 % -44.48 % -27.49 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3rd week: Control 102.70 ± 7. 50 159.94 ± 11.42 50.03 ± 3.03 337.30 ± 13.95 56.46 ± 2.05 154.73 ± 6.20---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 49.66 ± 5.60** 59.53 ± 3.30** 26.00 ± 2.19** 111.57 ± 3.76** 20.27 ± 1.52** 77.59 ± 4.92**

% Change -51.65 % -62.78 % -48.03 % -66.92 % -64.09 % -49.85---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4th week: Control 103.64 ± 8.39 155.63 ±7.40 60.75 ± 1.74 342.70 ± 11.80 54.99 ± 2.17 153.61 ± 7.02---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 45.92 ± 3.84** 40.66 ± 3.48** 23.42 ± 0.42** 78.27 ± 3.85** 12.85 ± 0.74** 57.34 ± 3.83**

% Change -55.69 % -73.87 % -61.45 % -77.16 % -76.63 % -62.67 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1st week withdrawal: 107.23 ± 9.22 173.23 ± 11.01 53.79 ± 3.49 340.23 ± 15.11 55.23 ± 1.79 150.73 ± 10.47 Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 91.82 ± 6.54 131.11 ± 11.45** 46.75 ± 0.42 233.91 ± 6.88** 31.70 ± 1.74** 123.43 ± 6.95% Change -14.37 % -24.31 % -13.09% -31.25 % -42.60 % -18.11 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week withdrawal: 107.23 ± 9.22 173.23 ± 11.01 53.79 ± 3.49 340.23 ± 15.11 55.23 ± 1.79 150.73 ± 10.47 Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 111.69 ± 5.36 169.57 ± 8.87 51.26 ± 1.36 256.87 ± 5.99** 48.66 ± 1.33* 144.37 ± 7.18% Change 4.16 % -2.11 % -4.70 % -24.50 % -11.89 % -4.22 %Data are represented as mean ± S.E.Statistical analyses were carried out between control (6) and treated (6) animals according to student t-test.* Significant at p<0.05 and ** highly significant at p<0.01.

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Table 6: Effect of daily oral administration of Rosmarinus officinalis extract (582.4 mg/kg, b.wt.) on chloride ion content in different brainareas of adult male albino rats at different time intervals.

Brain Areas Cerebellum µg/g Striatum µg/g Cerebral Cortex µg/g Hypothalamus µg/g Brainstem µg/g HippocampusTime Intervals µg/g1st week: Control 14.82 ± 1.09 22.00 ± 0.31 19.50 ± 0.92 26.53 ± 1.13 23.72 ± 1.29 39.64 ± 2.29---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 14.98 ± 0.28 39.15 ± 2.33** 25.67 ± 2.61* 47.08 ± 3.55** 37.85 ± 1.90** 44.67 ± 2.57% Change 1.08 % 77.95 % 32.10 % 77.46 % 59.57 % 12.69 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week: Control 14.30 ± 1.03 24.33 ± 1.10 19.77 ± 0.83 21.73 ± 2.13 23.91 ± 1.73 42.46 ± 2.91---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 17.48 ± 0.40** 39.92 ± 2.72** 29.24 ± 2.49** 57.10 ± 3.00** 44.99 ± 2.52** 55.01 ± 4.48*

% Change 22.24 % 64.08 % 47.90 % 162.77 % 88.16 % 29.56 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3rd week: Control 12.83 ± 0.85 22.14 ± 0.59 20.80 ± 0.65 22.12 ± 1.72 17.53 ± 0.37 50.83 ± 3.37---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 25.93 ± 0.74** 63.95 ± 4.53** 31.96 ± 1.52** 60.03 ± 3.88** 51.85 ± 1.60** 64.98 ± 2.84**

% Change 102.10 % 188.84 % 53.65 % 171.38 % 128.92 % 27.84 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4th week: Control 11.51 ± 0.73 23.14 ± 0.98 20.10 ± 1.37 21.97 ± 1.37 19.62 ± 1.17 36.14 ± 2.24---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 35.12 ± 1.24** 80.46 ± 5.67** 33.34 ± 1.24** 111.90 ± 6.78** 76.55 ± 3.93** 135.90 ± 3.72**

% Change 205.13 % 247.71 % 65.87 % 409.33% 290.16 % 276.04 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1st week withdrawal: 14.00 ± 0.49 31.30 ± 1.61 20.33 ± 0.58 24.60 ± 1.59 21.39 ± 1.14 46.37 ± 2.63 Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 33.98 ± 1.80** 75.56 ± 4.81** 27.32 ± 1.70** 96.85 ± 6.18** 28.21 ± 1.04** 72.40 ± 5.88**

% Change 26.65 % 141.41 % 34.38 % 293.69 % 31.88 % 56.14 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week withdrawal: 14.00 ± 0.49 31.30 ± 1.61 20.33 ± 0.58 24.60 ± 1.59 21.39 ± 1.14 46.37 ± 2.63 Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 33.26 ± 1.62** 55.92 ± 3.21** 20.69 ± 2.51 61.96 ± 3.40** 25.70 ± 2.37 54.50 ± 3.08*

% Change 23.97 % 78.66 % 1.77 % 151.87 % 20.15 % 17.53 %Data are represented as mean ± S.E. Statistical analyses were carried out between control (6) and treated (6) animals according to student t-test. * Significant at p<0.05 and ** highly significant at p<0.01.

Table 7: Effect of daily oral administration of Rosmarinus officinalis extract (582.4 mg/kg, b.wt.) on acetylcholine esterase activity in differentbrain areas of adult male albino rats at different time intervals.

Brain Areas Cerebellum µg/g Striatum µg/g Cerebral Cortex µg/g Hypothalamus µg/g Brainstem µg/g HippocampusTime Intervals µg/g1st week: Control 0.51 ± 0.030 2.43 ± 0.190 0.18 ± 0.010 3.36 ± 0.180 0.47 ± 0.020 1.26 ± 0.110---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 0.36 ± 0.026** 1.59 ± 0.090** 0.12 ± 0.005** 1.96 ± 0.060** 0.31 ± 0.015** 0.47 ± 0.026**

% Change -29.41 % -34.57 % -33.33 % -41.67 % -34.04 % -62.27 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week: Control 0.25 ± 0.020 1.79 ± 0.160 0.14 ± 0.010 1.49 ± 0.030 0.27 ± 0.006 0.62 ± 0.030---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 0.15 ± 0.016** 0.86 ± 0.031** 0.07 ± 0.004** 0.85 ± 0.044** 0.15 ± 0.012** 0.20 ± 0.014**

% Change -40.00 % -51.96 % -50.00 % -42.95 % -44.44 % -67.74 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------3rd week: Control 0.31 ± 0.020 1.63 ± 0.020 0.17 ± 0.009 2.11 ± 0.120 0.39 ± 0.030 0.84 ± 0.070---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 0.12 ± 0.011** 0.74 ± 0.027** 0.05 ± 0.004** 1.11 ± 0.066** 0.17 ± 0.006** 0.12 ± 0.006**

% Change -61.29 % -54.60 % -70.59 % -47.39 % -56.41 % -85.71 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4th week: Control 0.37 ± 0.010 1.95 ± 0.140 0.16 ± 0.010 2.32 ± 0.110 0.38 ± 0.020 0.92 ± 0.080---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 0.10 ± 0.006** 0.71 ± 0.081** 0.04 ± 0.003** 1.02 ± 0.072** 0.16 ± 0.010** 0.14 ± 0.001**

% Change -72.97 % -63.59 % -75.00 % -56.03 % -57.89 % -84.78 %

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Table 7: Continue1st week withdrawal:0.75 ± 0.070 2.45 ± 0.090 0.19 ± 0.01 3.20 ± 0.180 0.45 ± 0.020 1.21 ± 0.030Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 0.31 ± 0.019** 1.16 ± 0.053** 0.09 ± 0.003** 1.87 ± 0.120** 0.26 ± 0.020** 0.43 ± 0.006**

% Change -58.67 % -52.65 % -52.63 % -41.56 % -42.22 % -64.46 %---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2nd week withdrawal: 0.75 ± 0.07 2.45 ± 0.090 0.19 ± 0.01 3.20 ± 0.180 0.45 ± 0.020 1.21 ± 0.030 Control---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Treated 0.50 ± 0.032** 2.22 ± 0.094 0.12 ± 0.008** 2.11 ± 0.140** 0.36 ± 0.023** 0.58 ± 0.020**

% Change -33.33 % -9.39 % -36.84 % -34.06% -20.00 % -52.07 %Data are represented as mean ± S.E. Statistical analyses were carried out between control (6) and treated (6) animals according to student t-test. * Significant at p<0.05 and ** highly significant at p<0.01.

Peana et al. [24] reported that linalool could exertpart of its antinociceptive effect by inducing adenosinerelease or inhibiting adenosine uptake which there is aprobable involvement of adenosine receptors type A1

and A2A. At the presynaptic level, activation of A1

receptors induces a decrease in cAMP productionSawynok and Liu [25] and it is well known that, cAMPinfluenced the intra- and extra-cellular catecholaminelevels not only by stimulating its synthesis but also byregulating vesicular monoamine transport [26].

Santos and Rao [27] demonstrated that 1, 8 cineole(a constituent of Rosmarinus officinalis) caused arelease of histamine and serotonin in vitro. In addition,Kako et al. [28] found that 1,8 cineole stimulateddopamine release from rat striatal slices. Abdel-Rahmanand Fathi [29] investigated that Rosmarinus may be usedas a novel antidepressant substance. It was reportedthat the antidepressant drugs block neuronal uptake of5-HT and NE Baldessarini [30] and desensitize D2

dopamine autoreceptors [31]. According to Codignola etal. [32] and Sher et al. [33] diosmetin inhibited 3H-dopamine uptake ‘in a dose-dependent manner’.Furthermore, diosmetin inhibited 3H-serotonin uptake.

Most antidepressants exert important actions on themetabolism of monoamine neurotransmitters and theirreceptors [34]. Apigenin (a constituent of Rosmarinus) iswidely distributed in many fruits and vegetables [35]; itis known to pass the blood–brain barrier and to exertCNS-effects [36]. In addition, Nakazawa et al. [37]

suggested that it may have an antidepressant-likeactivity which induced alterations in turnover of thedopaminergic system, while the turnover of NE and 5-HT did not show any significant changes. It inducedincrease of the dihydroxyphenylacetic acid level in theamygdala, thalamus and hypothalamus; the effect ofapigenin on behavioural changes may be related todopaminergic neurotransmission. So, the decrement inmonoamine contents in this study may be due to thedecrease in reuptake or induce their turnover.

Since Rosmarinus officinalis was demonstrated asan anticonvulsant Brinckmann, [38]; several studiesclarified and showed anticonvulsants block persistent

sodium currents in CNS neurons at therapeuticconcentrations and may selectively target the persistentcurrent fraction [39, 40].

It was known that, voltage-gated sodium channelsare essential for the propagation of action potentialsalong axons and also contribute to controllingmembrane excitability [41]. Thus, voltage-gated sodiumchannel blockers are used to treat chronic pain [42-44].

Hence, González-Trujano et al. [4] reported thatRosmarinus officinalis produces a significantantinociceptive effect in different kinds of induced painin rodents. So, the Na+ ion content was decreased indifferent brain areas in the present study.

The present result revealed that, the daily oraladminstration of Rosmarinus officinalis aqueous extractshowed a significant decrease in GABA content whileit caused a significant elevation in Cl- ion content in allstudied brain areas under investigation throughout theexperimental period.

GABA is the major inhibitory neurotransmitter inthe brain, is essential for the over all balance betweenneuronal excitation and inhibition that is vital to normalbrain function [45]. GABA produces neuronal inhibitionby acting on an amazing diversity of membrane-boundreceptors. These receptors can be divided into twomajor types: ionotropic receptors that are ligand-gatedion channels (GABAA and GABAC receptors) andmetabotropic receptors that are G-protein coupledreceptors (GABAB receptors) that act via secondmessengers [46,47]. GABAA and GABAC receptors areGABA-gated chloride ion channels causing inhibitionof neuronal firing. Agents acting on GABAA receptorshave widespread therapeutic use as anaesthetics,anticonvulsants, anxiolytics and sedative-hypnotics [45].

There is now an impressive array of naturalproducts of plant origin that are known to influence thefunction of ionotropic receptors for GABA. The majorchemical classes of such natural products areflavonoids, terpenoids and phenols. Flavonoids andterpenoids can influence GABAA receptors in a mannerdependent of classical benzodiazepine sites [48].

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Flavonoids have a range of activities on GABAA

receptors, acting as positive and negative modulators ofGABAA receptor function Marder and Paladini [49] andhave been described as a “new family ofbenzodiazepine receptor ligands”. Benzodiazepines arethe most widely prescribed class of psychoactive drugsin current therapeutic use which exert theirpharmacological effects by potentiating GABA-inducedCl-currents. The most prominent effects are sedation,hypnosis, decreased anxiety, muscle relaxation,anterograde amnesia, and anticonvulsant activity [50]

Apigenin has been used for anxiolytic and sedativeeffects [51,52,34]. On the other hand, Wei et al. [53]; Violaet al. [54] and Wysong [55] indicated that it hasanxiolytic activity without incidence of sedation ormuscle relaxation effects and it binds to GABAreceptors which have affinity for benzodiazepines andbarbiturates. Thus, Viola et al. [54]; Salgueiro et al. [56]

and Avallone et al., [51] reported that it has a centrallyacting benzodiazepine ligand which shows CNS effectsafter systemic administration. On the same line,Campbell et al. [57] recorded that apigenin has beenshown to potentiate the effect of the benzodiazepinediazepam. Moreover, Jäger et al. [36] concluded that ithas been used clinically to control convulsions. While,Viola et al. [54] showed that apigenin-treated ratsshowed a significant reduction in locomotor activity incomparison with vehicle-treated rats this effect was notascribed to an interaction with GABAA-benzodiazepinereceptor but to other neurotransmission system.

Kang et al. [58] reported that quercitrin has asedative action in mice. Marder et al. [59] showed thathesperidin with sedative-hypnotic effects. In addition,Fernández et al. [60] recorded that glycosides are easilymetabolized by the organism and it could be possiblethat secondary metabolites may activate GABAA

receptors to mediate sedative effects. Moreover,diosmin possess sedative, sleep-enhancing effects,reduced the spontaneous locomotor activity in micefollowing i.p. injection which caused a generalinhibition of neuronal activity in the CNS and theseactions do not involve binding to the central GABAA

receptor, at least not directly.Luteolin is a widespread flavonoid which is

considered to have sedative effect [61]. Raines et al. [62]

suggested that luteolin degrades rat motor movementpotentially by modulation of the CNS or neuromuscularsystem. Furthermore, it is not an anxiolytic which notmodulate the benzodiazepine receptor site Viola et al.,[63]; Raines et al. [62]. On the other hand, despite its lowaffinity for the benzodiazepine-receptor, seems to haveanxiolytic-like effects or, at least, to interact withdifferent neurotransmitter systems, such as 5-HT toinduce CNS effects [64-66]. Several reports showed thatborneol modulate GABAA channels [67-69]. Rosmarinus

officinalis extract enhanced the receptor sensitivity forendogenous GABA preventing epileptic incidences [70].

Rutherford et al. [71] indicated that carnosic acidand carnosol bind to the chloride channel of theGABA/benzodiazepine receptor complex in brain tissue.It was known that the enhancement of GABAA receptorthat is gated by Cl- ion content, leading to increase therelease of GABA and influx of Cl- ion causing adecrease in GABA content and increase in Cl- ioncontent in brain, as a result GABA content wasdecreased and Cl- ion content was increased in brainareas in this study. Thus, Rosmarinus constituents wereconsidered as active inhibitory agents in the centralnervous system.

The present results demonstrated that, the dailyoral gavage of Rosmarinus officinalis aqueous extractinduced an inhibition of acetylcholine esterase activityin all examined brain areas throughout the experimentalperiod. These results are in agreement with the studyof Perry et al. [72] which investigated that Rosmarinusofficinalis has an anti-acetylcholine esterase activity.Inhibition of acetylcholine esterase (the key enzyme inthe breakdown of acetylcholine) is considered one ofthe treatment strategies against several neurologicaldisorders such as Alzheimer disease; the purpose oftreatment is to inhibit acetylcholine esterase resulting ina higher synaptic acetylcholine that will enhance thecholinergic transmission [73, 74].

It was known that, Rosmarinus officinalis has avery old reputation for improving memory and hasbeen used as a symbol of remembrance in Europe andacts as mood stabilizer [75]. Moreover, a potentialsource of acetylcholine esterase inhibitors is certainlyprovided by the abundance of plants in nature. Plantsbelonging to family Lamiaceae have been reported tohave acetylcholine esterase inhibitory potential. Majorclasses of compound reported to have such activity arethe terpenoids, glycosides and coumarins [76]. Accordingto Chung et al., [2] ursolic acid is a natural and potentacetylcholinesterase inhibitor.

Several reports attributed the inhibitory effect ofRosmarinus oil on acetylcholine esterase to somecomponents such as 1, 8 cineole, camphor, α-pineneand 4-terpineol [72,77,78,74]. In addition, Nasel et al., [79]

showed that 1, 8 cineole inhalation increases cerebralblood flow and enhances cortical activity. Moreover,ferulic acid is of interest since it ameliorated thereduction in acetylcholine levels in the cortex and theinflammatory responses in the hippocampus induced byβ-amyloid in mice and it also significantly improvedcognitive function [80].

Flavonoids possess phytoestrogen mimickingactions and have been shown to have positive effectson the cognitive function in females [81]. Luteolin maybe able to traverse the blood brain barrier and bind

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covalently to estrogen receptor in the brain [82]. Tsai etal., [83] deduced that luteolin related to the activation ofthe central cholinergic nervous system and may berelated to memory processes. Rui et al., [84] reportedthat luteolin increased brain-derived neurotrophic factor(BDNF) level. BDNF is well known for influencingcholinergic cell survival and maintenance, axonalgrowth, regeneration, synaptic plasticity, andstimulation of choline acetyltransterase activity,acetylcholine synthesis and release [85,86]. Thus, Rui etal., [84] reported that oral administration of luteolinhelped to modulate synaptic transmission by BDNF inβ-amyloid25-35- induced amnesic mice and was related tothe protection of modification of neurotransmission inthe central cholinergic neuronal system.

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