INTRODUCTION:

Rosemary (Rosemary officinalis) is a woody, perennial herb with fragrant evergreen needle like leaves. It is native to the Mediterranean region and common house hold plant grown in many parts of the world. It belongs to the family Laminaceae. It is used as a flavouring agent, as a beverage ¹, as well as in cosmetics. In folk medicine it used as an antispasmodic for renal, colic and dysmenorrheal and in relieving respiratory disorders and to stimulate growth of hair. Traditionally, Rosemary has been used by herbalists to improve memory, relieve muscle pain and spasm. Rosemary has reported much health benefits, in particularly as an antioxidant ², antimicrobial ^3,4 and anticarcinogenic activity ^5,6. Rosemary components have protective effect on cancers by decreasing the enzymes that can convert pro carcinogens to a more potent carcinogen and increasing enzymes that can inactivate carcinogens ⁷. Extract of rosemary relaxes smooth muscles of trachea and intestine and has choleretic, hepatoprotective activity.

R.officinalis helps to stimulate blood circulation in the skin and to relieve skin congestion. R. officinalis also stimulate the CNS, respiratory and locomotor’s activity.

In the present study, quantification of phytochemicals in Rosemary officinalis (AERO and MERO) and their oxidant scavenging capacity has been described.

MATERIALS AND METHODS:

Commercial rosemary was bought from E.L.Industries Pvt. Ltd., (India).All other reagents used were of analytical grade.

Sample Preparation:

Crude extract was obtained after maceration with distilled water and methanol at room temperature for 72 hrs, and repeated till exhaustion of the material. Thereafter, the combined crude extracts was filtered, distilled, evaporated and dried under reduced pressure to yield both the aqueous and methanol extract of Rosemary.

Preliminary phytochemical analysis:

The preliminary phytochemical analysis of Rosemary officinalis was carried out in both AERO and MERO as described by Sofowara (1993)⁸, Trease and Evans (1989)⁹ and Harborne (1973)¹⁰.

Estimation of Total Phenol:

Total phenol was estimated by the method of Folin- ciocalteau method ¹¹.To 0.4ml of rosemary extract was taken in a volumetric flask (25 ml) containing 9.0 ml of distilled water. 1.0 ml of folin – ciocalteau reagent was added to the volumetric flask and was shaken well. The reaction mixtures were then incubated at room temperature for 5 minutes. 10.0 ml of 7% sodium carbonate was added to the volumetric flask and mixed. The volumes in all the flask were made up to 25.0 ml with distilled water and mixed thoroughly. The flasks were then incubated for 90 minutes at 23º C and the blue color developed was read at 750 nm against the reagent blank using a spectrophotometer. A set of standards were treated similarly as mentioned above.

The amount of total phenol present in rosemary extracts were expressed in Gallic acid equivalents (GAE) per gram of rosemary.

Estimation of Flavonoids:

Total Flavonoids was by the method of Chang C et al., (2002)¹² . To, 1.0ml of rosemary extract was taken in a test tube containing 4.0 ml of distilled water. At time zero, 0.3 ml of 5% sodium nitrite was added to the tube. At 5^th minute, 0.3 ml of 10% aluminium chloride was added and at 6^th minute, 2.0 ml of 1M sodium hydroxide was added. The volume in the tube were immediately diluted with 2.4 ml of distilled water and mixed well. The absorbance of pink color developed was read at 510 nm against the reagent blank.

The total flavonoid content of rosemary extract was expressed in milligrams per gram of epicatechin equivalents (ECE).

Determination of Oxidant Scavenging Capacity:

The following test for oxidant scavenging capacity has been done in 3 groups as follows:

Group A: 0.1 ml of platelet

Group B: 0.1 ml of platelet + 0.5 ml of ADP (ADP activates the platelets)

Group C: 0.1 ml of platelet + 1.0 ml of rosemary extract + 0.5 ml of ADP

Platelet was isolated by the method of Aster and Jandl (1964)¹³. Lipid peroxide content was determined by the method of Yagi.et al., (1978)¹⁴. Lipid hydroperoxide content was determined by the method of Jiang et al., (1992)¹⁵. Conjugated dienes were assayed by the method of Rao and Recknagel(1968)¹⁶ .

Statistical analysis:

The values are expressed as mean ± SD. Statistical analysis was done by students’t’ test. ‘p’ value was arrived at to assess the statistical significance of changes observed.

RESULTS AND DISCUSSION:

Table 1 shows the presence of various phytochemicals in both methanol and aqueous extracts of Rosemary officinalis

TABLE 1: QUALITATIVE ANALYSIS OF BOTH AQUEOUS AND METHANOL EXTRACT OF ROSEMARY

PHYTOCHEMICALS	AQUEOUS EXTRACT OF ROSEMARY	METHANOL EXTRACT OF ROSEMARY
Tannin	+	+
Saponin	+	+
Cardiac glycosides	+	+
Terpenoids	+	+
Flavonoids	+	+
Carotenoids	+	+

Table 2 depicts the level of total phenols present in both methanol and aqueous extracts of Rosemary officinalis . Phenolic compounds are also effective hydrogen donors which makes them good antioxidants¹⁷. Current research has shown that polyphenols contribute to the prevention of cardiovascular disease, cancer, and osteoporosis and have a role in the prevention of neurodegenerative disease and diabetes mellitus ^{18, 19, 20,21}.

TABLE 2: LEVEL OF TOTAL PHENOL IN BOTH AQUEOUS AND METHANOL EXTRACT OF ROSEMARY

PARTICULARS	AQUEOUS EXTRACT OF ROSEMARY	METHANOL EXTRACT OF ROSEMARY
GAE IN GRAMS/100g OF ROSEMARY (MEAN)	2.41	3.64
STANDARD DEVIATION	0.17	0.31
STUDENT’S t TEST	p < 0.001	p < 0.001

From the table, it was observed that, MERO has 3.64g of GAE/100g of rosemary leaves and the AERO has 2.41g of GAE/100g of rosemary leaves.

Ivor .E.Dreosti (2000)²² has reported that the complex structure of flavonoids involves them in physiological process which may have a favourable effect on the risk of cancer and cardiovascular disease and immune function, vasodilation, inflammation, bacterial and viral infections, prostanoid metabolism, enzyme activation, receptor binding, cell-cell communication, cell proliferation and in a hormonal role as phyto estrogens.

The beneficial effects of fruits, vegetables and tea or even red wine have been attributed to flavonoid compounds rather than to known nutrients and vitamins. In the present study the MERO and AERO has shown the presence of 3.68and 3.39g of CE of flavonoids/100g of rosemary leaves respectively and it was depicted in table 3.

TABLE 3: LEVEL OF FLAVONOIDS IN BOTH AERO AND MERO OF ROSEMARY

PARTICULARS	AQUEOUS EXTRACT OF ROSEMARY	METHANOL EXTRACT OF ROSEMARY
CE IN GRAMS/ 100g OF ROSEMARY (MEAN)	3.36	3.66
STANDARD DEVIATION	2.0	2.72
STUDENT’S t TEST	p < 0.001	p < 0.001

Table 4 shows the oxidant scavenging capacity of rosemary extracts (MERO and AERO).The oxidant scavenging antioxidant potential was studied by considering three groups.

TABLE 4: OXIDANT SCAVENGING OF ROSEMARY OFFICINALIS (IN PERCENTAGE)

OXIDANTS	% OF SCAVENGING
CONJUGATED DIENES	63%
LIPID PEROXIDE	66%
LIPID HYDROPEROXIDE	62%

Normal platelets were taken as control (group1) and it was activated by ADP (1μg/ml) (group2) to generate oxidative stress. Rosemary extracts (MERO and AERO) were added to the activated platelets (group3) and the effect of rosemary extracts on suppressing the oxidants generation were studied.

Both the MERO and AERO has shown significant oxidant scavenging capacity. In this study, a good correlation between the antioxidant activities and total phenol content in the extracts were found. Since the phenol content of MERO was higher than AERO (table2), the oxidant scavenging potential of MERO was also higher than that of AERO and it was depicted in the table 4 and in fig.1, 2 and 3.

Fig 1.LEVELS OF LIPID PEROXIDES IN THREE GROUPS

A: platelet; B: platelet + ADP; AERO: aqueous extract of Rosemary officinalis; MERO: methanolic extract of Rosemary officinalis

Fig 2. LEVELS OF LIPID HYDRO PEROXIDES IN THREE GROUPS

A: platelet; B: platelet + ADP; AERO: aqueous extract of Rosemary officinalis; MERO: methanolic extract of Rosemary officinalis

Fig 3. LEVELS OF CONJUCATED DIENES IN THREE GROUPS

A: platelet; B: platelet + ADP; AERO: aqueous extract of Rosemary officinalis; MERO: methanolic extract of Rosemary officinalis

Chipault et al., (1952)²³ and Inatanic et al., (1983)²⁴ has reported that the leaves of Rosemary officinalis is a naturally occurring antioxidant and their extracts can be commercially used for their antioxidant activity. Chang et al., (1997) ²⁵ was reported that the extracts have been used to prevent the lipid oxidation and to inhibit the oxidation of both animal fats and vegetable oils. From the results, it can be concluded that the methanol extract of Rosemary officinalis (MERO) has potent antioxidant property than AERO.

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Received on 27.07.2011 Modified on 09.08.2011

Research J. Pharm. and Tech. 4(10): Oct. 2011; Page 1588-1590