INTRODUCTION:

All natural resources have some potential medicinal value. Plants can provide biologically active molecules and lead structures for the development of modified derivatives with enhanced activity and reduced toxicity. Cinnamomum malabatrum is a small tree which grows upto 15 metres height^1,2. This belongs to the family lauraceae which includes large number of plants that are well known for their antioxidant properties. The purpose of this study was to investigate the antioxidant potential of the plant after estimating the phenolics present in it. The free radicals are formed usually as byproduct of biochemical reactions taking place in the body. They have strong tendency to impair the proper functioning of the immune system which leads to infection and also cause degenerative disease including heart disease and cancer. Free radicals are also responsible for aging.

The reason that antioxidants are important to human physical well being comes from the fact that oxygen is a potentially toxic element since it can be transformed by metabolic activity into more reactive forms such as superoxide, hydrogen peroxide, singlet oxygen and hydroxyl radicals, collectively known as active oxygen or Reactive oxygen species (ROS). This ROS can cause oxidative stress and produce many degenerative disorderd like arthritis, diabetes, inflammation, genotoxicity and even cancer^3,4. Antioxidant molecules act by getting oxidized thereby they are involved in the scavenging of free radicals. Phenolic compounds are commonly found in both edible and non-edible plants, and they have been reported to have multiple biological effects, including antioxidant activity.

MATERIALS AND METHODS:

Collection and authentification of the sample

Cinnamomum malabatrum leaves used in the present study were collected from Anchal, Kerala and authentified by the botanist, Department of Environmental science, M.G University, Athirampuzha, Kottayam, Kerala, India. Fresh leaves were collected from the tree and dried at room temperature to remove moisture, and size reduced.

Apparatus and Chemicals

This study involved the use of a UV-VIS spectrophotometer (Shimadzu UV-1601 UV-VISIBLE Spectrophotometer). All sovents used were AR grade. Gallic acid, Hudrogen peroxide (Nice Chemical Private Ltd, New Delhi), Quercetin and Gallic acid ( Chemika-Biochemika reagents, Mumbai), Beta-carotene, linoleic acid, Tocopherol, Butylated hydroxyl Toluene (BHT), Sodium nitroprusside, Greiss reagent (Merck Specialty Private Ltd. Mumbai).

Extraction of Cinnamomum malabatrum leaves

Extraction of the fresh leaves of Cinnamomum malabatrum was carried out by successive solvent extraction using solvents of increasing polarity, n-hexane, alcohol and water. Extraction of leaves of Cinnamomum malabatrum with n-hexane was carried out by simple maceration technique. Extraction of marc (after n-hexane extraction) using alcohol was carried out by hot continuous extraction method using soxhlet extractor. The extract obtained was collected and concentrated by gentle heating. Aqueous extraction was carried out with the remaining marc by reflux method using a reflux condenser⁵.

Estimation of Total Phenolic Content

Folin Ciocalteau method is used for estimating the total phenolic contents of above extracts. Standard graph was generated using different concentration of gallic acid. From each extract 1ml was pipetted out and 5ml of Folin Cio-calteau reagent was added. After 5 minutes, 4ml of sodium carbonate solution was added to all and incubated at room temperature for 2 hours. Then, absorbances were measured at 750nm and the values obtained were interpreted in the standard graph of gallic acid to get the milligram equivalents of gallic acid^4.6.7.

Estimation of Flavonoid Content

Aluminium chloride colorimetrc method using quercetin as standard was used for the study. Standard graph was generated using different concentration of quercetin. Sample solution was prepared at a concentration of 1mg/ml in methanol. 1ml was pipetted out, from each dissolved samples and 4ml of water followed by 0.3ml of sodium nitrate was added. After 5minutes, 0.3ml of 10 % aluminum chloride solution and after five minutes 2ml of 1M sodium hydroxide was added. Mixed well and absorbance was measured at 510nm and the values were interpreted in the standard graph of Quercetin to get the milligram equivalents of Quercetin.^6,7

Antioxidant Activity Studies

Nitric oxide scavenging assay, Hydrogen peroxide scavenging assay and Beta carotene linoleic acid emulsion assay method was used to find out the antioxidant activity of the plant extracts.

Nitric Oxide Radical Inhibition Assay

The standard used in nitric oxide radical inhibition assay was Quercetin. Sample solution was prepared from n-hexane, ethanolic and aqueous extract of Cinnamomum malabatrum leaves in phosphate buffer. The concentration of sample solutions were 50,100,150,200 μg/ml solution. In the assay, the reaction mixture (3ml) containing sodium nitropruside (2ml), phosphate buffer saline (0.5ml) and Standard solution (0.5ml) or Sample was incubated at 25° C for 2.5 hours. After incubation, 0.5ml of the reaction mixture containing nitrite ions was pippeted out and mixed with 1ml of Greiss reagent (1ml 1% sulphanilimide and 3% phosphoric acid) and allows to stand for 5 minutes to complete diazotization. Then add 1 ml of 1% naphthylethylene diamine and allowed to stand for 0.5 hours. A pink coloured chromophore was formed in diffused light. A control was also prepared by the above solutions with out sample or standard. The absorbance of the solution was measured at 540 nm against the corresponding blank solution The percentage inhibition of nitrogen peroxide free radical by the sample and standard compound were determined by the given formula^8,9,10

Percentage inhibition = A_c- A_sx 100

A_c

Where, Ac is the absorbance of the control and As is the absorbance of the standard/sample solution. Then IC₅₀ value was calculated from graph plotted using percentage inhibition verses concentration.

Hydrogen Peroxide Radical Scavenging Activity

The standard solution (1mg/ml) α- tocopherol prepared in phosphate buffer. Sample solution was prepared from n-hexane, ethanolic and aqueous extract of Cinnamomum malabatrum leaves in phosphate buffer. Each test tube containing 1 ml of the solution was added with 0.6 ml hydrogen peroxide solution, and it made upto 5ml with phosphate buffer. After 10 minutes the absorbance of the solution was measured at 230nm using UV spectrophotometer against a blank solution containing phosphate buffer without using 2mM/L hydrogen peroxide. All samples were assayed in triplicates. The percentage scavenging of hydrogen peroxide for sample and standard compound were determined by the given formula^11,12

Percentage inhibition = A_c- A_sx 100

A_c

Where, Ac is the absorbance of the control and As is the absorbance of the standard/sample solution. The IC₅₀ calculated for each samples and the standard was compared and interpreted in the graph.

Beta Carotene Linoleic Acid Emulsion Assay

Standard used in Beta carotene method was Butylated hydroxyl toluene (BHT). The sample used is n-hexane, ethanolic and aqueous extract of Cinnamomum malabatrum leaves at a concentration of 10 mg/ml. 0.2 ml of each sample solution and standard solution were placed in different test tube. To each test tube add 5 ml of Beta-carotene emulsion. Then add 0.2 ml of ethanol. The reaction mixture is shaken gently and placed at 45 ºC in a water bath for 60 minutes. A control containing 0.2 ml of ethanol was used for the assay. The absorbance of the standard, sample and control was measured at 470 nm using UV/VISIBLE spectrophotometer against a blank, consisting of an emulsion without Beta carotene. The measurement was carried out at initial time (t=0) and successively at 0, 30 and 60 minutes. All samples were assayed in triplicates and averaged. The antioxidant activity was measured in terms of successful bleaching of Beta carotene, and they can be calculated from the formula^3.12

AA =1- A₀-A_tx 100

A₀⁰-A_t^o

Where, AA = Percentage antioxidant activity

A_0,A₀⁰ = Absorbance values measured at the initial incubation time for standard/sample and control respectively.

A_t, A_t⁰ = Absorbance values measured for the standard/sample and control respectively at 60mts.

RESULTS AND DISCUSSIONS:

The fresh leaves of Cinnamomum malabatrum extracted and the calculated percentage yield of the N- hexane extract was 0.42%, Alcoholic extract was 8.25% and Aqueous extract was 3.4%. Preliminary phytochemical screening of three extracts were carried out and showed the presence of phenolics and flavonoids in large amount. Then the total phenolic content in the extract was determined and expressed in gallic acid equivalents. The standard graph plotted is shown in figure I and the calculated phenolic content is shown in table I.

Table I. Total Phenolic Content of different extracts

Extract

Amount of extract

(in ml)

Mean absorbance

(nm)

Concentration from the graph (mcg/ml)

n-Hexane

Alcoholic

Aqueous

0.0152

0.056

0.0292

15.2

56.00

29.2

The flavonoid content in the extracts was determined by aluminium chloride colorimetric method. A standard graph of quercetin was plotted as shown in fig II and the flavonoid content was determined and expressed in Quercetin equivalents as shown in Table II.

Table II. Flavonoid content of different extracts

Extract

Amount of extract

(in ml)

Mean absorbance

(nm)

Concentration from the graph (mcg/ml)

n-Hexane

Alcoholic

Aqueous

0.183

0.457

0.298

10.26

44.21

24.48

It is well-known that phenolic serve in plant defense mechanisms to counteract reactive oxygen species (ROS) in order to survive and prevent molecular damage and damage by microorganisms, insects, and herbivores. In Nitric oxide radical inhibition assay, the absorbance of the reaction mixture for different concentrations of standard and samples and control were recorded in triplicate. . The percentage inhibition obtained in the different concentration of sample extracts were compared to the percentage inhibition obtained with the standard and expressed in graph as shown in Fig III.

IC₅₀value was calculated for each extract of Cinnamomum malabatrum and standard from graph as shown in table III. IC₅₀value of hexane extract was not obtained at the above mentioned concentrations.

Table III. IC₅₀ value of various extracts

Sl. No.

Sample

IC₅₀ value(mcg)

Alcoholic extract

Aqueous extract

Standard (quercetin)

86.04

89.26

43.03

From the Table III, IC₅₀ was less for alcoholic extract. So the alcoholic extract has comparatively good nitric oxide scavenging ability when compared with that of other extracts

In Hydrogen peroxide scavenging assay method, the absorbance of standard, sample and control is measured and recorded. The percentage inhibition obtained in the presence of different concentration of extract and standard were calculated and expressed graphically as shown in fig IV.

IC₅₀value was calculated for each extract of Cinnamomum malabatrum and standard from graph. The values are tabulated in table IV. IC₅₀value of hexane extract was not obtained at the above mentioned concentrations.

Table IV. IC₅₀ value of various extracts

Sl no:

Sample

IC₅₀ value(mcg)

Alcoholic extract

Aqueous extract

Standard(tocopherol)

67.69

127.18

34.46

From the table, IC₅₀ was less for alcoholic extract. So the alcoholic extract has comparatively good nitric oxide scavenging ability when compared with that of other extracts.

In Beta carotene linoleic acid emulsion assay method, the absorbance of the Standard, Sample and Control is measured at 540nm using a UV-Visible spectrophotometer and the percentage activity was calculated and the values are tabulated in the Table V.

CONCLUSION:

In conclusion the phenolic content and antioxidant property of Cinnamomum malabatrum have been estimated. Studies on total phenolics and flavonoid contents on various extracts of leaves indicates maximum concentration in alcoholic extract. All the extracts are having antioxidant activity against Nitric oxide, hydrogen peroxide and lipid peroxide free radicals. The highest antioxidant activity was shown by alcoholic extract.

Table V. Antioxidant activity of various extracts at 30^th and 60^th minutes using Beta carotene linoleic acid emulsion assay method

Extract	Concentration (mg/ml)	Abs (at 0 min)	Abs (at 30 min)	Abs (at 60 min)	Antioxidant activity (at 30 min)	Antioxidant activity (at 60 min)
Control	---	1.110±0.004	1.065±0.004	1.024±0.022
Alcoholic extract	1	1.553±0.006	1.541±0.002	1.530±0.006	73.04	73.26
Aqueous extract	1	1.243±0.012	1.226±0.021	1.198±0.009	42.23	46.77
n-hexane extract	1	1.148±0.018	1.116±0.032	1.090±0.011	28.89	32.56
Standard (BHT)	1	1.242±0.008	1.234±0.012	1.227±0.012	82.30	83.19

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Received on 13.04.2012 Modified on 20.05.2012

Asian J. Research Chem. 5(5): May 2012; Page 628-632