In Vitro Anti-Oxidant Activity of the Various Extracts of Cassia auriculata L. Flower by UV Spectrophotometer

 

M. Elayarani1, P. Shanmuganathan1 and P. Muthukumaran2*

Department of Biochemistry and Microbiology, Meenakshi Chandrasekaran College of Arts and Science, Pattukkottai, Thanjavur, Tamil Nadu

*Corresponding Author E-mail: kumaran.bio82@yahoo.com; muthubabi_p@yahoo.co.in

 

ABSTRACT:

The present study was carried out in In-vitro antioxidant effects of the Petroleum Ether, Ethanol, and Methanolic flower extracts of Cassia auriculata L . The powder of flower of Cassia auriculata L was extracted sequentially with petroleum ether, ethanol and methanol using Soxhlet extractor and it was subjected to preliminary phytochemical studies for the identification of phytoconstituents by chemical tests. Antioxidant activity of the extract was evaluated by using Diphenyl picryl hydrazyl (DPPH) radical scavenging, and reducing power methods. The extracts of Cassia auriculata L had shown good DPPH (1, 1-diphenyl-2-picryl-hydrazyl) radical scavenging activity. BHA was used as standard antioxidant and positive control. The DPPH radical scavenging activity of the extracts was increased with the increasing concentration, the reducing power of extracts was carried out with ascorbic acid as a standard reducing agent. The Methanolic extract of Cassia auriculata L exhibited higher scavenging and reducing power than the other extracts. All the analysis was made with the use of UV Visible Spectrophotometer (Systronics 117, India). The observation suggest that the flower of Cassia auriculata L   extracts has potent dose dependent anti oxidant activity, thus the Ethanobotanical claim of the plant being used in the cardio vascular disorder, muscle build up, nephro protection, anti inflammatory and as an aphrodisiac may be in part due to the antioxidant activity and also the presence of saponins and traces of Flavanoids ascertains that the Anti-oxidant activity

 

KEYWORDS: Cassia auriculata L , antioxidant, reducing Power, DPPH, BHA, Ascorbic Acid

 

INTRODUCTION:

Antioxidants are radical scavengers which protect the human body against free radicals that may cause pathological conditions such as ischemia, anaemia, asthma, arthritis, inflammation, neurodegeneration, Parkinson's diseases, mongolism, ageing process and perhaps dementias1 Flavonoids and flavones are widely distributed secondary metabolites with antioxidant and antiradical properties2. The medicinal actions of plants are unique to particular plant species or groups are consistent with this concept as the combination of secondary products in a particular plant is taxonomically distinct. Antioxidant-based dugs/formulations for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, Alzheimer's disease, and cancer have appeared during the last 3 decades3.

 

This has attracted a great deal of research interest in natural antioxidants. Subsequently, a worldwide trend towards the use of natural phytochemicals present in berry crops, tea, herbs, oilseeds, beans, fruits, and vegetables has increased. Several herbs and spices have been reported to exhibit antioxidant activity, including rosemary, sage, thyme, nutmeg, turmeric, white pepper, chili pepper, ginger, and several Chinese medicinal plants extracts4. The majority of the active antioxidant constituents are flavonoids, isoflavones, flavones, anthrocyanins, coumarins, lignans, catechins, and isocatechins. In addition to the above compounds found in natural foods, vitamins C and E, beta-carotene, and tocopherol are known to possess antioxidant potential5. With this background and abundant source of unique active components harbored in plants. The present study was taken up on one medicinal plant namely of Cassia auriculata L belongs to the Caesalpiniaceae family.

 

 

MATERIALS AND METHODS:

Plant Material:

Plant material the leaves of Cassia auriculata L were collected from in and around Thanjavur, Tamilnadu India.. Dried flower samples were ground into a uniform powder using a blender and stored in polythene bags at room temperature.

 

Preparation of Extracts:

The flowers of Cassia auriculata L were air-dried, pulverized. Sieve the powder was extracted sequentially with petroleum ether, ethanol and methanol using soxhlet extractor and concentrated under reduced pressure using flash evaporator and stored in screw cap vials.

 

Phytochemical Screening:6- 8

Phytochemical screenings were performed Using standard procedures. Phytochemical studies reveal the presence of Sterols, Saponins, and Flavanoids9.

 

Antioxidant Assay:

The antioxidant activity of Plant extracts was determined by different in-vitro methods such as, the DPPH free radical scavenging assay and reducing power methods. The different extracts were dissolved in methanol at the concentration of 2mg/ml. all the assays were carried out in triplicate and average value was considered.

 

a. DPPH Radical scavenging activity:

DPPH scavenging activity of the plant extract was carried out according to the method of Koleva .I et al.,10,11. 0.2 ml of methanolic solution of plant extract samples at different concentration (50- 250μg/ml) was mixed with 0.8 ml of Tris HCL buffer (100Mm, pH 7.4). One ml DPPH (500 M in methanol) solution was added to above mixture. The mixture was shaken vigorously and incubated for 30min in room temperature. Absorbance of the resulting solution was measured at 517nm UV-Visible Spectrophotometer (Systronics UV-Visible Spectophotometer 117, INDIA). All the assays were carried out in triplicates with BHA (Butylated Hydroxy Anisole) as a positive control. Blank was prepared with the addition of DPPH and for control 0.2 ml of methanol (without plant extract) was added. Percentage of DPPH scavenging activity determined as follows

 

                                (Absorbance of control – Absorbance

% DPPH radical =                 of test Sample) x 100

-scavenging            ----------------------------------------------

                                         Absorbance of control

 

Decreased absorbance of the reaction mixture indicates stronger DPPH radical-scavenging activity. In this study, Petroleum Ether, Ethanol and Methanolic flower extracts of Cassia auriculata L were used.

 

b. Reducing Power:

This was carried out as described previously [12]. 1 ml of plant extract solution (final concentration 100- 500 mg/l) was mixed with 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide [K3Fe (CN6)] (10g/l), and then mixture was incubated at 50 degree C for 20 minutes. Two and one-half, 2.5 ml of trichloroacetic acid (100g/l) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Finally, 2.5 ml of the supernatant solution was mixed with 2.5 ml of distilled water and 0.5 ml Fecl3 (1g/l) and absorbance measured at 700nm in UV-Visible Spectrophotometer (Systronics UV-Visible Spectophotometer 117, INDIA). As a control, ascorbic acid was used (final concentration 10 mg/ml). Increased absorbance of the reaction mixture indicates stronger reducing power. In this study, petroleum ether, ethanol and methanolic flower extracts of Cassia auriculata L were used.

 

Statistical analysis:

All the values of the experimental results were expressed as Mean ±S.D

 

RESULT AND DISCUSSION:

Phytochemical Screening:

Phytochemical studies reveal that the presence of Sterols, Saponins, and Flavonoids in Methanolic and Ethanolic extracts while only sterols are present in petroleum ether extract (Table 1).

 

Antioxidant Assay:

a.       DPPH Scavenging Activity:

The Scavenging effect of Petroleum ether, Ethanolic, Methanolic extracts of Cassia auriculata L   and BHA on the DPPH radical is illustrated in Table No: 2. Methanolic and Ethanolic extracts have significant scavenging effect on DPPH, it was increased with the increasing concentration from 50- 250μg/ml but the scavenging activity of all extracts was lower than that of standard.

 

b. Reducing Power:

Different extracts of Cassia auriculata L exhibited good reducing power. The reducing power of the plant extract was determined by the method of Yildirim, A., et al. High absorbance indicates high reducing power. Reducing power of the Methanolic and Ethanolic extracts was dose dependent but the petroleum ether extract had shown negligible effect and is presented in Table 3.

 

Table No: 1. Phytochemical Screening of Cassia auriculata L

Extract

Sterols

Saponins

Flavonoids

Alkaloid

Petroleum Ether flower extracts of Cassia auriculata

+

-

-

-

Ethanol, flower extracts of Cassia auriculata

+

+

+

-

Methanolic flower extracts of Cassia auriculata

+

+

+

-

 

 

Table No: 2. Radical Scavenging activity of various extracts of Cassia auriculata L by DPPH Method

Conc. of μg/ml

% Inhibition by concentration

petroleum ether extract of Cassia auriculata

% Inhibition by concentration ethanolic    extract of Cassia auriculata

% Inhibition by concentration methanolic  extract of Cassia auriculata

% Inhibition by BHA

50

4.33±5.25

18.66±5.84

18.85±4.85

38.59±4.78

100

6.79±2.31

37.21±3.97

39.47±5.23

60.84±4.35

150

9.12±6.50

45.94±5.24

46.31±6.55

70.06±2.54

200

10.21±3.95

50.56±1.32

51.43±4.73

76.54±5.12

250

12.45±3.39

61.67±5.98

65.42±3.33

83.27±4.11

Data are expressed as mean±SD of triplicate tests

 

Table No: 3. Reducing Power of various extracts of Cassia auriculata L

Conc. Of μg/ml

Reducing power of

petroleum ether extract of Cassia auriculata

Reducing power of concentration ethanolic    extract of Cassia auriculata

Reducing power of concentration methanolic  extract of Cassia auriculata

Reducing power of Ascorbic acid

50

10.23±1.25

43.16±4.18

43.44±3.89

51.94±2.78

100

18.48±2.18

51.34±2.64

52.14±4.31

59.22±3.25

150

20.89±6.25

65.98±3.21

67.08±5.36

70.18±2.14

200

21.64±4.11

80.14±3.90

82.19±3.18

90.48±3.81

250

25.56±4.17

85.12±2.41

90.49±4.21

96.00±4.81

Data are expressed as mean ± SD of triplicate tests

 

 

CONCLUSION:

Based on the results described, it may be concluded that the petroleum ether extract shown less significant scavenging and reducing power may be due to the absence of saponins and Flavonoids. The anti oxidant potential of Methanolic and Ethanolic extracts may be attributed to the presence of saponins and Flavonoids.

 

ACKNOWLEDGEMENTS:

This work is carried at the Department of Biochemistry and Microbiology,  Meenakshi Chandrasekaran  College of Arts and Science, Pattukkottai, Thanjavur(Dt), Tamilnadu. Authors are highly thankful to the Secretary and the Principal for providing the facilities.

 

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Received on 19.07.2011          Accepted on 22.08.2011        

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Asian J. Pharm. Tech. 1(3): July-Sept. 2011; Page 70-72