22 Phcog J | Dec 2012 | Vol 4 | Issue 34

O R I G I N A L A R T I C L EP H C O G J

ABSTRACT

Clausena excavata

C. excavata

50 = 201.3 μ50 = 450.6 μ

C. excavata

to be antioxidants.

Keywords:

Evaluation of antioxidant activity of the methanolic leaf extract of Clausena excavata Burm. f.(Rutaceae) using

the lipid peroxidation modelC Guntupalli,1* G Siva Kumar,2 Adapa Satish Kumar3 and Tejaswi Tubati4

1,2Department of Pharmacognosy, Gitam Institute of Pharmacy, Gitam University Gandhinagar Campus, Rushikonda Visakhapatnam-530045. 3,4Department of Pharmacology, Mother Teresa Pharmacy college, Kothuru, Sathupally, Khammam Dist-507303, A.P, India.

INTRODUCTION

Ethnobotanical screening of species of the Rutaceae family in several continents has shown that they have important medicinal properties. Clausena excavata is a wild shrub of the Rutaceae family that is distributed in South Asia. It is also cultivated in some areas and grows up to 1.5 m high. It has been used in folk medicine for the treatment of cancer and several disorders in the eastern parts of Thailand.[1–4] Its leaves and stem are also used to treat colic, cough, headache,[3] rhinitis, sore, wounds, and

[3] The leaves are particularly used in traditional medicine to

cure cold, abdominal pain, malaria,[5] dysentery, and anti-platelet aggregation.[6] The plant is also reported to have insecticidal, tonic, and vermifugal properties.[6] C. excavata has been reported to contain carbozole alkaloids, couma-rins, limnoids,[4] etc. The sap of the leaves is applied on the affected area to treat all kinds of muscular pain.[3] A decoction of the root is given to patients with malaria;[5] an infusion of the stem is given in patients with colic. The leaves and stem bark are also used locally as a diuretic, tonic, astringent, and antinociceptive agent.[3] The cou-marins derived from the leaves have been found to have inhibitory effects on tumor progression.[7–8]

Several pharmacologically active constituents of plants

phenylpropanoids,[9] [10] furoquinoline alkaloids,[11] coumarins,[12] and acridone alkaloids.[13] How-ever, the chemical components of species of this family are yet to be fully elucidated. We chose to study the phy-tochemical properties of extracts of C. excavata because it is known to have antioxidant properties. Cellular damage arising from reactive oxygen species (ROS) has been shown to play a part in the development and

*Corresponding author. Dr. Chakravarthi GuntupalliMother Teresa Pharmacy college,Kothuru, Sathupally, Khammam Dist-507303, A.P, India.Land line No: 08761-215689Mobile No: 7893075068

E-mail: chakra_varthi@rediffmail.com

DOI: 10.5530/pj.2012.34.4

C Guntupalli, et al.: Evaluation of antioxidant activity of the methanolic leaf extract of Clausena excavata Burm. f.(Rutaceae) using the lipid peroxidation model

Phcog J | Dec 2012 | Vol 4 | Issue 34 23

pathophysiology of neurodegenerative disorders (e.g., Alzheimer disease, Parkinson disease, multiple sclerosis,

autoimmune pathologies, and digestive system disorders [14] We

therefore focused our investigation on the antioxidant potential of C. excavata.

The aim of the present investigation was to separate, characterize, and identify the major chemical constitu-ents of C. excavata and to explore the antioxidant poten-tial of its methanolic extracts and isolated constituents. The thiobarbituric acid reactive species (TBARS) assay was performed using standard methods with melon-dialdehyde (MDA) equivalents derived from tetrae-thoxypropane. MDA and other aldehydes have been

with TBA to yield a pink product that shows absorption at 532 nm.[15]

MATERIALS AND METHODS

Plant material

Leaves of C. excavata were collected from the northern regions of Malaya in June-July 2005, and their identity was was further authenticated by Bowen, from the Depart-ment of Pharmacognosy, University of Sunderland, and a voucher specimen sample was deposited in the herbar-ium. The collected plant materal were dried well under shade and powdered using elecric blender.

Soxhlet extraction

The powdered leaf material (100 g) was successively extracted with petroleum ether, hexane, chloroform, dichloromethane, ethanol, and water. The volume of the solvent used in each case was 500 ml. Before extraction with each solvent, the powdered material was air dried and weighed. The largest quantity of extract was obtained with methanol. Further fractionation of the extracts was therefore carried out on the methanolic leaf extract by high-performance liquid chromatography.

Preliminary phytochemical screening

The different qualitative tests can be performed for estab--

position. The freshly prepared extracts were subjected to standard phytochemical analysis to test for the presence of the phytoconstituents like tannins, saponins, alka-

terpenoids. Chemical tests were carried out on methanol and aqueous extracts of the powdered specimens using standard procedures for the detection of saponins, alka-loids (Mayer’s reagent and Dragendorff ’s reagent test), carbohydrates (Molish’s and Benedict’s test), glycosides (Borntragor’s and Legal’s tests), phenolics and tan-

(alkaline reagent test), terpenoids (Salkowski test and Trichloro acetic acid test) to identify the phytochemical constituents.

Chemicals

1 % Thiobarbituric Acid (TBA), Propylgallate (0.1 mM), ascorbic acid (1 mM), eCl3 (1 mM), 2 % butylated hydroxy toluene (BHT) in ethanol, nBovine brain extract (Sigma product: B 3635), HCL (25% Hydrochloric acid), Glass balls (about 2mm diameter) were used.

Preparation of liposomes and extracts

Brain bovine extract (50 mg; B 3635, Sigma) was placed in a clean universal container with about 7 glass balls (diame-ter, 2 mm) and 10 ml of phosphate-buffered saline (PBS), and sonicated in an ice-water bath until a homogenous and milky lipid suspension was obtained.[16] The metha-nolic extract (50 mg) was dissolved in 10 ml of methanol

Procedure for the assay

The liposomes were prepared as a 5 mg/mL suspension of bovine brain extract (Sigma) in PBS and stored at –80°C. Ascorbic acid and ferric chloride aqueous solu-tions (1 mM) were freshly prepared before each test. Seven concentrations of methanolic extract were pre-pared (5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.07861 mg/ml). Four replicates were carried out for each concentration of the methanolic extract and without the methanolic extract.

Blank: Distilled water (0.4 mL) and 0.5 mL of PBS were

Free reaction mixture: Liposomes (0.2 mL), 0.5 mL PBS, 0.1 mL FeCl3 solution, and 0.1 mL of ascorbic acid solu-tion were added in the second row of tubes (4). The positive control tubes(4) contained 0.1 mL propyl gallate, 0.2 mL liposomes, 0.5 mL PBS, 0.1 mL FeCl3 solution, and 0.1 mL ascorbic acid solution, resulting in 68 tubes ( Sterilin). Distilled water is used as balnk whereas Propyl gallate is used as standard compound. The tubes were

C Guntupalli, et al.: Evaluation of antioxidant activity of the methanolic leaf extract of Clausena excavata Burm. f.(Rutaceae) using the lipid peroxidation model

24 Phcog J | Dec 2012 | Vol 4 | Issue 34

heated in a water bath (Gallenkamp) at 85–90°C for 30 min, and after they had cooled, 2.5 mL of n-butanol was added. They were shaken individually with a vor-tex mixer (Gallenkamp) and centrifuged at 3500 rpm (MISTRAL-3000i) at room temperature for 10 min. The top n-butanol layer (2.5 mL) from each tube was trans-ferred to a cuvette, and its absorbance was measured with a UNICAM UV/VIS spectrophotometer operating at 532 nm, using n-butanol as the internal standard.[17–18] The data were processed with the aid of Microsoft Excel 2000, and the percentage inhibition of each compound at a given concentration was calculated. The IC50 values were obtained from LOWESS analysis of percentage inhibition of peroxidation versus the logarithmic concen-tration of each inhibitor, using PRISM GraphPad. The results are shown in Table 2.

RESULTS AND DISCUSSION

Free radicals such as the superoxide radical, hydroxy radical, peroxyl radical, and singlet oxygen have been implicated in the pathophysiology of many diseases. Herbal drugs containing radical scavengers are gaining

Table 1. Phytochemical test results for secondary metabolites.

S r. No. Plant Part of the

plantSolvent for extraction

Secondary metabolitesS A F P G Sa

1 Clausena excavata Leaves Methanol – – ++ ++ + –2 Clausena excavata Leaves Methanol – – + + – –S: Steroids; A: Alkaloids; F: Flavonoids; P: Phenolics; G: Glycosides; Sa: Saponins; +: Positive; –: Negative

Table 2. Antioxidant activity according to the lipid peroxidation assay.Percentage inhibition by the methanolic extract of Clausena excavata

Conc mg/ml

Conc mM/L Absorbance at 532 nm (n = 4) Mean S.D % R.S.D %

InhibitionBlank 0.0880 0.0480 0.0910 0.1040 0.0828 0.0242 29.2266FRM 0.2810 0.2930 0.2660 0.2630 0.2758 0.0139 5.0543Propyl gallate 0.1000 0.1690 0.1210 0.1810 0.1170 0.1470 0.0327 22.2731 84.31Substance test 5.0000 18.7041 0.1270 0.1740 0.1500 0.1740 0.1563 0.0225 14.4284 62.9534Substance alone 5.0000 18.7041 0.0020 0.0040 0.0010 0.0010 0.0020 0.0014 70.7107Substance test 2.5000 9.3521 0.1980 0.1750 0.1860 0.1920 0.1878 0.0098 5.2254 56.9948Substance alone 2.5000 9.3521 0.0030 0.0760 0.0070 0.0020 0.0220 0.0361 163.9307Substance test 1.2500 4.6760 0.2180 0.2480 0.2580 0.2150 0.2348 0.0215 9.1587 33.1606Substance alone 1.2500 4.6760 0.0810 0.0060 0.0030 0.0020 0.0230 0.0387 168.2783Substance test 0.6250 2.3380 0.3140 0.1560 0.2600 0.2350 0.2413 0.0657 27.2345 32.3834Substance alone 0.6250 2.3380 0.0910 0.0110 0.0070 0.0030 0.0280 0.0421 150.4528Substance test 0.3125 1.1190 0.2974 0.3035 0.2721 0.2291 0.2755 0.0338 12.2689 20.1554Substance alone 0.3125 1.1690 0.0295 0.0349 0.0421 0.0478 0.0386 0.0080 20.8136Substance test 0.1563 0.5845 0.3139 0.3160 0.2600 0.2350 0.2812 0.0403 14.3183 10.0027Substance alone 0.1563 0.5845 0.0191 0.0266 0.0234 0.0298 0.0247 0.0046 18.4852Substance test 0.0781 0.2923 0.2673 0.3126 0.2842 0.2420 0.2765 0.0297 10.7224 3.8342Substance alone 0.0781 0.2923 0.0093 0.0070 0.0011 0.0150 0.0081 0.0058 71.3744S.D: Standard Deviation, R.S.D: Relative Standard DeviationSustance test: With methanolic extract concentration Substance alone: Without methanolic extract concentration

importance for the treatment of such diseases. Many

to their phenolic constituents.[19–22]

Our results indicated that the methanolic leaf extract of C. excavata showed marked antioxidant activity (Table 2), whereas the aqueous leaf extract of C. excavata showed moderate antioxidant activity. The IC50 value (Fig. 1) of the methanolic leaf extract was found to be 201.3 μg/ml, whereas the IC50 value for the aqueous extract of C. excavata was found to be 450.6 μg/ml. The methanolic leaf extract was used for the preliminary phytochemical screening to identify secondary metabolites/active con-

in the extracts (Table 1). The extended degree of antioxi-dant activity displayed by the methanolic extract could be

[19] gallic acid[20–21] and angelicin,[22] which are known to be antioxidants.

extract of leaves of C. excavata -oxidant activity. This warrants further investigation to identify the active constituents accountable for its anti-oxidant activity.

C Guntupalli, et al.: Evaluation of antioxidant activity of the methanolic leaf extract of Clausena excavata Burm. f.(Rutaceae) using the lipid peroxidation model

Phcog J | Dec 2012 | Vol 4 | Issue 34 25

COMPETING INTERESTS

The authors declare that they have no competing interests associated with this study.

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Figure 1. Dose-response curve for percent inhibition versus concentration of the extract.