In-vitro antioxidant activity of Premna serratifolia Linn

 

P. Muthukumaran*, S. Salomi  and R. Umamaheshwari

P.G .Department of Biochemistry, Meenakshi Chandrasekaran College of Arts and Science, Pattukkottai-614 626, Thanjavur, Tamil Nadu

 

ABSTRACT:

The aim of this study was to investigate the antioxidant effect of Premna serratifolia Linn. The antioxidant activity was evaluated by various antioxidant assays, including 1, 1-diphenyl-2- picrylhydrazyl (DPPH), 2, 2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), and hydrogen peroxide scavenging method. The antioxidant activities were compared to standard antioxidant ascorbic acid. Premna serratifolia Linn wood extract showed a significant antioxidant activity in DPPH, ABTS and H2O2 scavenging methods. The findings of the present study suggest that Premna serratifolia Linn  could be a potential source of natural antioxidant that could have greater importance as therapeutic agent in preventing or slowing oxidative stress related  degenerative diseases.

 

KEYWORDS: Premna serratifolia Linn, antioxidant activity

 

INTRODUCTION:

Oxygen derived free radicals, such as the superoxide anion and hydroxyl radicals are cytotoxic and promote tissue injury. Antioxidants act as a major defence against radical mediated toxicity by protecting the damages caused by free radicals. Furthermore although medicinal plants are used as ‘antioxidants’ in traditional medicine, their claimed therapeutic properties could be due, in part, to their capacity for scavenging oxygen free radicals. Reactive oxygen species (ROS) including free radicals such as superoxide anion radicals, hydroxyl radicals, singlet oxygen and non-free-radical species such as hydrogen peroxide are various forms of activated oxygen and often generated by oxidation product of biological reactions or exogenous factors.

 

ROS have aroused signicant interest among scientists in the past decade. Their broad range of effects in biological and medicinal systems has drawn on the attention of many experimental works. In living organism, various ROS can form by different ways.

 

Normal aerobic respiration stimulates polymorphonuclear leukocytes and macrophages, and Peroxisomes appear to be the main endogenous sources of most of the oxidants produced by cells. Exogenous sources of ROS include tobacco smoke, certain pollutants, organic solvents, and pesticides (1-3). ROS can cause lipid peroxidation in foods, which leads to the deterioration of the food (4,5). In addition, it is well known that ROS induce some oxidative damage to biomolecules like lipids, nucleic acids, proteins, amines, deoxyribonucleic acid and Carbohydrates. Its damage causes ageing, cancer, and other many diseases (6, 7). As a result of this, ROS have been implicated in more than hundred diseases, including malaria, acquired immunodeciency syndrome, heart disease, stroke, arteriosclerosis, diabetes,


and cancer. ROS are continuously produced during normal physiologic events, and removed by antioxidant defence mechanisms (8). There is a balance between generation of ROS and antioxidant system in organisms. In pathological condition, ROS are overproduced and result in lipid peroxidation and oxidative stress. The imbalance between ROS and antioxidant defense mechanisms leads to oxidative modication in cellular membrane or intracellular molecules (9). Various endogenous antioxidant defence mechanisms play an important role in the elimination of ROS and lipid peroxides to protect the cells against toxic effects of ROS and lipid peroxides (10).

 

Many antioxidant compounds, naturally occurring from plant sources, have been identied as free radical or active oxygen scavengers (11, 12). Recently, interest has increased considerably in nding naturally occurring antioxidant for use in foods or medicinal materials to replace synthetic antioxidants, which are being restricted due to their side effects such as carcinogenicity. Natural antioxidants can protect the human body from free radicals and retard the progress of many chronic diseases as well as retard lipid oxidative rancidity in foods (13).

 

Premna serratifolia Linn., (Verbenaceae) is an important plant belonging to the family Verbenaceae, and is one of the most widespread large shrubs in the forests of India, usually occurring in deciduous forests. The whole plant possesses medicinal properties, useful in the treatment of cardiovascular diseases, skin diseases, inflammatory diseases, arthritis, gonorrhea, rheumatism, anorexia and jaundice. It is an important Ayurvedic medicinal herb and its synonym is Premna integrifolia. It is popularly known as“Munney” in Tamil, and “Agnimantha” in Ayurvedic system of medicine. Root forms an ingredient in well known Ayurvedic formulation “Dasamula” which is used for variety of affections (14). It is widespread throughout tropical Pacific and tropical Asia. It is common along the Indian and Andaman coasts. Infusion of the leaves is administered with pepper in cold and fever. Leaves are used to cure "weakness of limbs" and the leaves and leaf sap were used to alleviate headache (15).

 

Premna serratifolia Linn, has cardiotonic (16), anti-coagulant(17), anti-inflammatory(18), anti hyperglycaemic (19), anti-parasitic (20), antioxidant (21) and antimicrobial (22) properties. Most of the plant parts of Premna serratifolia Linn., have been used in the traditional system of medicine in India to treat various infectious diseases.  Therefore we undertook the present investigation to examine the antioxidant activity of ethanolic extract of  Premna serratifolia Linn., through various in vitro models.

 

 

MATERIALS AND METHODS:

Plant material

Fresh wood (without bark) of Premna serratifolia Linn., were collected in April, 2012, from the waste lands in the villages of Thanjore , Tamil Nadu, India

 

Preparation of the extract

The freshly collected wood was chopped, shade dried and coarsely powdered (40 mesh size). The powder was defatted with petroleum ether (60 - 80 °C) and then extracted with 90% ethanol in a Soxhlet extractor. The extract was dried under reduced pressure  using a rotary vacuum evaporator and the percentage yield was 7.90% w/w. The obtained ethanol extract was suspended in 5% gum acacia for the pharmacological screening.

 

Chemicals

1 , 1 - diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were obtained from Sigma Aldrich Co, St Louis, USA. Ascorbic acid and rutin were obtained from SD Fine Chemicals Ltd., Mumbai, India. Ethanol and dimethyl sulphoxide were obtained from Ranbaxy Laboratories Ltd., Punjab, India. Hydrogen peroxide (30%) was obtained from Qualigen Fine Chemicals, Mumbai, India. All chemicals used were of analytical grade.

 

Phytochemical studies

Freshly prepared Premna serratifolia Linn wood  extract was subjected to phytochemical screening tests for the detection of various constituents using conventional protocol.

 

DPPH radical scavenging activity

Free radical scavenging activity of extracts of pericarp of Premna serratifolia Linn were tested by its ability to bleach the stable 1,1-diphenyl 2-picryl-hydrazyl (DPPH) radical. A stock solution of DPPH 0.3mM in methanol) was prepared such that 1ml of it in 3ml methanol gave an initial absorbance of 0.9.Decrease in absorbance in the presence of Ethanolic extract at different concentration(50-500 mg/ml) were noted after 15 min. scavenging activity was expressed as the %inhibition. (23,24),

 

Radical scavenging activity (%)  =   OD control - OD sample x 100

                                                                   OD control

 

ABTS radical cation decolourisation assay

ABTS (54.8 mg) was dissolved in 50 ml of distilled water to 2 mM concentration and potassium

persulphate (17 mM, 0.3 ml) was added. The reaction mixture was left to stand at room temperature overnight in dark before use. To 0.2 ml of various concentrations of the extracts or standards, 1.0 ml of distilled DMSO and 0.16 ml of ABTS solution was added to make a final volume of 1.36ml.Absorbance was measured spectrophotometrically, after 20 min at 734 nm. The assay was performed in triplicate (25, 26).

 

Scavenging of hydrogen peroxide

It can be generated through a dismutation reaction from superoxide anion by superoxide dismutase. It can generate the hydroxyl radical in the presence of metal ions and superoxide anion (27).

 

O2 + H2O2     → OH- + OH+ + O2

 

A solution of hydrogen peroxide (20mM) was prepared in phosphate buffered saline (PBS, pH 7.4).Various concentrations of 1ml of the extracts or standards in methanol were added to 2 ml of hydrogen peroxide solutions in PBS. The absorbance was measured at 230 nm, after 10 min against a blank solution that contained extracts in PBS without hydrogen peroxide (28).

 

RESULTS:

The results of the preliminary phytochemical screening of the ethanol extract of Premna serratifolia Linn., revealed the presence of phytoconstituents such as alkaloids, steroids, flavonoids, phenolic compounds, tannins and glycosides specifically iridoid glycosides.

 

The antioxidant activity of Premna serratifolia Linn was evaluated by DPPH, ABTS and Hydrogen peroxide radical scavenging methods. Premna serratifolia Linn showed a dose dependent scavenging activity and free radical inhibition of DPPH, ABTS and H2O2 comparable to free radical scavenging activity of ascorbic acid. (Table 1-3, Fig 1).

 

 

Table 1.DPPH free radical Scavenging activity of ethanolic wood extract  Premna serratifolia Linn

Sample

Concentration  μg /mL

Percentage Inhibition

IC50

 

Ethanolic Extract

100

150

200

300

500

43.25

50.84

57.80

72.89

81.64

155 μg/ ml

 

Ascorbic acid                                              

16μg/ml  

 

 

Table 2.ABTS Scavenging activity of Premna serratifolia Linn

 Sample

Concentration

μg /mL

Percentage Inhibition

IC50

 

Ethanolic Extract

100

150

200

300

500

20.30

43.34

52.72

54.47

68.4

211 μg /ml

Ascorbic acid                                                                  258μg/ml       

 

Table 3.Hydrogen peroxide scavenging activity of Premna serratifolia Linn

Sample

Concentration

μg /mL

Percentage Inhibition

IC50

Ethanolic Extract

100

150

200

300

500

18.82

33.26

46.12

58.26

72.48

619 μg/ ml

Ascorbic acid                                                                    405μg/ml  

 

 

DISCUSSION:

Reactive oxygen species are continuously formed in cells as consequence of oxidative biochemical reactions and external factors. However they become harmful when they are produced in excess under certain abnormal conditions such as inflammation, ischemia and in presence of iron ions. Under these conditions the endogenous antioxidants may be unable to counteract ROS formation. Reactive oxygen species formed may cause cellular damage and this damage may involve in etiology of diverse human diseases. Exogenous antioxidant supplement is helpful to overcome this severe problem of free radicals, which may scavenge these free radicals.

 

T he free radical scavenging activity of natural compounds can be evaluated through their ability to quench the synthetic free radicals, in which the absorbance of the reaction mixture is taken in visible range to know whether the compound is having antioxidant activity.

 

DPPH assay is based on the measurement of the scavenging ability of antioxidant towards the stable DPPH radical. DPPH is relatively stable nitrogen centered free radical that can accept an electron or hydrogen radical to become a stable diamagnetic molecule. DPPH radicals react with suitable reducing agent as a result of which electron become paired off forming the corresponding hydrazine. The solution therefore looses color stoichiometrically depending on the number of electrons consumed which is measured spectrometric ally at 517 nm (29). From the results it may be postulated that the wood extract of Premna serratifolia Linn have hydrogen donors, thus scavenge the free radical DPPH.

 

ABTS assay is relatively recent one, which involves a more drastic radical, chemically produced and, is often used for screening complex antioxidant mixture such as plant extracts, beverages and biological fluids. The solubility in both the organic and aqueous media and the stability in a wide pH range raised the interest in the use of ABTS radical for the estimation of the antioxidant activity. The principle behind the technique involves the reaction between ABTS and potassium persulphate to produce the ABTS radical cation (ABTS+) a blue green chromogen. In the presence of antioxidant reductant, the colored radical is converted back to colorless ABTS, the absorbance of which is measured at 734nm.

 

The extract of Premna serratifolia Linn possessed an antioxidant activity with IC50 value being 211 μg /ml; suggest the free radical scavenging activity of Premna serratifolia Linn wood extract.

 

 

Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membrane rapidly. Once inside the cell, H2O2 can probably react with Fe2+and possibly Cu2+ to form hydroxyl radical and this may be the origin of many of its toxic effects. It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate. The decomposition of H2O2 by root extract of Premna serratifolia Linn results from its antioxidant and free radical scavenging activity.

 

CONCLUSION:

The results obtained in the present study indicate that Premna serratifolia Linn wood extract exhibits free radical scavenging activity. The overall antioxidant activity of Premna serratifolia Linn wood extract might be attributed to its polyphenolic content and other phytochemical constituents. The findings of the present study suggest that Premna serratifolia Linn could be a potential source of natural antioxidant that could have greater importance as therapeutic agent in preventing or slowing oxidative stress related degenerative diseases.

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Received on 18.09.2012

Modified on 14.10.2012

Accepted on 25.10.2012     

© A&V Publication all right reserved

Research J. Pharmacology and Pharmacodynamics. 4(6): November –December 2012, 336-367