INTRODUCTION:

Reactive oxygen species (ROS) are generated continuously in the body by endogenous and exogenous factors like normal aerobic respiration, stimulated polymorpho-nuclear leukocytes, macrophages and exposure to various pollutants¹. This may be harmful to the body and may cause peroxidation of lipids, aggression of tissue membranes or proteins, damage to DNA and enzymes². These can be related to the pathogenesis of disorders such as cancer, hypertension, Alzheimer’s disease, Parkinson’s disease, ischemia, liver disorder, neural disorder, metal toxicity and pesticide toxicity^3,4. Antioxidants can prevent undesirable oxidation processes by reacting with free radicals, chelating with free catalytic metals and also by acting as oxygen scavengers⁵. Several studies have demonstrated that plants produce potent antioxidants and represent an important source of natural antioxidants^1-6. Therefore, the antioxidants of natural origin have been the centre of attraction for modern researchers.

Simaruba glauca DC. (Paradise tree) belongs to the family Simarubaceae. It is found growing under a wide range of conditions in Central America, Kenya and Burundi in Central Africa.

In India, it is cultivated in Orissa and also at an introductory stage of plantation in other states like Andhra Pradesh, Karnataka, Maharashtra, Tamil Nadu, and Kerala⁷. The bark, wood and leaves of Simarouba have been used for their amoebicide, analgesic, anthelmintic, antibacterial, antidysenteric, antimalarial, astringent, febrifuge, stomachic, and vermifuge properties. The main active groups of phytochemicals in Simarouba are the quassinoids like ailanthinone, glaucarubinone and holacanthone. Other chemical constituents include benzoquinone, canthin, dehydroglaucarubinone, glaucarubine, glaucarubolone, melianone, simaroubidin, simarolide, simaroubin, simarubolide, sitosterol and tirucalla⁸.

MATERIALS AND METHODS:

Plant material

Leaves of Simaruba glauca were collected from the herbal garden of Navajyothi Sree Karunakara Guru Research Centre for Ayurveda and Siddha, Uzhavoor, Kottayam, Kerala. The plant was identified and voucher specimens were deposited in the herbarium of the institution for future reference. The leaves were washed and dried in an oven at 45°C. Powdered samples were extracted in a soxhlet apparatus using methanol (65°C) for 16 hours. The extract was concentrated and stored in refrigerator (4°C). The yield of the extract was 17%.

Chemicals

2, 2-diphenyl- 2- picryl-hydrazyl (DPPH), 2- deoxy-D-ribose, ascorbic acid, curcumine, nitroblue tetrazolium (NBT) and thiobarbituric acid (TBA) were obtained from Sigma Chemicals Co., St. Louis, MO, USA. Butylated hydroxyl anisole (BHA), sulphanilamide and potassium ferricyanide were procured from Merck, KGOA, Germany. All other chemicals and solvents were of analytical grade available commercially.

Preliminary phytochemical analysis:

The preliminary phytochemical screening of the methanolic extract of S. glauca was done to find out the presence or absence of different phyto-constituents⁹.

Evaluation of radical scavenging activity:

Radical scavenging activity of MESG was determined by measuring reducing power, inhibition of DPPH, superoxide, nitric oxide and hydroxyl radicals. The MESG was re-dissolved in a minimum amount of methanol and made up to the required volume in 1% gum acacia¹⁰. For all the assays, different dilutions of MESG such as 5µg/ml, 10µg/ml, 25µg/ml, 50µg/ml, 100µg/ml and 250µg/ml were used.

Determination of reducing power:

The reducing power of the extract was determined according to the method of Oyaizu¹¹. Various concentrations of MESG in 1.0ml deionized water were mixed with 2.5ml phosphate buffer (0.2M, pH 6.6) and 1% potassium ferricyanide (2.5ml). The mixture was incubated at 50°C for 20 min. Aliquots (2.5ml) of 10% TCA were added to the mixture, which was then centrifuged at 1000 x g for 10 min. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and 0.5ml freshly prepared 0.1% FeCl₃solution. The absorbance was measured at 700 nm in a spectrophotometer (Shimadzu UV VIS-1700). Ascorbic acid was kept as a reference standard. Higher absorbance of the reaction mixture is an indication of the greater reducing power.

DPPH radical scavenging activity:

The DPPH radical scavenging activity of the extract was measured by the method of Blois¹². DPPH (1ml, 0.1mM) was added to 3.0 ml of the different concentrations of MESG. It was incubated at room temperature for 45 min and the absorbance was measured at 517 nm. BHA was taken as the reference standard.

Superoxide radical scavenging activity:

Superoxide radical scavenging activity was determined by NBT reduction method ¹³. The reaction mixture contained EDTA (6 mM), NaCN (3 mM), riboflavin (2 mM), NBT (50 mM), various concentrations of the extract and phosphate buffer (67 mM, pH 7.8) in a final volume of 3 ml. The systems were uniformly illuminated with an incandescent lamp for 15 min. Before and after illumination, the optical density of the systems was measured at 560 nm. Ascorbic acid was kept as a reference compound.

Nitric oxide radical scavenging activity:

Nitric oxide generated from sodium nitroprusside was measured by the Griess reagent ¹⁴. Various concentrations of the extract was treated with sodium nitroprusside (1 mM) in phosphate buffered saline (PBS, pH 7.4) in a final volume of 3ml and incubated for 150 min at 25°C. After incubation, 0.5 ml of the solution was removed and diluted with 0.5 ml of Griess reagent (1% sulphanilamide, 2% orthophosphoric acid, and 0.1% naphthylethylenediamene dihydrochloride). The absorbance was read at 546 nm. Curcumine was kept as a reference compound.

Hydroxyl radical scavenging activity:

Hydroxyl radical scavenging activity was measured by using the method of Kunchandy and Rao¹⁵. The reaction mixture (1ml) contained 100µl 2- deoxy - D- ribose (28mM) in phosphate buffer,(20mM pH 7.4), 500µl plant extract in different concentrations in phosphate buffer (20mM, pH 7.4), 100µl H₂O₂ (1.0mM), 100µl ascorbic acid (1.0mM) and a mixture of 200µl EDTA (1.04mM) and 200µM FeCl₃(1:1v/v). The system was incubated at 37°C for one hour. One ml of 1% thiobarbituric acid and 1.0ml of 2.8% TCA acid were added to the reaction mixture and incubated at 100°C for 20 min. After cooling, the absorbance was measured at 532 nm. BHA was used as a positive control.

Decreased absorbance of the reaction mixture indicated increased radical scavenging activity against DPPH, superoxide, nitric oxide and hydroxyl radicals. The percentage inhibition of each parameter was calculated using the following equation: percentage of inhibition = (A_c– A_s) / A_c X 100, where A_cis the absorbance of the control and A_sis the absorbance of the sample.

Estimation of total phenol content:

Total Phenolic compounds in the extract were estimated with Folin-Ciocalteau reagent according to the method of Slinkard and Singleton¹⁶. Gallic acid was treated as reference standard. Absorbance of the blue color developed was read at 760 nm.

Statistical analysis:

The results are expressed as mean ± SE of three parallel measurements. The data was analysed by one way ANOVA. P <0.05 was regarded as significant.

RESULTS AND DISCUSSION:

Reactive oxygen species (ROS) readily combine and oxidize biomolecules such as carbohydrates, proteins and lipids, thus making them inactive with subsequent damage to cells, tissues and organs. Body’s innate immune system has many enzymes and non- protein compounds that protect it from free radicals and reactive oxygen species produced inside the body during normal metabolism as well as those due to external stimuli¹⁰.

Reactive oxygen metabolites (ROMs), such as superoxide anions (O₂^.-) hydroxyl radical, (·OH) and nitric oxide (NO) are directly or indirectly involved in the multistage process of carcinogenesis. They act as initiator or promoter in carcinogenesis. However, NO plays a dual role in cancer. At high concentrations, it kills cancer cells, but at low concentration it promotes tumor growth and metastasis¹⁷.

Phytochemical compounds present in MESG:

Phytochemical screening of the crude MESG revealed the presence of alkaloids, flavanoids, sterol/terpenoids, quinines, anthroquinones, glycoside, reducing sugar, carbohydrate, volatile oil, and phenolic compounds. However, coumarins, saponins, tannins and resins were absent in the extract.

Reducing power activity:

Fig. 1 depicts the reductive effect of MESG. The reducing power of MESG enhanced in a concentration dependant manner. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity¹⁸. The reducing power of the extract might be due to their hydrogen donating ability which can be attributed to the reductants present^11,19. The phytochemicals present in MESG could act as good reductants which could react with free radicals leading to the stabilization and termination of radical chain reaction.

Fig. 1 Reducing power of MESG

DPPH radical scavenging activity:

DPPH radical has been widely used to test the radical scavenging ability of various natural products. It has been accepted as a model compound for free radicals originating in lipids¹. When the odd electrons become paired off in the presence of a free radical scavenger, the absorption reduces and the DPPH solution is decolorized as the colour changes from deep violet to light yellow. The degree of reduction in absorbance is indicative of the antioxidant power of the extract²⁰. Scavenging effects of different concentrations of MESG and BHA on the DPPH radical are illustrated in Fig.2. MESG had significant free radical scavenging effects on DPPH radical, which increased with increasing concentration from 5-250 µg/ml. The highest percentage of inhibition of MESG and BHA were found to be 83.87 and 92.16 respectively in a concentration of 250µg/ml. IC₅₀ of MESG was found to be 21µg/ml. The results showed that MESG possesses statistically significant DPPH radical scavenging activity (P<0.05).

Fig. 2 DPPH radical scavenging activity of MESG

Superoxide radical scavenging activity:

Superoxides are produced from molecular oxygen due to oxidative enzymes of the body as well as via non-enzymatic reaction such as auto-oxidation by Catecholamine. The scavenging activity towards the superoxide radical (O₂^.-) is measured in terms of inhibition of generation of O₂^{. -}⁴. Fig. 3 shows the superoxide radical scavenging activity of 5-250 µg/ml of MESG in comparison with same concentration of Ascorbic acid. The Superoxide radical scavenging activity of the extract increased markedly with the increase in concentration. The highest percentages of inhibition of MESG and reference compound were found to be 76.25 and 95.15 respectively in a concentration of 250µg/ml. IC₅₀ of MESG was found to be 27µg/ml. The results suggested that MESG had statistically significant superoxide radical scavenging activity (P<0.05).

Fig. 3 Superoxide radical scavenging activity of MESG

Nitric oxide radical scavenging activity:

Nitric oxide is a free radical generated by endothelial cells, macrophages, neurons etc and involved in the regulation of various physiological processes. Sodium nitroprusside serves as a chief source of free radicals⁵. The absorbance of the chromophore formed during diazotization of the nitrite with sulphanilamide and subsequent coupling with napthylethylene diamine is used as the marker for NO scavenging activity²¹. MESG significantly inhibited nitric oxide in a concentration dependant manner (Fig.4). The highest percentages of inhibition of MESG and reference compound were found to be 70.43 and 96.13 respectively in a concentration of 250µg/ml. IC₅₀ of MESG was found to be 32µg/ml. The results showed that MESG had statistically significant nitric oxide radical scavenging activity (P<0.05).

Fig. 4 Nitricoxide radical scavenging activity of MESG

Hydroxyl radical scavenging assay:

Hydroxyl radical scavenging activity of MESG was measured by studying the competition between deoxy ribose and the test compound for hydroxyl radicals generated by Ferric³⁺ascorbate-EDTA-H₂O₂ system (Fenton reaction)²². The percentage of inhibition of the different concentrations of the extract showed potent inhibition of hydroxyl radicals in a concentration dependent manner (Fig 5). The highest percentages of inhibition of MESG and reference compound were found to be 80.19 and 94.13 respectively in a concentration of 250µg/ml. IC₅₀ of MESG was found to be 26µg/ml. The results proved that MESG had statistically significant hydroxyl radical scavenging activity (P<0.05).

Amount of total phenolic compounds:

Phenolics are the most widespread secondary metabolite in plant kingdom. These diverse groups of compounds have received much attention as potential natural antioxidant in terms of their ability to act as both efficient radical scavengers and metal chelators. It has been reported that, the antioxidant activity of phenol was due to their redox properties, hydrogen donors and singlet oxygen quenchers²³. Therefore, in the present study, total phenolic content present in the extract was estimated. The result of the present study indicated that, the extract had 24.6 mg phenols per gram.

Fig. 5 Hydroxyl radical scavenging activity of MESG

CONCLUSION:

The results obtained in the present study indicated that MESG exhibited significant reducing power and free radical scavenging activity. Therefore, the leaves of Simaruba glauca could be a potential source of natural antioxidant that could have great importance in nutraceuticals and pharmaceutical preparations.

ACKNOWLEDGMENTS:

The authors are grateful to Swami Gururethnam Jnana Thapaswi, Director, Navajyothi Sree Karunakara Guru Research Centre for Ayurveda and Siddha, Uzhavoor, Kottayam, Kerala for providing the necessary facilities for the study. Technical assistance rendered by Sri. Janeesh MP is also acknowledged.

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Received on 16.11.2009 Modified on 29.12.2009

Asian J. Research Chem. 3(2): April- June 2010; Page 312-315