Comparative Study of Antioxidant Activity of Ethanol and Aqueous Extracts of Different Parts of Nyctanthes arbor-tristis Linn .

Background: Antioxidants play a major role in curing degenerative disease. In present time many synthetic antioxidants are commonly used but due to their toxic and carcinogenic effects, their use has been restricted. So the search for natural antioxidants is important. Studies on natural compounds is of interest due to their satisfactory health benefits with low toxicity. Objective: The antioxidant activity of ethanol and aqueous extract of shoot, seed and leaf of an important medicinal tree Nyctanthes arbor-trist is (Harsingar) were evaluated by DPPH scavenging antioxidant assays. Materials and Methods: Different concentrations 25, 50, 75, 100% of the aqueous and ethanol extracts were prepared with standard solutions (ascorbic acid 0.01, 0.03, 0.05, 0.07, 0.09, 0.1 mg/ml). To 1.5 ml solution of DPPH (0.1 mM), 0.5 ml of various concentrations of the extract and standard were added. Results: In the present study a comparative evaluation of antioxidant activity of seed, shoot and leaf of Nyctanthes arbor-tristis Linn was performed for their aqueous and ethanol extracts. Ethanol and aqueous extracts of shoot revealed more antioxidant activity then seed and leaf. Conclusion: All aerial parts of Nyctanthes arbor-trist is Linn possess antioxidant activity. The plant may be functioned out for its anticancerous properties in future.


INTRODUCTION
Antioxidants are vital substances which possess the ability to protect the body from damage caused by free radical induced oxidative stress.Free radicals are highly reactive molecules containing one or more unpaired electron; they donate or take electron from other molecule in an attempt to pair their electron and generate a stable species. 1 Antioxidants are both natural and synthetic compounds able to scavenge free radicals and inhibit oxidation processes. 2Many synthetic antioxidants such as butylatid hydroxyl anisole (BHA) and butylatid hydroxyl toluene (BHT) are very effective and used for industrial processing but they possess some side effect and toxic properties to human health therefore, there is an increasing interest in natural antioxidants, eg.Polyphenols, present in medicinal and dietary plants. 3Antioxidants play an important role in defending the body against free radical damage.Epidemiological studies specify that intake of fruits and vegetables have the ability to inhibit the damaging behavior of free radicals in the human body. 4There were many plants other than those taken by human being to fulfill their food demand possesses important medicinal properties remain undiscovered.Nyctanthes arbor-tristis Linn is native to India one of the most useful traditional medicinal plants.It is a night flowering tree of family Oleaceae, it is well known in india and its neighbouring countries as one of the most versatile medicinal plant having a wide spectrum of biological activities, it is widely cultivated in tropical and subtropical regions all over the world.6][7] Recent studies have been shown that the leaves and stem of N. arbor-tristis possesses potential source of natural antioxidant. 8The antioxidant activity of phenolic compound is mainly due to their redox properties, which can play an important role in absorbing and neutralizing free radicals. 9In present study a comparative approach has been made to analyse the antioxidant properties of shoot, leaf and seeds of N. arbor-tristis.

Phytochemical analysis
Phytochemical analysis of different parts of N. arbor-trist is was carried out applying various phytochemical tests. 10,11st for anthraquinone 0.5 ml of the extract was boiled with 1 ml of sulphuric acid and filtered while hot.The filtrate was shaken with 1 ml of chloroform.The chloroform layer was pipetted out another test tube and 1 ml of dilute ammonia was added.The resulting solution was observed for color change.
Test for terpenoids To 0.5 ml each of the extract was added 2 ml of chloroform.Concentrated sulphuric acid (3 ml) was carefully added to form a layer.A reddish brown coloration of the interface indicates presence of terpenoides.

Test for flavonoids
Diluted ammonia (2 ml) was added to a portion of an aqueous filtrate of the extract.Concentrated sulphuric acid (2 ml) was added.Yellow coloration that disappears on standing indicates the presence of flavonoids.

Plant material
Fresh leaf, shoot and seed of N. arbor-tristis were obtained from reserve forest area of Gwarighat.The sample was thoroughly rinsed and air dried.They were kept in hot air oven on 150ºC for 4 hours for drying.The dried plant material was grind to fine powder.

Preparation of extracts
To prepare ethanol extractof shoot, leaf and seed of N. arbor-tristis, 2.5 g of each plant sample was soaked in 25 ml.ethanol, and kept in orbital shaker (sonar) on 150 rpm for 2 days for extraction.Plant parts (leaf, shoot, and seed) each of 2.5 g were boiled on 100ºC in 50 ml water for 20 minutes.The extracts were filtered through what men filter paper and filtrate were used for antioxidant activity.

Spectrophotometric measurements
Spectrophotometric measurements were performed by uv-visible double beam spectrophotometer (EI 2375) on wavelength 514 nm.

Determination of antioxidant activity-DPPH radical scavenging activity
The DPPH radical scavenging activity of standard (ascorbic acid) and sample were determined by applying the method described by Mensor et al. 2001 12 with some modifications.Different concentrations 25, 50, 75, 100% of the aqueous and ethanol extracts and standard solution (ascorbic acid 0.01, 0.03, 0.05, 0.07, 0.09, 0.1 mg/ml) were prepared.To 1.5 ml solution of DPPH (0.1 mM), 0.5 ml of various concentration of the extract was added.Then the mixture was shaken vigorously and allowed to stand for 30 min at room temperature.The absorbance of resulting solution was measured at 514 nm with uv visible spectrophotometer.Inhibition of free radical DPPH in percent antioxidant activity (%AA) was calculated in following way-

Abs-Absorbance
Antioxidant activity of two extracts of shoot, leaf and seeds were compared statistically by calculating ± standard deviation.Enhancement in the antioxidant activity, with increasing concentrations of extract were calculated by fold enhancement in comparison to lowest concentration (25%) and expressed in result section.

Phytochemical analysis of leaf, shoot and seed of aqueous extract of N. arbor-tristis
Test for anthraquinone: Color change was observed when sample were tested for anthraquinone test which was an indication of presence of anthraquinone in samples.Least color intensity of anthraquinone was found in seed extract of N. arbor-tristis, whereas in leaf extract color intensity was slightly more than seed extract.In shoot extract maximum intensity of color change was observed, this is an indication of presence of high anthraquinone (Table 1, Figure 1).Test for flavonoids: Yellow color was observed which showed the presence of flavonoids in all samples.It was found maximum in leaf extract of N. arbor-tristis (Table 1, Figure 2).Test for terpenoids: Reddish brown coloration of the interface in all samples indicates presence of terpenoids.Minimum color intensity was found in seed extract and maximum in leaf and shoot extracts of N. arbor-tristis.It was clear from above observations that shoot and leaf contain more terpenoids than seed (Table 1, Figure 3).Antioxidant activity was analyzed for shoot, leaf and seeds of N. arbor-tristis.The free radicals scavenging activity of different parts of N. arbor-tristis was studied by their ability to reduce the DPPH, stable free radicals.
Comparison of two extracts (aqueous and ethanol) of shoot, seed and leaf were made to study their antioxidant activity.Antioxidant activity of both extracts was analyzed by applying different concentrations (25, 50, 75 & 100%).Antioxidant activity increased with increasing concentration of both plant extract.Ethanol extract of shoot revealed more antioxidant activity then seed and leaf (Table 2) (DPPH scavenging activity in shoot was ranging from 34(25%) to 91 (100%).The enhancement of antioxidant activity was 57 folds in shoot ethanol extract (Figure 4), whereas in leaf it was ranging from 47 to 88folds (Figure 5), and in seed scavenging activity was ranging from 470.03 (in 25%) to 89 0.03 (in 100%) 42 folds (Figure 6), although the maximum antioxidant activity was recorded on lowest concentration (25%) of seed ethanol extract (Graph 1).In aqueous extract the leaf extract exhibited more antioxidant activity then shoot and seed.DPPH scavenging activity in leaf ranging from 12  The enhancement of antioxidant activity% was 66 folds in leaf extractwhereas in shoot it was ranging from 40 Aqueous extract of seed has the minimum antioxidant activity occurred only 24 folds.24.

DISCUSSION
The importance of medicinal plant is due the presence of phytochemicals present inside them.Phytochemicals are naturally occurring substances of medicinal plants that have defense mechanism and protect the plant from various diseases.Phytochemicals are primary and secondary compounds.Chlorophyll, proteins and common sugars are included in primary constituents and secondary compounds have terpenoid, alkaloids and phenolic compounds.In the present study phytochemical analysis has been made to examine the presence of terpenoids, flavonoids and anthraquinone in the aqueous extract of shoot, leaf and seed of N. arbor tristis, out of them terpenoids and flavonoids were found maximum in the aqueous extract of leaf. 13Also reported the same observation in the methanolic extract of leaf of N. arbor-tristis.Presence of flavonoids in Terminalia bellerica (leaf) and terpenoids in Xanthium strumarium (leaf) in the Ricinus communis (leaf) are reported earlier. 14ntioxidant activity is a measure of anticancerous properties of any plants.Recently the antioxidant nature of many plants has beendiscussed.Researchers have got the data about antioxidant activity of many mysterious plants. 15,16N. arbor-tristis is a well-known medicinal plant from ancient time, leaves of this plant was used to cure fever, arthritis, sciatica. 17Different plant part hasbeen used to cure many diseases similarly, powder of seeds is used to cure piles and skin disease. 18But the antioxidant nature of this plant remains undiscovered.There are very few reports regarding the antioxidant activity of different parts of this plant. 19Reported that n-butenol extract of flower of N. arbor-tristis exhibited antioxidant activity.Ethanol bark extract of N. arbor-tristis was also observed to possess antioxidant activity. 18Present investigation is a comparative analysis of antioxidant activity of three parts with their

CONCLUSION
This study established a fact that the plant N. arbor-tristis is a good source of antioxidant compounds and different plant organs responded differently regarding the nature of solvent used to extract the metabolites from them.