INTRODUCTION:

An herb known as priyal is a drug of the ayurveda and the Unani system of medicine. Buchanania lanzan Spreng. belonging to family Anacardiaceae, commonly known as Chironji. The leaves are reported to be valued for their tonic, cardiotonic properties, their powder is a common medicine for wounds and also used in the treatment of skin diseases^1-5. A new glycoside Myricetin 3’-Rhamnoside-3-Galactoside was identified from leaves of chironji⁶. It is a medium evergreen deciduous tree, growing 50 ft tall. It is a commercially useful tropical plant. It bears fruits each containing a single seed, which is a popular edible nut, known as chironji. It is common in India mostly in eroded lands. It has tickly leathery leaves which are broadly oblong, with blunt tip and rounded base^7,8. Leaves have 10-20 pairs of straight parallel veins and are pubescent. All parts of the plant are used for the treatment of various disorders. The oil from the seeds is used to reduce granular swelling of the neck^9,10. Ointment is made from the kernel which is used to relieve itch and prickly heat.

The oil is used as a substitute for olive and almonds oil in indigenous medicine. The oil on direct inter-esterfication yields a product which may be suitable as a coating material for delayed-action tablets. The oil also appears to be a promising commercial source of palmitic and oleic acids⁵. The gum from the bark used for treating diarrhea and intercostals pains and leaves are used for promoting wound healing^{11, 12}.Oxygen consumption inherent in cell growth leads to the generation of a series of reactive oxygen species (ROS)¹³. They are continuously produced by the body’s normal use of oxygen such as respiration and some cell-mediated immune functions. ROS include free radicals such as superoxide anion radicals (O₂^•−), hydroxyl radicals (OH^•) and non-free radical species such as hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂)¹⁴. ROS are continuously produced during normal physiologic events and can easily initiate the peroxidation of membrane lipids, leading to the accumulation of lipid peroxides. ROS are also capable of damaging crucial biomolecules such as nucleic acids, lipids, proteins and carbohydrates and may cause DNA damage that can lead to mutations. If ROS are not effectively scavenged by cellular constituents, they lead to disease conditions. ROS have been implicated in more than 100 diseases¹⁵. Antioxidants can protect the human body from free radicals and ROS effects. They retard the progress of many chronic diseases as well as lipid peroxidation^16,17. Hence, at the present time, the most commonly used antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propylgallate and tert-butyl hydroquinone. However, BHA and BHT have been suspected of being responsible for liver damage and carcinogenesis^18,19. Therefore, there has been intensive research on natural antioxidants derived from medicinal parts. Medicinal plants contain a wide variety of natural antioxidants, such as phenolics acids, flavonoids and tannins which possess more potent antioxidant activity than common dietary plants²⁰.

MATERIALS AND METHODS:

Plant material and Extraction:

Fresh leaves of Buchanania lanzan Spreng. Procured from uttan van-ausadhi sanshodhan sanstha, Bhayander, Thane, authenticated from Department of life science, Ruia College, Mumbai. The leaves were shade dried. Dried leaves were ground to coarse powder. Powder was extracted with ethanol, which is further evaporated to dryness to obtain alcoholic extract.

Free Radical Scavenging Activity (DPPH Assay):

The ability of the test extract to scavenge the free radicals was determined by an in vitro assay using method using a stable free radical DPPH²¹. To methanolic solution of DPPH, equal volume of test extract of different concentration was added. After 20 min, the decrease in absorbance of test (due to quenching of DPPH free radical) was read at 517 nm. Results were expressed in percentage inhibition and concentration of extracts required to cause a 50% decrease in the absorbance was calculated (IC₅₀)

Reducing Power Assay:

The reducing power of the ethanolic extract of Buchanania lanzan (EEBL) was determined according to the method of Oyaizu (1986)²². Different concentrations of the extract in 1.0 ml of deionised water were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferrocyanide (2.5 ml,1%). The mixture was incubated at 50^oC for 20 min. A portion of trichloroacetic acid (2.5 ml, 10%) was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1%) and the absorbance was measured at 700 nm and compared with standards. Increased absorbance of the reaction mixture indicated increased reducing power.

Scavenging of Hydrogen peroxide:

The ability of the extract to scavenge hydrogen peroxide was determined according to the method of Ruch, Cheng and Klaunig (1989)²³. A solution of hydrogen peroxide (2 mmol/l) was prepared in phosphate buffer (pH 7.4). Extract of different concentration were added to hydrogen peroxide solution (0.6 ml). Absorbance of hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer without hydrogen peroxide. The percentage scavenging activity of hydrogen peroxide by extract was calculated.

Nitric oxide radical scavenging activity:

Nitric oxide was generated from nitroprusside and measured by the Griess reaction Marcocci (1994)²⁴. Sodium nitroprusside (5 mM) in phosphate–buffered saline (PBS) was mixed with 3.0 ml of differentconcentrations (25-1000 μg /ml) of the drugs dissolved inthe suitable solvent systems and incubated at 25^oC for150 min. The samples from the above were reacted withGriess reagent (1% sulphanilamide, 2% H₃PO₄ and 0.1%napthylethylenediamine dihydrochloride). Theabsorbance of the chromophore formed during thediazotization of nitrite with sulphanilamide andsubsequent coupling with napthylethylenediamine wasread at 546 nm and referred to the absorbance at standardsolutions of potassium nitrite, treated in the same waywith Griess reagent.

Hydroxy radical scavenging activity:

The scavenging capacity for hydroxyl radical was measured according to the modified method of Halliwell (1987)²⁵. The assay was performed by adding 0.1 ml of 1mM EDTA, 0.01 ml of 10 mM FeCl₃, 0.1 ml of 10 mM H₂O₂, 0.36 ml of 10 mM deoxyribose, 1.0 ml of different dilutions of the extract (25 – 1000 μg/ml) dissolved in distilled water, 0.33 ml of phosphate buffer (50 mM, pH 7.4) and 0.1 ml of ascorbic acid in sequence. The mixture was then incubated at 37 °C for 1 h. A 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of 10% TCA and 1.0 ml of 0.5% TBA (in 0.025M NaOH containing 0.025% BHA) to develop the pink chromogen measured at 532 nm.

RESULTS AND DISCUSSION:

Free Radical Scavenging Activity (DPPH Assay):

The DPPH test provided information about the activity of test compounds with a stable free radical. The stable radical DPPH has been widely used for the determination of primary antioxidant activity, that is, the free radical scavenging activities of pure antioxidant compounds, plant and fruit extracts and food materials. Antioxidant molecules can quench DPPH free radical and convert them to a colorless/bleached product i.e. 2,2- diphenyl-1-hydrazine or a substituted analogous hydrazine, resulting in decrease in absorbance at 517 nm band²⁶. Hence, the more rapidly the absorbance decreases, the more potent the antioxidant activity of the extract in terms of hydrogen donating capacity. Figure 1 and Table-1 shows the dose-response curve of DPPH radical scavenging activity of the EEBL, compared with ascorbic acid, as standard.

Fig.1. Free radical scavenging activity of EEBL and Ascorbic acid (n=3)

Table 1; Free radical scavenging activity of EEBL and ascorbic acid.

Concentration in µg/ml	EEBL (Mean±SEM) % INHIBITION	ASCORBIC ACID (Mean±SEM) % INHIBITION
25	40.94±0.8500	55.41±0.744
50	56.97±0.6571	65.24±0.970
100	63.46±3.3050	76.20±0.197
200	70.34±0.2040	83.53±0.677
400	77.08±1.1920	86.96±0.361
800	86.16±0.7090	92.47±0.563
1000	90.50±0.7050	98.40±0.597

From above result IC₅₀ values of EEBL and ascorbic acid are 22.55 µg/ml and 30.52 µg/ml respectively. Though the DPPH radical scavenging abilities of the extracts were less than those of ascorbic acid at 1000 μg/ ml, the study showed that the extracts have the proton-donating ability and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants.

Reducing Power Assay:

The reducing power of the extracts was measured by the direct electron donation in the reduction of [Fe (CN)₆]^3-to [Fe(CN)₆]^4-. The product was visualized by addition of free Fe³⁺ions after the reduction reaction, by forming the intense Prussian blue color complex, (Fe³⁺)₄[Fe²⁺(CN^-)₆]₃, and quantified by absorbance measurement at 700 nm. A direct correlation between antioxidant activity and reducing power of EEBL was observed. The reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking free radical chain by donating a hydrogen atom²⁷. The presence of reductants (i.e. antioxidants) in the plant extract causes the reduction of the Fe³⁺/ferrocyanide complex to the ferrous form. Therefore, the Fe²⁺ can be monitored by measuring the formation of pearl’s Prussian blue at 700 nm. Figure 2 and Table-2 shows the reductive capabilities of the plant extract compared to ascorbic acid. The reducing power of EEBL was very potent and the power of the extract was increased with quality of sample.

Fig.2. Reducing power assay results of EEBL and Ascorbic acid (n=3)

Table 2; Reducing power assay results of EEBL and Ascorbic acid

Concentration in µg/ml	EEBL(Mean±SEM) % INHIBITION	ASCORBIC ACID (Mean±SEM) % INHIBITION
25	0.293±0.0023	0.418±0.0017
50	0.429±0.0050	0.591±0.0020
100	0.611±0.0040	0.709±0.0023
200	0.685±0.0025	0.778±0.0017
400	0.723±0.0028	0.813±0.0022
800	0.774±0.0051	0.848±0.0052
1000	0.818±0.0060	0.892±0.0054

Scavenvenging of Hydrogen Peroxide:

Hydrogen peroxide itself is not very reactive, but can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cells²⁸. Scavenging of H₂O₂ by extracts may be attributed to their phenolics, which can donate electrons to H₂O₂, thus neutralizing it to water. The EEBL was capable of scavenging hydrogen peroxide in a concentration-dependent manner. Figure 3 and Table-3 shows that MELC shows less scavenging activity (H₂O₂) than that of Ascorbic acid. The IC₅₀ value for scavenging of for EEBL 27.87 μg/ml while IC₅₀ value for ascorbic acid was 25.73 μg/ml.

Fig.3. Hydrogen Peroxide Scavenging Activity of EEBL and Ascorbic acid (n=3)

Table 3; Hydrogen Peroxide Scavenging Activity of EEBL and Ascorbic acid

Concentration in µg/ml	EEBL (Mean±SEM) % INHIBITION	ASCORBIC ACID (Mean±SEM) % INHIBITION
25	44.84±1.0040	48.58±0.60680
50	55.70±0.3454	58.96±0.89400
100	67.04±1.5410	70.68±0.30510
200	79.00±0.6108	83.65±1.30300
400	84.20±1.6860	88.55±0.16600
800	90.36±0.5106	94.42±0.96000
1000	93.68±0.9350	98.05±0.71050

Nitric Oxide Scavenging Activity

Nitric oxide or reactive nitrogen species, formed during their reaction with oxygen or with superoxides, such as nitrogen dioxide, dinitrogen trioxide, peroxynitrite, etc. are very reactive species. These compounds are responsible for altering the structural and functional behavior of cellular components, they are also implicated for inflammation, cancer and other pathological conditions²⁹. Therefore, EEBL was evaluated for nitric oxide scavenging activity. From Figure 4 and Table 4, EEBL showed moderately good nitric oxide scavenging activity between 25 and 1000 μg/ml. The percentages of inhibitions were increased with increasing concentration of the extracts. IC₅₀ value for scavenging of nitric oxide for EEBL was 236.85 μg/ ml while IC₅₀ value for ascorbic acid was 208.33 μg/ ml.

Fig.4. Nitric Oxide Scavenging Activity of EEBL and Ascorbic acid (n=3)

Table 4. Nitric Oxide Scavenging Activity of EEBL and Ascorbic acid

Concentration in µg/ml	EEBL(Mean±SEM) % INHIBITION	ASCORBIC ACID (Mean±SEM) % INHIBITION
25	21.53±0.0670	25.45±0.103
50	33.29±0.0689	35.96±0.103
100	39.96±0.1030	41.53±0.854
200	42.22±0.1030	48.00±0.103
400	61.65±0.4520	66.31±0.178
800	70.59±0.5870	75.86±0.890
1000	86.59±0.9870	91.52±0.542

Hydroxy radical scavenging activity:

The hydroxyl radical is extremely reactive in biological systems and has been implicated as highly damaging species in free radical pathology, capable of damaging biomolecules of living cells. Hydroxyl radical produced in the body is very reactive and one of the strongest oxidizing agents that reacts with almost all biomolecules found in living cells at a high rate constant, and is involved in many pathophysiological processes including DNA strand breakage and K⁺ loss from the cell membrane³⁰. In the present investigation from Figure 5 and Table 5, the extract inhibited ^.OH radical generation as can be seen with IC₅₀values 103.71 μg/ml and 95.42 μg/ml for EEBL and Ascorbic acid respectively.

Fig. 5. Hydroxyl Radical Scavenging Activity of EEBL and Ascorbic acid (n=3)

Table 5; Hydroxyl Radical Scavenging Activity of EEBL and Ascorbic acid

Concentration in µg/ml	EEBL(Mean±SEM) % INHIBITION	ASCORBIC ACID (Mean±SEM) % INHIBITION
25	15.25±0.111	19.93±0.981
50	22.46±0.176	27.80±0.021
100	48.21±0.121	52.40±0.328
200	53.10±0.687	61.05±0.212
400	62.30±0.985	67.66±0.546
800	69.17±0.185	73.28±0.892
1000	82.17±0.912	89.90±0.654

CONCLUSION:

The results obtained in the present study clearly demonstrate that the leaf extract under study contain certain antioxidant compounds, which can effectively scavenge various reactive oxygen species/free radicals under in-vitro conditions. However, the components responsible for the antioxidative activity are currently unclear. Therefore, further investigations need to be carried out to isolate and identify the antioxidant compounds present in the plant extract. Furthermore, the in-vivo antioxidant activity of this extract needs to be assessed prior to clinical use. Plant secondary metabolites are characterized by an enormous chemical diversity currently one-fourth of all prescribed pharmaceuticals compounds in developed countries are directly or indirectly (semi-synthetic) derived from plants. As plants produce a huge amount of antioxidants they can represent a source of new compounds with antioxidant activities

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Received on 06.02.2011 Modified on 08.03.2011

Research J. Pharm. and Tech. 4(6): June 2011; Page 920-924