INTRODUCTION:

Huge amount of biologically active compounds are found in plant kingdom ^[1]. In the world about 50% of drugs used in medicine and 25% of prescribed drugs are of plant Origin^[2,
3]. Natural products and related drugs are used to treat 87% ofall categorized human diseases including bacterial infection,cancer and immunological disorders ^[4].

In aerobic life, undoubtedly, oxygen is an indispensable part. However, in some circumstances, through the formation of both free radical and non-free radical reactive oxygen species (ROS), it can seriously affect our well being ^{[5, 6]}. Antioxidants are added to a various foods to prevent or deter free radical induced lipid oxidation ^[7]. If free radical production rate exceeds the normal capacity of the antioxidant defense mechanisms, substantial tissue injury may occur ^[8].

The amount of free radicals increases due to continuous exposure to chemicals and contaminants and this causes irreversible oxidative damage including biological damage, DNA damage, diabetes, respiratory tract disorders, carcinogenesis and cellular degeneration related to aging ^[9,10]. Antioxidant status improvement plays an important role to minimize the oxidative damage ^[11]. In recent years, the interest in plant origin natural antioxidants has greatly increased as the possibility of toxicity of synthetic antioxidants has been criticized ^[12].

Trichosanthes dioica Roxb is an annual or perennial herb under the family Cucurbitaceae distributed in tropical Asia, Polynesia, and Australia. Over 20 species are recorded in India of which two are cultivated as vegetable. In Bangladesh the local name is patol and is one of the important vegetables ^[13]. The fruits and leaves are cooked in various ways either alone or in combination with other vegetables or meats and these are the edible parts of the plant ^[14]. It has been reported that the leave juice is used as tonic, febrifuge, and in subacute cases of enlargement of liver and spleen ^[15]. For the treatment of alcoholism and jaundice leaves and fruits are used and in edema and in alopecia only leaves are used ^[16]. Benincasa hispida is a plant under family cucurbitiaceae. It is probably a native of Japan and Java, it is also cultivated more or less throughout India and in warm countries. In Bangladesh locally it is known as Chichinga and is traditionally used as a laxative, diuretic, tonic. It is also used to control urinary discharges; removes foul taste from the mouth; heart tonic. From the Sanskrit texts it is known that it is useful in insanity, epilepsy, constipation, piles dyspepsia and other nervous diseases ^{[17, 18]}. Trichosanthes cucumerina linn. is also a plant under the family cucurbitiaceae and widely found in Asian countries including Bangladesh , where the local name of this is Chalkumra ^[19]. The whole plant including root, fruit, leaf, and seed contain medicinal values. T. cucumerina has anti-inflammatory and antidiabetic properties ^{[20, 21]}, the anti-inflammatory activity is mainly shown by aerial parts ^[22].The fruit pulp of T. cucumerina has the antioxidant activity ^[23]; both hepatoprotective ^[24] and gastroprotective ^{[25, 26]} activities are shown by the aerial parts of T. cucumerina. It can also act as a natural antibiotic, expectorant, laxative, cure constipation and jaundice^[27] and it is also used in the treatment of cold, bronchitis, cough and asthma ^[28]. Some components contained by the root and fruits of this plant are cytotoxic to some cancer cell lines ^[29]. Among the south Asian countries Bangladesh has a rich and prestigious heritage of herbal medicines, out of 500 species of medicinal plants about 250 species are used for the preparation of traditional medicines in Bangladesh but majority of these plants have not yet undergone chemical, pharmacological and toxicological studies to investigate their bioactive compounds ^[30]. Therefore much current research devoted to the phytochemical screening for different types of biological activity. As part of the endeavor for search of medicinal properties in local floristic resources we herein report a study of antioxidant and cytotoxic activity of the above mentioned three medicinal plants of Bangladesh.

MATERIALS AND METHODS:

Collection and Identification of Plant material:

The plants namely Trichosanthes dioica Roxb (Local name: Patol), Benincasa hispida (Local name: Chichinga), Trichosanthes cucumerina linn. (Local name: Chalkumra) were collected from Savar kancha Bazar, Dhaka and Bhoirab, Bramhanbaria, Bangladesh in December 2011 and was identified at the Bangladesh National Herbarium, Mirpur, Dhaka where the voucher specimen no: 33767 have been deposited for future reference. The specimen samples were kept in the Laboratory of Natural Products research in the Department of Pharmacy, Jahangirnagar University, Bangladesh.

Chemicals and drugs:

All chemicals and reagents used were of analytical grade. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) radical was obtained from Sigma Aldrich Co. (St. Louis, USA). Molisch reagent, sodium hydroxide and Fehling’s solution, Mayer’s reagent, Hagers reagent, Wagners reagent, Dragendroffs reagent, sulphuric acid, ferric chloride, concentrated HCl.

Drying and Pulverization:

The fresh leaves of the plants were first washed with water to remove adhering dirt and then cut into small pieces, sun dried for 4 days. After complete drying, the entire portions were pulverized into a coarse powder with the help of a grinding machine and were stored in an airtight container for further use. The powder was stored in an airtight container and kept in a cool, dark and dry place until analysis commenced.

Cold extraction:

From the three plants about 200 g of dried, ground plant material was taken in a clean, flat bottomed 3 glass container and soaked in 500 ml of 95% methanol. The container with its contents was sealed and kept for a period of 7 days accompanying occasional shaking and stirring. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton material. Then it was filtered through Whatman filter paper (Bibby RE200, Sterilin Ltd., UK).

Extraction with Methanol:

The concentrated methanol extract was made slurry with water. The slurry was taken in a separating funnel and few ml Methanol (50 ml) was added to the aqueous solution and the mass was shaken vigorously in a separating funnel. Then the funnel was allowed to stand for few minutes for the complete separation of the layers. The organic layer was collected. The process was repeated two times.

Extraction with petroleum ether:

Petroleum ether (50 ml) was added to the Methanolic aqueous solution and the mass was shaken vigorously in a separating funnel. Then the funnel was allowed to stand for few minutes for the complete separation of the layers. The organic (upper layer) layer was collected. The process was repeated two times.

The filtrate (methanol and petroleum ether extract) obtained was evaporated with rotary evaporator at 40⁰ C. The extract was transferred to a closed container for further use and protection.

Phytochemical screening:

Testing the presence of different chemical groups in the plant extract is the preliminary phytochemical studies. To identify the chemical constituents of plant extracts, standard procedures were followed. In each test 10% (w/v) solution of extract in methanol and Pet ether extracts were taken unless otherwise mentioned in individual test.

Determination of Carbohydrates:

Two drops of molisch’s reagents were added to about 5 mg of the extract in 5 ml aqueous solution in a test tube. 1 ml of conc. H₂SO₄ was allowed to flow down the side of the inclined test tube so that the acid formed a layer beneath the aqueous solution without mixing. A red ring was formed at the common surface of the two liquids which indicated the presence of carbohydrate. On standing or shaking a dark-purple solution was formed. Then the mixture was shaken and diluted with 5 ml of water. Dull violet precipitate was formed immediately.

Determination of Glycosides: A small amount of an alcoholic extract of the fresh or dried plant material was taken in 1ml of water. Then, a few drops of aqueous sodium hydroxide were added. A yellow color was considered as an indication for the presence of glycosides.

A small amount of an alcoholic extract of the plant material was taken in water and alcohol and boiled with Fehling’s solution. Brick-red precipitate was considered as an indication for the presence of glycosides.

Determination of Alkaloids:

Mayer’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Mayer’s reagent was added. Yellow color precipitate was formed and that was indicated as the presence of alkaloids.

Hager’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Hager’s reagent was added. Yellow crystalline precipitate was formed and that was indicated as the presence of alkaloids.

Wagner’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Wagner’s reagent was added. Brownish-black precipitate was formed and that indicates the presence of alkaloids.

Dragendroff’s test: 2 ml solution of the extract and 0.2 ml of dilute hydrochloric acid were taken in a test tube. Then 1 ml of Dragendroff’s reagent was added. Orange brown precipitate was formed and that indicates the presence of alkaloids.

Determination of Steroids:

Sulphuric acid test:

1 ml solution of chloroform extract was taken and then added1ml Sulphuric acid. Red color indicates the presence of steroid.

Determination of tannins:

Ferric Chloride Test: 0.5 ml solution of the extract was taken in a test tube. Then 1 ml of 5%Ferric chloride solution was added. Greenish black precipitate was formed and indicated the presence of tannins.

Determination of Flavonoids:

Added a few drops of concentrated hydrochloric acid to a small amount of an alcoholic extract of the plant material. Immediate development of a red color indicates the presence of flavonoids.

Determination of Saponins:

0.5 ml solution of the extract was diluted with distilled water to 20 ml and shaken in a graduated cylinder for 15 minutes. One centimeter layer of foam indicates the presence of saponins.

Determination of Antioxidant Potentials:

DPPH radical scavenging activity:

The free radical scavenging capacity of the extracts was determined using DPPH [31, 32]. The absorbance was read at 515 nm using a spectrophotometer. Ascorbic acid was used as a standard. The inhibition curve was plotted and IC₅₀ values were calculated.

Nitric oxide scavenging assay:

Nitric Oxide Scavenging assay was carried out according to the procedure of Alisi CS and Onyeze, 2008 ^[33]. The method is based on the generation of NO from sodium nitroprusside and subsequent estimation of nitrite ions using Griess reagent produced by the reaction of NO with oxygen in aqueous solution at physiological pH (7.2). In a test tube 4 ml of plant extract or standard of different concentrations were mixed with 1.0 ml of Sodium nitroprusside (5mM) solution. Then the test tube was incubated for 120 minutes at 30°C. After incubation 2 ml of solution was withdrawn from the mixture and mixed with 1.2 ml of griess reagent (1% Sulfanilamide in 5% H₃PO₄ and 0.1 % Naphthylethylene diamine dihydrochloride). Absorbance of the chromophore formed during diazotization of the nitrite with sulphanilamide and subsequent coupling with Naphthylethylenediamine dihydrochloride was measured at 550 nm using a spectrophotometer against a blank. The percent (%) inhibition of nitrite formation was calculated from the following equation.

{(A0 – A1)/A0} X 100

Where A0 is the absorbance of the Control and A1 is the absorbance of the extract or standard. The half maximal inhibitory concentration (IC50) was calculated by linear regression method.

Determination of total antioxidant capacity:

The determination of antioxidant capacity in the plant extract was assessed by the phosphomolybdenum method as described by Prieto et al., 1999 ^[34]. The method is based on the reduction of Mo (VI)–Mo (V) by means of the extract followed by the formation of a green phosphate/Mo (V) complex at acid pH. A 0.3 ml of crude extract and 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) were mixed together. The resulting solution was incubated at 95⁰C for 90 min. Then the absorbance of the solution was measured at 695 nm using UV spectrophotometer against a blank after cooling to room temperature. Chloroform or Pet. ether (0.3 ml) instead of plant extract is used as the blank. The antioxidant activity is expressed as the number of equivalents of ascorbic acid (AAE) and was calculated by the following formula:

A = (c x V)/m

Where A is the total content of antioxidant compounds, mg/g plant extract, in Ascorbic acid;

c represents the concentration of Ascorbic acid established from the calibration curve, mg/ml;

V is the volume of extract in ml and m is the weight of pure plant extract in g.

Determination of total phenolic content:

The content of total phenolic compounds in plant extracts was determined by Folin–Ciocalteu Reagent (FCR) using UV spectrophotometer (UV–1501PC SHIMADZU, Japan) described by the method McDonald et al., 2001 ^[35].0.5 ml of diluted plant extract and standard of different concentrations solution were taken in the test tube followed by adding 5 ml of Folin –ciocalteu (Diluted 10 fold with water) and 4 ml of Sodium carbonate (1 M) respectively. Solutions were then incubated for 15 minutes at 45⁰C in the water bath. The absorbance was measured colorimetrically at 765 nm to determine the total phenol content. Various concentrations of Gallic acid (25, 50, 100, and 200 μg/ml) were used to prepare the standard curve and the total content of Phenolic compounds in the crude extracts was calculated according to the following formula:

C = (c x V)/m

Where C is the total content of phenolic compounds in mg/g plant extract;

c is the concentration of Gallic acid established from the calibration curve in mg/ml;

V is the volume of extract in ml and m is the weight of Methanol or Pet. ether extract in g. The value of total content of phenolic compounds is expressed as GAE (Gallic Acid Equivalent) in mg/g extract ^[36].

Determination of total flavonoids content:

The total flavonoid in the crude extracts was measured using the Aluminum Chloride Colorimetric Method as described by Kumaran and karunakaran, 2007 ^[37]. To 1 ml of plant extract or standard of different concentrations 3 ml Methanol or Pet. ether, 0.2 ml of 10% aluminum chloride, 0.2 ml potassium acetate (1M) and 5.6 ml of distilled water were added. Then the solution was incubated for 30 minutes at room temperature. The absorbance was measured at 415 nm using UV spectrophotometer against a blank. Standard curve was prepared using quercetin by dissolving it in Chloroform or Pet. ether followed by serial dilution to 25, 50, 100, 200 μg/ml.

Reducing power:

The reducing power was determined according to the method previously described by Oyaizu, 1986 ^[38]. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The presence of reductants such as antioxidant substances in the antioxidant samples causes the reduction of the Fe3+/Ferricyanide complex to the ferrous form. Therefore, Fe2+ can be monitored by measuring the formation of Perl’s Prussian blue spectrophotometrically ^[39].To 1 ml of crude extract 2.5 ml phosphate buffer (0.2 M, pH 7.0) and 2.5 ml of potassium Ferricyanide [K3Fe (CN6)] solution, 1 %( w/v) were added. The resultant mixture was incubated for 30 minutes at 50⁰C to complete the reaction and added 2.5 ml (10% w/v) trichloroacetic acid. Then the total mixture was centrifuged at 1800 rpm for 10 min. Further 2.5 ml of the supernatant solution was withdrawn from the mixture and mix with 2.5 ml of distilled water and added 0.5 ml (0.1% w/v) FeCl₃ solution. The absorbance of the solution was then measured at 700 nm using a spectrophotometer against blank. Ascorbic acid and phosphate buffer were used as the standard and blank respectively. Increased absorbance of the reaction mixture represents increased reducing power.

Determination of Cytotoxicity Potential:

Brine shrimp lethality bioassay was used to determine the cytotoxic activity of the plant extract ^[40]. It is a recent development in the assay procedure of bioactive compounds, which indicates cytotoxicity as well as a wide range of pharmacological activities (e.g. anticancer, antiviral, insecticidal, pesticidal etc.) of the compounds ^[41]. The assay is considered as a useful tool for preliminary assessment of toxicity and it has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacterial toxins, pesticides and Cytotoxicity testing of dental materials.

The eggs of Brine Shrimp ware hatched in a tank at a temperature around 37⁰C with constant oxygen supply. Two days were allowed to hatch and mature the nauplii. Stock solution of the sample was prepared by dissolving required amount of extract in specific volume of pure dimethyl sulfoxide (DMSO). With the help of a pasteur pipette nauplii were exposed to different concentrations of the extracts.

Preparation of test groups:

All the test samples were taken and dissolved in 200 µl of pure dimethyl sulfoxide (DMSO) in vials to get stock solutions. Then 100 µl of solution was taken in test tube each containing 5ml of simulated seawater and 10 shrimp nauplii. Thus, final concentration of the prepared solution in the first test tube was 400 µg/ml. Then a series of solutions of varying concentrations were prepared from the stock solution by serial dilution method. In each case 100 µl sample was added to test tube and fresh 100µl DMSO was added to vial. Thus the concentrations of the obtained solution in each test tube were as-500 µg/ml,200 µg/ml,100 µg/ml,50 µg/ml,20 µg/ml,10 µg/ml,5 µg/ml,1 µg/ml.

Counting of nauplii:

The test tubes were kept at room temperature for 24 hours, numbers of nauplii were counted and the results were noted. From this, the percentage of mortality of brine shrimp nauplii was calculated at each probit analysis method as the measure of concentration for each sample. The median lethal concentration (LC₅₀) and 95% confidence interval was determined using toxicity of the plant extract.

RESULTS:

Phytochemical screening:

The crude extract was subjected for chemical group tests and identified various types of important chemical constituents. Results of different group tests are given in table 1.

From the results it is observed that carbohydrates are present in every extract except PM but flavonoids are found only in PM; alkaloids are absent in CE and CHE; Steroids, tannins and saponins are present in three extracts; but glycosides are absent in all the extracts.

DPPH radical scavenging activity:

The results of DPPH free radical scavenging activity on the two crude extracts of three plants and of ascorbic acid (standard) are shown in Table 2, where maximum radical scavenging activity (IC₅₀ value 858.41 μg/ml) was shown by Trichosanthes cucumerina linn. methanolic extract (CHM) and The minimum radical scavenging activity (IC50 value 18.822 μg/ml) was shown by Trichosanthes dioica Roxb pet ether extracts (PE), while the IC50 value of Ascorbic Acid was 15.707 μg/ml, which is a well known antioxidant. DPPH radical scavenging activity of the plant extracts are arranged in the following descending order: CHM > CM > PM for Methanol extracts and CE > CHE >PE for Pet ether extracts respectively. The DPPH radical contains an odd electron, which is responsible for the absorbance at 515-517 nm and also for a visible deep purple color.

Nitric oxide scavenging assay:

The different extracts of the different plants exhibited dose dependent scavenging of nitric oxide (Table 2) with an IC₅₀ value of (2.588, 0.6311μg/ml); (37.30, 1021.9μg/ml); (0.0256, 0.1078 μg/ml respectively for the Methanol and Pet Ether extracts of Trichosanthes cucumerina linn (PM,PE), Benincasa hispida (CM,CE), Trichosanthes cucumerina linn. (CHM, CHE) plants compared to 82.642 μg/ ml which was the IC₅₀ value for the reference ascorbic acid.

The highest NO scavenging activity was shown by Benincasa hispida pet ether extracts (CE) and lowest by Trichosanthes cucumerina linn. Methanol extracts (CHM).

Table 2: DPPH radical and NO scavenging activity

Name of the Extracts	IC₅₀ (µg/ml)
Name of the Extracts	DPPH Method	NO Method
PM	386.22±24.01235	2.588± 0.0247
PE	18.822± 0.07320	0.6311± 0.1793
CM	566.26±11.00271	37.30± 4.35701
CE	782.43±5.491461	1021.9± 16.235
CHM	858.41±30.12729	0.0256± 0.0036
CHE	552.83±3.05438	0.1078± 0.0722
Ascorbic acid	15.707± 1.1812	82.642± 1.040

Data are expressed as Mean ± SD of triplicate experiments

Total antioxidant activity:

Table 3 represents the total antioxidant capacities of the studied plant extracts and expressed as an ascorbic acid equivalents where PE showed the highest antioxidant capacity (873.3mg ascorbic acid/g plant extract) while the cheapest activity was found to be (352.85mg ascorbic acid/g plant extract) for CHM.

Table 3: Total antioxidant activity

Name of the Extracts	No. of ascorbic acid equivalents (mg/g)
PM	785.1±14.00071
PE	873.3±60.38692
CM	543.3±208.8793
CE	734±26.02153
CHM	352.85±50.27529
CHE	594.5±101.1163

Data are expressed as Mean ± SD of triplicate experiments

Total Phenol and Total flavonoids:

The results of the phenol and flavonoid content of crude extracts are given in Table 4. The total phenolic contents of plant extracts was determined using the Folin–Ciocalteu assay and was expressed as Gallic acid equivalents (GAE). The phenolic content of the studied plant extracts was varied from 49.375 to 259.375 mg/g of the dry weight. Total phenolic content of the plant extracts is arranged in the following descending order: PM > CE > CHM > CM > PE > CHE. On the other hand the total flavonoid content of the extracts was evaluated by aluminum colorimetric assay in which quercetin was used as an internal standard where the total flavonoid content ranged from 14 to 52.95 mg/g. Total flavonoid content of the plant extracts is arranged in the following succession: PM > CE > CM > PE > CHM > CHE. The Total flavonoid content of Trichosanthes cucumerina linn is lowest both for Methanol and Pet ether extracts.

Table -4: Total Phenol and Total flavonoids content

Name of the Extracts	Total Phenol mg/g	Total Flavonoid mg/g
PM	259.375±61.87184	52.95±0.212132
PE	71.25±4.419417	30 ±0.0019
CM	73.125±2.65165	37.15±0.0707
CE	84.6875±25.19068	39±0.00781
CHM	73.40625±3.049398	22±0.00534
CHE	49.375±0.883883	14±0.01423

Data are expressed as Mean ± SD of triplicate experiments

Reducing power:

Figure 1 shows the reductive capabilities of the plant extracts compared to Ascorbic acid; determined using the potassium Ferricyanide reduction method. The reducing power the extracts were strong and dose dependent. The activity was found to increase with increasing concentration of the plant extracts and serves as a significant indicator of their potential antioxidant activity. However, PE displayed the highest reducing power.

Cytotoxicity Potential:

In the present study (Table 5, 6, 7), each of the test samples showed different mortality rates at different concentrations, the percentage mortality increased with an increase in concentration.

Table 5: Cytotoxicity potential of Trichosanthes dioica

Test solution	Conc. (µg/ml)	Log Conc.	% Mortality	LC₅₀
Methanol	1	0	0	3.005
	5	0.69897	0
	10	1	10
	20	1.30103	10
	50	1.69897	90
	100	2	40
	200	2.30103	60
	500	2.69897	100
Pet ether	1	0	0	3.73
	5	0.69897	10
	10	1	20
	20	1.30103	20
	50	1.69897	10
	100	2	20
	200	2.30103	80
	500	2.69897	100
Vincristine Sulphate	0.078125	-1.10721	20	0.66
	0.15625	-0.80618	30
	0.3125	-0.50515	40
	0.625	-0.20412	50
	1.25	0.09691	60
	2.5	0.39794	80
	5	0.69897	90
	10	1	100
	20	1.30103	100
	40	1.60206	100

Table 6: Cytotoxicity potential of Benincasa hispida

Test solution	Conc. (µg/ml)	Log Conc.	% Mortality	LC₅₀
Methanol	1	0	0	1591.1
	5	0.698970004	20
	10	1	90
	20	1.301029996	20
	50	1.698970004	10
	100	2	10
	200	2.301029996	30
	500	2.698970004	80
Pet ether	1	0	0	4.22
	5	0.698970004	0
	10	1	10
	20	1.301029996	0
	50	1.698970004	20
	100	2	10
	200	2.301029996	20
	500	2.698970004	100
Vincristine Sulphate	0.078125	-1.10721	20	0.66
	0.15625	-0.80618	30
	0.3125	-0.50515	40
	0.625	-0.20412	50
	1.25	0.09691	60
	2.5	0.39794	80
	5	0.69897	90
	10	1	100
	20	1.30103	100
	40	1.60206	100

The variation in results may be due to the difference in the amount and kind of cytotoxic substances (e.g. tannins, glycosides, steroids) present in the crude extracts. It was observed that five of the six extracts showed significant cytotoxicity against Brine Shrimp nauplii with LC₅₀ of methanolic extract of Trichosanthes cucumerina is 2.59 µg/ml, Trichosanthes dioica is 3.005 µg/ml and Benincasa hispida is 1591.1 µg/ml respectively.

And pet ether extract of Trichosanthes cucumerina is 4.86 µg/ml, Trichosanthes dioica is 3.73 µg/ml and Benincasa hispida is µg/ml respectively while the LC₅₀ of the reference drug Vincristine sulphate was 0.66 µg/ml.

Table 7: Cytotoxicity potential of Trichosanthes cucumerina

Test solution	Conc. (µg/ml)	Log Conc.	% Mortality	LC₅₀
Methanol	1	0	50	2.59
	5	0.698970004	50
	10	1	50
	20	1.301029996	100
	50	1.698970004	90
	100	2	100
	200	2.301029996	100
	500	2.698970004	100
Pet ether	1	0	10	4.86
	5	0.698970004	10
	10	1	10
	20	1.301029996	0
	50	1.698970004	50
	100	2	40
	200	2.301029996	60
	500	2.698970004	100
Vincristine Sulphate	0.078125	-1.10721	20	0.66
	0.15625	-0.80618	30
	0.3125	-0.50515	40
	0.625	-0.20412	50
	1.25	0.09691	60
	2.5	0.39794	80
	5	0.69897	90
	10	1	100
	20	1.30103	100
	40	1.60206	100

DISCUSSION:

It has been reported that the polyphenolic compounds, like flavonoids, tannins and phenolic acids, commonly found in plants and have multiple biological effects, including antioxidant activity ^[42-45].

For investigation of antioxidant potentials of the used plant extracts different in vitro antioxidant assays were used. Generally, extracts and/or compounds displaying high antioxidant activity by one method had good antioxidant activity by the other methods and likewise for compounds with low activity ^[46]. Because no single method can fully evaluate the total antioxidant capacity, different antioxidant compounds may work through different mechanisms. For this reason, multi-method approach is often used for studying the complex antioxidant activities; all methods did not give the same results for activity ^[47].Frankel and Warner ^{[48, 49]} suggested the antioxidant activity should be measured by using more than one method due to the limitations. In the present study the crude extracts showed different levels of antioxidant activity in different antioxidant techniques employed.

DPPH is a stable free radical potentially reactive with substance able to donate a hydrogen atom and thus useful to assess antioxidant activity of specific compounds of extracts ^[50].Various reports suggest that cysteine, glutathione, ascorbic acid, tocopherol, flavonoids, tannins, and aromatic amines by their hydrogen donating ability are reduce and decolorize DPPH ^[51].

Nitric oxide or reactive nitrogen species are very reactive which are responsible for altering the structural and functional behavior of many cellular components. In some physiological processes like smooth muscle relaxation, neuronal signaling, inhibition of platelet aggregation and regulation of cell mediated toxicity, Nitric oxide (NO) is a potent Pleiotropic mediator ^[52]. NO scavenging capacity of the extracts may help to arrest the chain of reactions initiated by excess generation of NO that are harmful to the human health ^[53]. In the present study the different extracts of the different plants exhibited dose dependent scavenging of nitric oxide, this may be due to the antioxidant principles in the extract, which compete with oxygen to react with nitric oxide thereby inhibiting the generation of nitrite.

The phosphomolybdenum method is an alternative to the methods already available for the evaluation of total antioxidant capacity. Moreover, it is a quantitative one, since the antioxidant activity is expressed as the number of equivalents of ascorbic acid ^[34]. Total antioxidant activities by the phosphomolybdenum method usually detect antioxidants such as ascorbic acid, some phenolics, a-tocopherol, and carotenoids. The assay is successfully used to quantify vitamin E in seeds and, being simple and independent of other antioxidant measurements commonly employed, it was decided to extend its application to plant extracts. The present study reveals that the antioxidant activity of the extracts is in the increasing trend with the increasing concentration of the plant extracts. The antioxidant activity of the plant extracts is mainly due to their redox properties, which play an important role in neutralizing free radicals, quenching singlet and triplet oxygen^{[34, 54]}.

Polyphenolic compounds (flavonoids, phenolic acids) found in plants have been reported to have multiple biological effects, including antioxidant activity ^[42-43]. Several studies have also revealed that the phenolic content in the plants are associated with their antioxidant activities, because of their redox properties, which allow them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers ^[55-57]. So it is assumed that the high phenolic content may be responsible for the free radical scavenging activity of the plant extracts.

Reducing power is a convenient and rapid screening method, which may serve as a significant indicator of its potential antioxidant activity ^[58]. It has been reported that there is a direct correlation between antioxidant capacity and reducing power of certain plant extracts ^[59]. The reducing properties are generally associated with the presence of reductones ^[60], which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom and may have great relevance in the prevention and treatment of diseases associated with oxidants or free radicals ^[61]. So the ferric reducing property of plant extracts (figure 1) implies that they are capable of donating hydrogen atom in a dose dependent manner.

The cytotoxicity bioassay against Artemia salina is a simple and inexpensive method to test cytotoxicity, to biodirect fractionation of natural products and as a predictor of antitumor and pesticidal activity ^[62]. The method allows the use of smaller quantity of the extracts and permits larger number of samples and dilutions within shorter time than using the original test vials ^[63]. In the present study the crude extracts showed prominent result in brine shrimp cytotoxicity assay. The inhibitory effect of the extracts might be due to the toxic compounds present in the active fraction that possess ovicidal and larvicidal properties. The metabolites either affected the embryonic development or slay the eggs ^[64]. So the cytotoxic effects of the plant extracts enunciate that it can be selected for further cell line assay because there is a correlation between cytotoxicity and activity against the brine shrimp nauplii using extracts ^{[64, 65]}.

CONCLUSION:

The observations found in the present study support this view that the medicinal plants possesses a wide variety of natural source for the discovery of natural-product pharmaceuticals and to be used as preventive agents in the pathogenesis of various diseases. Likewise detailed chemical studies for the purification and identification of the bioactive components followed by pharmacological investigations and toxicological assessment are still required to examine the mechanisms of action of these agents. In conclusion, both brine shrimp and antimicrobial bioassays should be used together to entirely identify the promising activity of plant crude extracts or compounds derived from them.

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Received on 30.06.2013 Modified on 07.07.2013

Research J. Pharm. and Tech. 6(9): September 2013; Page 1042-1050

Extracts	Chemical Groups
Extracts	Carbohydrates	Glycosides	Alkaloids	Steroids	Tannins	Flavonoids	Saponins
PM	-	-	+	-	-	+	+
CM	+	-	+	+	+	-	+
CHM	+	-	+	+	+	-	-
PE	+	-	+	-	-	-	-
CE	+	-	-	-	+	-	-
CHE	+	-	-	+	-	-	+