INTRODUCTION:

Free radicals mediated oxidative stress plays a vital role in the occurrence of many diseases and systemic disorders in humans. Free radicals are unstable molecules generated during cellular metabolism.¹ Free radicals are basically listed in two broad groups, reactive oxygen species (ROS) and relative nitrogen species (RNS). The most common ROS species include, hydrogen peroxide (H₂O₂), peroxyl (ROO^-), superoxide anion (O₂^-) and reactive hydroxyl (OH^-). The nitrogen based free radicals are nitric acid (NO^-) and peroxynitrite anion (ONOO^-). ² Free radicals are highly active agents which initiate several degenerative processes in humans. ³ They react with a various biological molecules like proteins, DNA, cell membranes resulting in several health problems. Humans have a defense mechanism to control oxidative stress caused by the free radicals, however it is affected by factors like diet, age and health status of individual. ⁴

Excessive generations of free radicals may take over the antioxidant defense mechanism and cause various disease such as cancer, diabetes, cardiovascular disease, aging and metabolic syndrome. ⁵ Free radicals are also found to cause lipid peroxidation in food and leads to their deterioration. ⁶ Free radicals possess sever deleterious effects on human body and responsible for several chronic diseases. Free radicals posses some beneficial effects as well viz, activation of immune system and destroying the pathogenic microorganisms.⁷

Antioxidant therapy has gained an utmost important in the treatment of disease. Antioxidant compounds interfere with the oxidative stress by reacting with free radicals, chelating, catalytic metals and also by acting as oxygen scavengers.⁸ Synthetic antioxidants such as Propyl Galllate (PG), Tertiary Butylated Hydroxy Quinone (TBHQ), Butylated Hydroxy Toluene (BHT) and Butylated Hydroxy Anisole (BHA) are used to reduce rancidity in food items. ⁹These synthetic substances have been reported to cause several side effects like liver enlargement, reduce food intake, inhibited growth etc. Currently, scientists are directing towards the traditionally known plants for the development o natural antioxidant compounds. In recent past several traditionally known medicinal plants are reported to contain high amount of antioxidant compounds like flavonoids, phenols and vitamin A, C and E. these compounds could be used for therapeutic purposes against several diseases. ¹⁰

Barleria mysorensis is a xerophytic plant and belongs to the family Acanthaceae. It is mainly found in India and Sri Lanka and commonly known as Barleria. It got its genus name as Barleria from the place Barleria from where it was first identified. It is a under shrub, perennial, branched, stems are terete with simple thorns and leaves are subsessile, elliptic-ovate or orbicular. Flowers are solitary, axillary, bract spinescent. ¹¹ This plant is use as Febrifuge (lowering of temperature during fever), and relieving of stomach pain. Leaf decoction is given for the treatment of cough. ¹² Leaf paste is applied locally as an antidote for scorpion bite.¹³

Furcraea foetida is an evergreen perennial, subshrub and mostly grown as ornamental plant in the gardens. F. foetida belongs to the family of Agavaceae. It is widely distributed in Caribbean and South America and commonly known as the giant cabuaya or green-aloe or Mauritius-hemp. ¹⁴In the past few years Foetida sp. has been reported for the isolation of steroidal compound- saponin, furcreastatin.¹⁵ F. foetida possess anti-inflammatory effects, wound healing activity.¹⁶ F. foetida is used for treatment of uterus problems, hepatitis and stomach ache, rheumatism, paralysis and recommended as diuretic in case of oedema.^{17, 18}

The aim of this study was to assess aqueous extract of B. mysorensis and F. foetida for phytochemical composition and antioxidant activity.

MATERIALS AND METHODS:

Chemicals

DPPH (1, 1, Diphenyl-2-Picryl hydrazyl) was purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). Methanol, Ferric chloride (FeCl₃), Ferrozine, Folin-Ciocalteau reagent, Ascorbic acid and Gallic acid were purchased from SRL Pvt. Ltd. (Mumbai, India). Sodium Carbonate (Na₂CO₃) was purchased from Himedia Laboratories Pvt.Ltd. (Mumbai, India).

Plant material

B. mysorensis (Shrub, Acanthaceae) was collected from the natural population growing in Andhrahalli (13°00’32 N 77°28’51 E), Bengaluru, Karnataka, India, during September 2011 and F. foetida (Subshrub, Agavaceae) was collected from the VIT University campus, Katpadi (12°58’06N 79°09’20E), Vellore, Tamil Nadu, India, during August 2011. The plant material was carried to the Molecular and Microbiology Research Laboratory, VIT University. Herbarium was maintained in our laboratory for the future reference.

Processing of plants

The leaves of B. mysorensis and F. foetida were collected and then washed thoroughly in tap water followed by distilled water. The leaves were shade dried and uniformly grinded using mechanical grinder to make fine powder. The leaves powder (10% w/v) was soaked in sterilized distilled water and loaded on a shaker at a speed of 120 rpm for 24 hours at room temperature. Mixtures were filtered by using Whatman number 1 filter paper followed by centrifugation for 5 minutes at 3000 rpm. The extracts was concentrated and dried in lyophilizer. Dried extracts were collected in air tight container and stored at room temperature for further use.

Phytochemical screening

Phytochemical screenings of the aqueous extract of B. mysorensis and F. foetida leaves was carried out by using the standard protocols. ¹⁹Extracts were screened for tannins, saponins, alkaloids, carbohydrates, phenolic compounds, proteins, phytosterols, flavonoids, glycosides, oil and fats.

Antioxidant activity

DPPH radical scavenging activity

The aqueous extract of the B. mysorensis and F. foetida leaves was diluted in distilled water to make 20, 40, 60, 80, 100 µg/ml dilutions. Two milliliters of each dilution was thoroughly mixed with 1 ml of DPPH solution (0.2 mM/ml in methanol). The mixture was incubated in dark at 20° C for 40 minutes. Absorbance was measured at 517 nm using UV-Vis spectrophotometer. Ascorbic acid was used as a positive control. Each experiment was performed in triplicates at each concentration.^{20, 21}

The percentage scavenging of DPPH by the extracts was calculated according to the following formula:

%DPPH Radical scavenging= [(A_c-At)/A_c] ×100

Here,

A_c is the absorbance of the control (DPPH),

A_t is the absorbance of test sample.

Metal chelating activity

The aqueous extract of B. mysorensis and F. foetida leaves were diluted in distilled water to make 100, 200, 400 and 500µg/ml dilutions. Two ml of sample was mixed with 100 µl of FeCl₂(2mM in distilled water) and 400 µl of Ferrozine (5 mM in distilled water) was added and mixed thoroughly. The mixture was incubated in room temperature for 10 minutes. Absorbance was measured at 562 nm using UV-Vis spectrophotometer with distilled water as blank. Reaction mixture without extract was used as control. EDTA was used as a positive control. ²²

The percentage of metal chelating activity was calculated using following formula:

% metal chelating activity= [1 - (A_t/A_c)] × 100

Here,

A_t is the absorbance of test sample

A_c is the absorbance of control

Estimation of total phenolic content

Total phenolic content of the aqueous extract of the B. mysorensis and F. foetida was dertermined by using Folin-Ciocalteau reagent method.²³ A volume of 100 µl (containing 125, 250, 500 and 1000 µg extract) was mixed with 2.5 ml of Folin-Ciocalteau reagent (1/10 dilution in purified water) and 2 ml of 7.5% Na₂CO₃. The mixture was incubated at 45^oC for 15 minutes. Na₂CO₃solution (2 ml of 7.5% Na₂CO₃ in 2.60 ml of distilled water) was used as blank. The absorbance was measured at 765 nm using UV-Vis spectrophotometer. Each experiment was performed in triplicates at each concentration.

Statistical Analysis

The result of DPPH radical scavenging activity, metal chelating activity and total phenolic content of the aqueous extract of B. mysorensis and F. foetida leaves are reported as mean ± standard deviation of three replicates. Statistical analyses were performed by two-way ANOVA. Significant differences between groups were determined at P<0.05. Results were analyzed statically by using Microsoft Excel 2007 and GraphPad Prism 5.

RESULTS AND DISCUSSION:

Medicinal plants are an important source of therapeutically useful compounds. Traditional and folk medicinal system includes several plants and herbs for the treatment of several diseases. Herbal medicines have offers an enormous contribution to primary health care and have shown a great potential in modern phytomedicine against numerous ailments and the complex diseases and ailments to the modern science. Medicinal plants have been widely reported for antimicrobial activity, anticancer activity, hemolytic activity, anti-inflammatory activity, larvicidal activity, antidiabetic activity, anthelmintic activity, hepatoprotective activity and antioxidant activity etc. ^24-32 The above mentioned reports support the medicinal potential of the plants. Therefore in this study two medicinal plants, B. mysorensis and F. foetida were selected and screened for the presence of phytochemical groups and antioxidant activity by various in vitro methods.

The selection of B. mysorensis and F. foetida for this study was based on their uses in the traditional medicinal system, where B. mysorensis is notified for its relief in abdominal pain and bowel disorders and F. foetida for cure in case of paralysis, rheumatism, ulcers, and wounds. Limitations of the existing drugs in terms of their toxicity, the method of synthesis, their side effects and the variability in their dosage have lead scientists to develop new drugs from a reliable and alternative sources such as plants, algae etc. In our previous study, aqueous extract of B. mysorensis and F. foetida leaves were found to possess poor antimicrobial activity. ³³ Whereas, this study was designed to evaluate the antioxidant activity of aqueous extracts of the B. mysorensis and F. foetida.

Percentage yield

100 gm of fresh leaves of B. mysorensis and F. foetida were dried and extracted in distilled water to obtain the crude extract. B. mysorensis resulted in 0.71% and 7.79% yield with respect to fresh mass and dry weight respectively, whereas, F. foetida resulted in 8.6% and 11.14% yield with respect to fresh mass and dry weight respectively. Percentage yield of plant extracts are reported in Figure 1.

Figure 1: Percentage yield of plant extracts

Here, A1 and A2 are the percentage yield of B. mysorensis extracts with respect to fresh mass and dry weight respectively. B2 and B2 are the percentage yield of F. foetida extracts with respect to fresh mass and dry weight respectively.

Phytochemical analysis

Phytochemical are non nutritive plant chemicals that have protective or disease preventive properties. Phytochemicals such as tannins, saponins, carotenoids, phenolic compounds, flavonoids and alkaloids etc possess several medicinal properties and determined the medicinal potential of the plant. ^{34, 35} In this study, phytochemical analysis for B. mysorensis showed the presence of tannins, phenolic compounds, saponins, flavonoids and phytosterols while F. foetida showed the presence of tannins, phenolic compound, saponins, flavonoids, carbohydrate and phytosterols. Few phytochemicals such as Oil and fats, proteins and amino acids, alkaloid and glycosides were found to be absent in both B. mysorensis and F. foetida plants. in preliminary phytochemical screening, both plants were found to be rich source of phenolic compounds and flavonoids, which are well accepted group of antioxidant compounds. ^{36, 37} Results are summarized in Table 1.

Table 1: Phytochemical analysis of B. mysorensis and F. foetida

Phytochemical test	*Barleria mysorensis*	*Furcraea foetida*
Tannins	+	+
Phenolic compound	+	+
Saponins	+	+
Oil and fat	-	-
Protein & amino acids	-	-
Flavanoids	+	+
Carbohydrate	-	+
Alkaloids	-	-
Glycosides	-	-
Phytosterol	+	+

(+) present; (-) absent

Antioxidant activity

DPPH radical scavenging activity

Antioxidant potential of the aqueous extract of B. mysorensis and F. foetida was measured by DPPH radical scavenging activity in a dose dependant manner. When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then the odd electron of DPPH radical pairs with the hydrogen atom which this give rise to the reduced color formation from purple to yellow at 517 nm. ³⁸Among the tested plants, F. foetida (IC₅₀= 62.62 µg/ml) showed significantly higher (p < 0.05) DPPH radical scavenging activity than that of B. mysorensis (IC₅₀= 130.19 µg/ml). Both extracts showed dose dependent increase in the DPPH radical scavenging activity. The results of DPPH radical scavenging activity are represented as mean ± standard deviation of three replicates and expressed as percentage DPPH radical scavenging (Figure 2).

Figure 2: Percentage DPPH radical scavenging activity of varying concentrations of aqueous extract of B. mysorensis and F. foetida leaves. Data is represented as mean ± standard deviation (n = 3 test) with ascorbic acid equivalence.

Metal chelating activity

Metal chelating agents which serve as an secondary antioxidant as they reduce redox potential inhibit radical generations by stabilizing transition metals, consequently reducing free radical damage. ³⁹Metal chelating activity is in direct relation with the amount of phenolic compound present in the extracts hence it illuminates the antioxidant property. Among the tested plants, aqueous extract of F. foetida (IC₅₀ value 351.05 µg/ml) showed significantly higher (p < 0.05) metal chelating activity than that of B. mysorensis (IC₅₀ value 1824.62 µg/ml). Both extracts showed a dose dependent increase in the metal chelating activity. The results of metal chelating activity are represented as mean±standard deviation of three replicates and expressed as percentage of metal chelating and summarized in Figure 3.

Figure 3: Percentage metal chelating activity of varying concentrations of aqueous extract of B. mysorensis and F. foetida leaves. Data is represented as mean ± standard deviation (n = 3 test) with EDTA equivalence.

Total phenolic content

Phytochemicals such as carotenes, xanthophylls, tannins, flavonoids and phenolics compounds belong to diverse group of polyphenolic compounds. They protects the plants from oxidative stress are widely known for antioxidant potential. ⁴⁰ Antioxidant properties of polyphenols arise as hydrogen and electron donor. Radicals derived from polyphenols possess the ability to stabilize and delocalize the unpaired electron of free radical. ⁴¹Therapeutic use of these antioxidant compounds could help in controlling free radical mediated disorders viz, coronary heart disease, inflammation, stroke, diabetes mellitus and cancer. ⁴² Among all polyphenolic compounds, phenolic compounds are the most abundant and well documented group of antioxidant compounds. Therefore in this study, amount of total phenolic content in the aqueous extract of B. mysorensis and F. foetida was determined by the Folin-Ciocalteu reagent method. Both extract exhibited good amount of phenolic compounds, however aqueous extract of F. foetida exhibited significantly high phenolic content (p < 0.05). Results are reported as mean ± standard deviation of three replicates and expressed as gallic acid exultance (GAE) in μg. Both the extract showed dose dependent increase in total phenolic content (Figure 4).

Figure 4: Total Phenolic content in varying concentrations of aqueous extract of B. mysorensis and F. foetida leaves. Data is given in mean ± standard deviation (n = 3 test) and expressed as Gallic acid equivalence (GAE) in μg.

CONCLUSION:

The results obtained in this study justify the antioxidant effects of the aqueous extract of F. foetida and B. mysorensis. We conclude that F. foetida showed maximum antioxidant activity. However, further studies are necessary to isolate and reveal the active compounds contained in the aqueous extract of F. foetida and to establish the mechanisms of action.

ACKNOWLEDGEMENT:

Authors are thankful to the Staff and the Management of VIT University, Vellore, Tamil Nadu, India for providing necessary facilities and support for the successful completion of this work.

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Received on 25.10.2012 Modified on 10.11.2012

Research J. Pharm. and Tech. 5(12): Dec. 2012; Page 1503-1508