In vitro antioxidant , total phenolic , membrane stabilizing and antimicrobial activity of Allamanda cathartica L . : A medicinal plant of Bangladesh

The methanol extract of the leaf of Allamanda cathartica L. as well as its hexane, carbon tetrachloride, chloroform and aqueous soluble partitionates were subjected to screening for antioxidant, membrane stabilizing and antimicrobial activities. The antioxidant potential was evaluated by 1,1-diphenyl-2picrylhydrazyl (DPPH) and Folin-Ciocalteau reagents using butylated hydroxytolune (BHT) and ascorbic acid as standards. The carbon tetrachloride soluble fraction revealed the highest free radical scavenging activity (IC50 = 47.5±0.11 μg/ml) which could be correlated to its total phenolic content of 59.31±0.47 mg of gallic acid equivalent (GAE)/g of extractives. In hypotonic solution and heat induced conditions, the aqueous soluble fraction inhibited haemolysis of human erythrocyte by 69.49±0.49 and 40.0±0.75%, respectively. Here, acetyl salicylic acid (0.1 mg/ml) was used as reference showing 72.79 and 42.12% of haemolysis of red blood cells (RBCs) in hypotonic solution and heat induced conditions, respectively. The carbon tetrachloride soluble fraction of A. cathartica demonstrated activity against microbial growth with zone of inhibition ranging from 5.0 to 8.5 mm. This fraction demonstrated 8.5 mm zone of inhibition against Bacillus megaterium.


Allamanda cathartica L. (Synonyms: Echites verticillata
Sessé & Moç, Orelia grandiflora Aublet, Allamanda grandiflora (Aublet) Poiret in Lam, Allamanda hendersonii W. Bull ex Dombrain.;Bengali name: Ghanta phul) commonly known as Golden Trumpet, Yellow Bell or Buttercup Flower, is a perennial shrub that can grow up to a height of 15 feet tall or more.The plant is native to Brazil, but widely cultivated throughout the tropics.A. cathartica is primarily used as an ornamental plant.The plant is used to relieve coughs and to clear the nasal passages.The leaves are also made into decoctions for use as a purgative.This plant has anti-bacterial and anticancer properties.It is also widely used in the treatment of jaundice.The root and stem of this plant contain two rare lactones which are active against polio virus and pathogenic fungi.Root is also used in various formulations to treat malarial symptoms.Sap is used to eliminate intestinal worms.The plant is also used as laxative and emetic (David, 1997).The leaves stem and branches of this plant are used against snake bite (Gomes et al., 2010).
As part of our ongoing investigations on medicinal plants of Bangladesh (Kaisar et al., 2011;Sharmin et al., 2012), the methanol extract of leaves of A. cathartica as well as its organic and aqueous soluble fractions were studied for the antioxidant potential in terms of total *Corresponding author.E-mail: sharminreza10@yahoo.com.phenolic content and free radical scavenging property, membrane stabilizing and antimicrobial activities for the first time and were reported, the results of our preliminary investigations.

Collection of plant and extraction
The leaves of A. cathartica were collected at their fully mature form in April 2011 from Mirpur Botanical Garden and a voucher specimen (DACB -36081) has been deposited in Bangladesh National Herbarium for future reference.
The collected plant materials were cleaned, sun dried and pulverized.The powdered plant material (700 g) was soaked in 2.0 L of methanol at room temperature for 7 days.The extract was filtered through fresh cotton bed and finally with Whatman filter paper number 1 and concentrated with a rotary evaporator at reduced temperature and pressure.An aliquot (5 g) of the concentrated methanol extract was fractionated by modified Kupchan (van Wagenen et al., 1993) partition protocol and the resultant partitionates were evaporated to dryness with rotary evaporator to yield hexane (HXSF, 1.0 g), carbon tetrachloride (CTCSF, 1.2 g), chloroform (CSF, 1.5 g) and aqueous (AQSF, 1.0 g) soluble materials.The residues were then stored in a refrigerator until further use.

Total phenolic content
The total phenolic content of the extractives was determined with Folin-Ciocalteau reagent by using the method developed by Harbertson and Spayd (2006).

DPPH free radical scavenging assay
Following the method developed by Brand-Williams et al. (1995), the antioxidant activity of the test samples was assessed by evaluating the scavenging activities of the stable 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical by using synthetic antioxidants, butylated hydroxytoluene (BHT) and ascorbic acid as positive controls.

Membrane stabilizing activity
The membrane stabilizing activity of the extractives was assessed by evaluating their ability to inhibit hypotonic solution and heat induced haemolysis of human erythrocytes following the method developed by Omale et al. (2008).

Antimicrobial screening
Antimicrobial activity was determined by disc diffusion method (Bayer et al., 1966).

Statistical analysis
For all bioassays, three replicates of each sample were used for statistical analysis and the values are reported as mean ± standard deviation (SD).

RESULTS AND DISCUSSION
The present study was undertaken to evaluate the antioxidant potential in terms of total phenolic content and free radical scavenging property, membrane stabilizing and antimicrobial activities of different organic and aqueous soluble materials of the methanol extract of A. cathartica leaves.
In DPPH free radical scavenging activity assay, all the fractions demonstrated mild to moderate free radical scavenging potential with IC 50 values ranging from 47.5 to 419.87 µg/ml.The highest free radical scavenging activity was demonstrated by the carbon tetrachloride soluble fraction (IC 50 = 47.5±0.11µg/ml) which could be correlated to its phenolic content 59.31±0.47mg of GAE/g of extractives (Table 1, Figures 1 and 2).
The membrane stabilizing activity of A. cathartica was also determined.All the extractives significantly protected the lysis of human erythrocyte membrane induced by hypotonic solution and heat induced conditions, as compared to the standard acetyl salicylic acid.In hypotonic solution and heat induced conditions, the aqueous soluble fraction inhibited 69.49±0.49and 40.00±0.75%haemolysis of RBCs, respectively as compared to 72.79 and 42.12% inhibition by acetyl salicylic acid (0.10 mg/ml), respectively (Table 2 and Figure 3).The antimicrobial activity of A. cathartica test samples was evaluated against five Gram positive and eight Gram negative bacteria and three fungi and the results were compared with standard antibiotic, Ciprofloxacin.The carbon tetrachloride soluble fraction displayed zone of inhibition ranging from 5.0 to 8.5 mm.This fraction revealed 8.5 mm zone of inhibition against Bacillus megaterium (Table 3).
It is clearly evident from the aforementioned findings that the test samples of A. cathartica have significant membrane stabilizing activity, mild to moderate antioxidant and weak antimicrobial potentials.Therefore, the plant is a good candidate for further systematic, chemical and biological studies to isolate the active principles.

Table 1 .
Total phenolic content and free radical scavenging activity of A. cathartica.

Table 2 .
Effect of different extractives of leaf of A. cathartica on hypotonic solution and heat induced haemolysis of erythrocyte membrane.