Collection of sample: The two varieties of fresh strawberries were collected from the field of Bangladesh Agricultural Research Institute (BARI), Pahartali, Chittagong, Bangladesh on February 2010 and used for the anti-microbial study and cytotoxic activity.
Procedure of nutrient analysis: The moisture and ash content of two varieties of strawberry were carried out by the standard methods. The other nutrient contents were determined by using the established reported methods, i.e., total sugar and starch content by the anthrone method, reducing sugar by dinitrosalicylic acid method, non-reducing sugar by the methods of Ranganna. The water soluble protein of two varieties of strawberry were determined by the method of Folin-Lowry and Total protein by Micro-Kjeldahl method by using 6.25 factor to calculate protein content from nitrogen content. Vitamin C content of strawberry was determined by the standard method while beta-carotene content was determined according to the procedure reported in the methods of vitamin assay. Lipid content of strawberry was determined by the method of Bligh and Dyer.
Determination of mineral content: The minerals present in the two varieties of strawberry were analyzed by the procedure as described in the Analytical methods.
Statistical analysis: The results obtained from the analysis were subjected to statistical analysis using the Statistical Package for Social Sciences (SPSS) Version 15. Means were used for the analysis of the result.
Preparation of extracts for anti-microbial and cytotoxic study: Three grams of two varieties of fresh strawberry were taken separately in a mortar and pasted well. After that 8-10 mL of sterile de-ionized distilled water was added to each sample and homogenized well. Homogenized sample was filtered by double layered muslin cloth. The mixture was then centrifuged at 2,000 rpm for 10 min at 4°C. The clear supernatant was transferred into a beaker and used for anti-microbial as well as cytotoxic study.
Biological screening
Bacteria and growth conditions: Three bacterial species, one gram positive and two Gram negative, such as Bacillus subtilis and Escherichia coli and Salmonella typhi, respectively were used as test organisms. Exactly 0.2 mL of overnight cultures of each organism was inoculated into 20 mL of sterile nutrient broth and inoculated for 3-5 h to standardize the culture to 108 cfu mL-1. Each of suspension was then transferred to the plate containing nutrient agar media.
Antimicrobial assay: The disc diffusion method was used to test anti-bacterial activity. Dried and sterilized filter paper discs (5 mm diameter) were soaked with known amounts of aqueous solution of two varieties of strawberry using micropipette. Discs containing the test materials were placed on Nutrient agar medium uniformly seeded with the test microorganisms. Standard antibiotic disc Amoxicilin (0.05 mg disc-1) and blank discs (soaked with distilled water) were used as a positive and negative control, respectively. These plates were then kept at low temperature in refrigerator (4°C) for 12 h to allow maximum diffusion of the compound present in strawberry samples in agar media before any growth of the organisms. The plates were then incubated at 37°C for 24 h to allow maximum growth of the microorganisms and a clear distinct zone of inhibition was visualized surround the medium. The antibacterial activity of the test samples were determined by measuring the diameter of zone of inhibition expressed in millimeter.
Cytotoxicity study: Brine shrimp lethality bioassay technique was applied for the determination of cytotoxic properties of aqueous extract of two varieties of strawberry.
Preparation of sea water: Thirty eight grams of NaCL was weighted, dissolved in one liter of distilled water and finally filtered through Whatman No. 1 filter paper.
Hatching of brine shrimp of eggs: Sea water was taken in a small tank and shrimp eggs were added to one side of the divided tank which was covered. The shrimps were allowed for 48 h to hatch and matured as shrimp larvae (nauplii).Then the hatched nauplii were harvested in a clean beaker.
Application of the test solution and nauplii in the vials: At room temperature 12.5, 25, 50, 100, 150 and 200 μL of the two varieties of the aqueous strawberry extracts were taken in the vials and 5 mL of the sea water was added to each vial containing 10 brine shrimp nauplii. So, the concentration of the sample in the vials were 750, 1,500, 3,000, 6,000, 9,000 and 12,000 μg mL-1, respectively. These vials were used for each concentration and 5 mL sea water containing 10 nauplii were used as negative control.
Counting of the nauplii: After 24 h, the vials were observed using a magnifying glass and the number of survived nauplii in each vial were counted and noted. From this data, the percentage of mortality of the nauplii was calculated for each concentration.