MATERIALS AND METHODS

Materials: Five unifloral honey such as honey from Litchi (Litchi shinensis), Kadom (Anthocephalus cadmba), Til (Seasamum indicum ), Plum (Zizyphus mauritiana) and Mustard (Brassica campestris) flowers were collected from BCSIR Laboratory, Rajshahi, Bangladesh.

Production of Unifloral Honey: The honey, produced in honeycomb from nectar and pollen collected by bees from particular kinds of flower, is said to be the unifloral honey. The honey bee- box was placed at the center of the cultivated land of particular kinds of flowers. The bee′s flying range for collection of nectar and pollen were equivalent to the radius of the land. At that time there was no other crops surrounding the experimental plot. Thus the nectar and pollen were collected from that flowers to obtain unifloral honey (Mottalib, 2001).

Antibacterial screening:
Primary assay: The agar diffusion technique (Bauer et al., 1966) was employed to conduct antimicrobial testing. Five pathogenic bacteria were selected for the test, three of them were gram negative and others were gram positive. The test organisms (Table 1) were collected from the Department of Microbiology, University of Dhaka, Bangladesh.

Table 1:	List of pathogenic bacteria

Secondary assay: The lowest concentration of antimicrobial agents requires to inhibiting the growth of the organism in vitro is referred to the minimum inhibitory concentration (MIC). The serial dilution technique (Hammond et al., 1978) was followed using nutrient broth medium to determine the MIC value of the chemotherapeutic agent.

Solubility of the Antibacterial principle of honey in organic Solvents:
The honey was treated with a number of organic solvents of various polarities and the antibacterial activities were compared with the zone of inhibition thus produced. For this purpose petroleum ether (C2H5-O-C2H5), ethyl acetate (CH3COOC2H5) and chloroform (CHCl3) are used for extraction. The 10ml of honey was taken in a separating funnel and treated with an equal quantity of each of the organic solvents. The organic layer was collected and tested for antibacterial activity against five test organisms by disc-diffusion method (Bauer et al., 1966). Tetracycline disc (30μg) was used as a standard for comparison of the antibacterial activity.

Extraction of Antimicrobial compounds from the honey: The 10ml of honey was taken separately in two separating funnels. These were extracted with CHCl3 (3×10 ml). The CHCl3 fraction thus obtained was evaporated under reduced pressure in a rotavapour at 450C, until a solid mass was obtained. Thus antimicrobial principles were obtained.

RESULTS AND DISCUSSION

Antibacterial effect of crude honey: The antibacterial activities of the crude honey were determined at a concentration of 6.75μg disc^-1 against the tested organisms. The results are shown in Table 2. It was found that the entire test sample showed various positive response against all the tested organism. Fig. 1 showed the antibacterial activity of crude honey against pathogenic organism. The variation of the activity may be due to the different sources of the unifloral honey. Both honey and sugar are used with good effect as dressings for wounds and ulcers. The good control of infection is attributed to the high osmolarity, but honey can have the additional antibacterial activity because of its content of hydrogen peroxide (H₂O₂), lysozyme and other unidentified substances from certain floral sources.

Table 2:	Antibacterial activity of the five brands of unifloral honey against the test pathogenic organisms

Antibacterial effect of diluted honey: The antibacterial activity of the honey diluted with 50% water (3.375μg disc^-1). It was found that the entire test sample showed positive response (Table 3). Fig. 2 showed the antibacterial activity of diluted honey against pathogenic organism.

Table 3:	Antibacterial activity of the diluted honey (50% water) against the test pathogenic organisms

Table 4:	MIC values of the test unifloral honey against five pathogenic organisms

Table 5:	The MIC of the Litchi honey against Shigella dysenteriae
Cs = Test tube with sample; Ci = Test tube with inoculum; Cm = Test tube with medium; G = Growth; N.G. = No growth; Number of cells = 107 cell ml-1

Minimum inhibitory concentration (MIC): The minimum inhibitory concentration (MIC) of the unifloral honey was determined against five bacteria by serial dilution technique (Hammond et al., 1978).

The minimum inhibitory concentration of the honey from Litchi, Mustard, Plum, Til and Kadom against S. dysenteriae, S. typhi, E. coli, S. aureus and B. megaterium are shown in Table 4. Among the tested sample of honey, Kadom showed most prominent minimum inhibitory concentration. Fig. 3 showed the MIC of test sample against pathogenic organism. Specific gravity of different brand of honey are: Litchi, Mustard, Plum, Til and Kadom honey are 1.36, 1.35, 1.33, 1.35 and 1.34, respectively (Motalib, 2001).

The minimum inhibitory concentration (MIC) of the Litchi honey against Shigella dysenteriae is shown in Table 5.

Antibacterial screening of the extract: The antibacterial activity of the CHCl3, CH3COOC2H5 and C2H5-O-C2H5 extract were tested against S. dysenteriae, S. typhi, E. coli, S. aureus and B. megaterium and the results obtained were compared with that of standard antibiotic disc such as Erythromycin-15, Tetracycline-30 and Doxycycline-30.

The unifloral honey from Litchi, Mustard, Kadom, Plum and Til showed significant antibacterial activity against the test pathogenic bacteria. The test sample also showed antibacterial activity even diluted with distilled water.

Table 6:	Antibacterial activity of the chloroform extract (CHCl3) of different brands of unifloral honey and standard antibiotics

Fig. 1:	Antibacterial activity of crude honey against pathogenic organisms

Fig. 2:	Antibacterial activity of diluted honey (50 %) against pathogenic organisms

Fig. 3:	MIC of the test sample against pathogenic organisms

To determine the cause or responsible compound of honey for antibacterial activity, honey was extracted with petroleum ether (C₂H₂-O-C₂H₅), chloroform (CHCl₃) and ethyl acetate (CH₃COOC₂H₅), and were tested for antibacterial activity. In every case promising activity was observed. The research work is in progress.