Antifungal Activity of Honey Samples from Khyber Pakhtunkhwa (Pakistan) as affected by Botanical Origin

The present study evaluated the antifungal activity of honey samples collected from different locations at Khyber Pakhtunkhwa (KPK, Pakistan). Disc diffusion method was used to test the antifungal potential of twenty-one (branded, unbranded, and natural comb) honey samples from the different botanical origins at different concentrations (undiluted, 10%, 30%, and 50%, w/v) against Candida albicans and Rhodotorula species. Branded, unbranded, and natural comb honey samples generate different inhibition zones (4-13 mm, 5-15 mm, and 8-17 mm) against Rhodotorula species. Candida albicans showed resistance for all tested honey samples. Minimum inhibitory concentration (MIC) against Candida albicans and Rhodotorula species were 53.33%-88.12% and 1.76%-90.22% for branded, 61.3% - 93.8% and 9.90% - 95.5% for unbranded, and 67.1%-96.8% and 6.39%-98.8% for natural comb honey. In conclusion, natural comb honey from Khyber Pakhtunkhwa may have antifungal therapeutic potential and could be a useful source for generating functional food.


InTRODUCTIOn
2][3][4][5] Honey contains 25 different carbohydrates, mainly glucose and fructose. 6,72][13] Using the agar well diffusion method, different concentrations of honey (10%, 25%, and 50% by mass per volume) showed antifungal activities against Penicillium crustosum, Penicillium expansum, Penicillium griseofulvum, Penicillium raistrickii, and Penicillium verrucosum. 14Honey inhibitory activity against microorganisms is due to hydrogen peroxide (H 2 O 2 ), released by the oxidase enzyme added by bees to nectar.Rates of H 2 O 2 production by glucose oxidase in honey vary greatly and increase disproportionately with different degrees of honey dilution.The rate of H 2 O 2 production per milliliter of the honey solution decreased at a higher concentration of honey. 15Furthermore, the antioxidants activity of honey protects wound tissues from oxygen radicals and may be produced by H 2 O 2 . 16Antimicrobial activity of the honey is due to its high acidity, and high osmotic concentration.Honey osmotic effect due to its high sugar content also inhibits microbial growth as the sugar molecules tie up water molecules and induce insufficient water for the microorganism's growth. 2,4,17,18 Daa regarding the antifungal activity of honey from various countries demonstrated different results, an aspect of accessibility of pollens or changing of location and types. 12,13,19 Nvertheless, up to date, no articles addressed the comparative assessment of antifungal activities of honey found in Khyber Pakhtunkhwa (KPK, Pakistan).The present study aimed to evaluate the antifungal activities of natural comb and farms honey samples from KPK (Pakistan).

Honey solution preparation
Sample solutions were prepared at different concentrations in distilled water (10%, 30%, and 50%, w/v), incubated at 37°C for 30 min using a shaking water bath in the absence of light.

Tested organisms and yeast strains
Candida albicans (American Type Culture Collection, ATCC Code 90028) and Rhodotorula sp.(PCSIR 001) were obtained from Food Microbiology Laboratory, PCSIR Laboratories Complex, Peshawar, Pakistan.

Preparation of inoculum suspension
Candida albicans and Rhodotorula sp. were maintained on Sabouraud dextrose agar (SDA; BioMerieux, Marcy 1Etoile, France) at 4°C.Subcultures of each species were achieved in the same media for 48 h at 35°C before each experiment.The stock of inoculum suspension was prepared in 5 mL of sterile saline water (0.85%).The suspension was accustomed to 0.5 McFarland turbidity standards.Dilution of the suspension was further sub-cultured on SDA to measure the quantity of cfu/mL.The adjusted inoculums were 1 x 107 cfu/mL

Antifungal activity
The antifungal activity of honey samples was evaluated using an agar disc diffusion method against tested organisms 20 .Fresh culture suspension (100 μL) of the tested microorganisms was spread on respective media Sabouraud dextrose agar (SDA) plates.The concentration of culture was 1×10 7 cfu/mL.For screening, sterile filter paper discs (5 mm diameter) were impregnated with 10 µL of honey equivalent to 0.1 mg of honey after being placed on the surface of inoculated media agar plates.The plate was placed at 4°C for 2 h before being incubated under a favorable condition at 37°C for 24 h.Around the disc, a clear inhibition zone (diameter in mm) indicates the antifungal activity of the honey.An equivalent amount of water was set up as controls.Honey samples were inoculated separately on standards nutrients media with no test organisms to evaluate the possible contamination.The results of all the samples were determined in triplicate with a calculated standard deviation.

Statistical Analysis
Means and standard deviations were calculated for three independent determinations for each variable using the SPSS program.

Minimum Inhibitory Concentration (MIC)
Minimum Inhibitory Concentration (MIC) determination was made by incorporating various honey dilutions (10%, 30%, 50%, w/v) into the nutrient broth to examine their competence against Candida albicans and Rhodotorula sp.Up to 0.2 mL of the cell suspension was inoculated into 4 mL volume of honey concentration in a test tube, while inoculation of 4 mL volume of nutrient broth with 0.2 mL of the cell suspension served as control.The optical density was determined and recorded in a spectrophotometer at 620 nm before incubation (T0), after which the cultures were incubated for 24 h in the dark at 37 °C with constant shaking to prevent adherence and clumping.After 24 h of incubation, the optical densities were again determined and recorded (T24).The optical density for each replicate at T0 was subtracted at determined using the formula: Percentage inhibition = 1 -(OD test/OD control) x 100.
Where the resulting measurement recorded a negative inhibition value (growth promotion), this was reported as stimulation using the formula: Percentage inhibition = (OD test/OD control) x 100

ReSUlTS
Antifungal activities of the branded, unbranded, and natural honey samples from Khyber Pakhtunkhwa (Pakistan) were studied.Undiluted and diluted honey samples (10%, 30%, and 50%, w/v) showed statistically significant antifungal activities against Rhodotorula sp.compared to Candida albicans (Table 1).The zone of inhibition of growth for Rhodotorula sp.surrounding branded honey samples (50%) was significantly larger (13 mm) for versatile honey  while significantly lower (4mm) for young's honey at 10% dilution (Table 1).All other branded samples showed moderate values against the Rhodotorula sp.In unbranded honey, the zone of inhibition at 50% dilution for big bee honey was the highest (15 mm) against Rhodotorula sp., while Bekerr honey (30%) exhibited the lowest zone of inhibition (5 mm).The remaining honey samples showed moderate antifungal activities (Table 2).In natural comb honey, big bee's honey (50% dilution) displayed the maximum zone of inhibition (17 mm), while sperkay honey (10% dilution) showed the minimum (8 mm) zone of inhibition against Rhodotorula sp.(Table 3).In contrast, Candida albicans showed significant resistance against all tested honey samples.

DISCUSSIOn
The incidence of fungal infections is growing worldwide. 12,13 he severe nature of the infections is due to their drug resistance efficiency.Due to lack of efficacy, side effect, and/or resistance related to the existing drugs, hive products such as honey have been rediscovered for antimicrobial actions.We evaluated various honeyfrom KPK (Pakistan) efficacy against clinically isolated Candida albicans and Rhodotorula sp.The result showed considerable variations.,22 High sugar concentration in honey leads to high osmolarity and thus produces antimicrobial potential.Previous data reported that undiluted honey might inhibit the growth of many species of Rhodotorula sp., but there was no effect against C. albicans. 19The MIC for branded, unbranded, and natural honey samples were comparatively assessed, and the result revealed thatMIC for natural honey was significantly lower (67.19%-96.83%)and (6.39%-98.87%)against C. albicans and Rhodotrula sp., respectively.

COnClUSIOn
It could be concluded that the alterations in the antifungal activity of honey samples are directly related to their floral origin.The current study highlighted that natural honey might significantly inhibit the growth of a fungus and possibly be established as a topical antifungal agent.However, further research is needed to isolate and identify the active compounds from different honey samples of KPK and standardize their product for healthy living.

Table 2 .Table 3 .
: not observed; * Results of the average of three replicates ± Standard Deviation.Antifungal activities of unbranded honey samples against C. albicans and Rhodotorula sp Zone Diameter Inhibition (mm) at different dilution of honey : not observed; * Results of the average of three replicates ± Standard Deviation.Antifungal activities of natural honey samples against C.albicans and Rhodotorula sp.Zone Diameter Inhibition (mm) at different dilution of honey : not observed; * Results of the average of three replicates ± Standard Deviation.Ur Rahman et al. | J Pure Appl Microbiol | 16(2):1147-1153 | June 2022 | https://doi.org/10.22207/JPAM.16.2.42

Table 1 .
Antifungal activities of branded honey samples against C. albicans

Table 4 .
Percent of growth inhibition (MIC) of C. albicans and Rhodotorula sp. by branded honey samples

Table 6 .
Percent of growth inhibition (MIC) of C. albicans and Rhodotorula sp. by natural honey samples