Research Article | Open Access
Khaliq Ur Rahman1 , Imdad Ullah2, Kamran Ullah3, Arshad Husain4, Shafqat Ullah4, Abdur Rauf1, Zainab Gul5, Halim Ur Rahman6 and Mohamed Fawzy Ramadan7,8*
1Department of Chemistry, University of Swabi, Anbar, Khyber Pakhtunkhwa, Pakistan.
2Department of Zoology, University of Swabi, Anbar, Khyber Pakhtunkhwa, Pakistan.
3Institute of Chemical Sciences, University of Peshawar Khyber Pakhtunkhwa, Pakistan.
4PCSIR Laboratories Complex Peshawar, Khyber Pakhtunkhwa, Pakistan.
5Institute of Physical Medicine and Rehabilitation, Khyber Medical University Peshawar, Pakistan.
6Department of Pharmacy, Riphah International University Islamabad, Pakistan.
7Deanship of Scientific Research, Umm Al-Qura University, Makkah, P. O. Box 715, Saudi Arabia.
8Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, Zagazig, Egypt.
J Pure Appl Microbiol. 2022;16(2):1147-1153 | Article Number: 7474
https://doi.org/10.22207/JPAM.16.2.42 | © The Author(s). 2022
Received: 09/12/2021 | Accepted: 31/03/2022 | Published online: 31/05/2022
Issue online: June 2022
Abstract

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.

Keywords

Bee products, biological activity, Candida albicans, Rhodotorula sp

Introduction

Honey is viscous nectar, collected by the honey bees, combined and converted with a definite substance, then stored to mature and ripen in honeycomb.1-5 Honey contains 25 different carbohydrates, mainly glucose and fructose.6,7 Before discovering bacteria, which is a causal agent of infection, honey has been for several wound infections treatment.4, 8-10

              Antifungal drugs are essential and encourage research on new natural product chemotherapeutic agents.11-13 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.14 Honey inhibitory activity against microorganisms is due to hydrogen peroxide (H2O2), released by the oxidase enzyme added by bees to nectar. Rates of H2O2 production by glucose oxidase in honey vary greatly and increase disproportionately with different degrees of honey dilution. The rate of H2O2 production per milliliter of the honey solution decreased at a higher concentration of honey.15 Furthermore, the antioxidants activity of honey protects wound tissues from oxygen radicals and may be produced by H2O2. 16 Antimicrobial 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 Data 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 Nevertheless, 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).

Materials and Methods

Samples collection
A total of twenty-one samples of honey, including natural honey beera (ziziphus), bekkarr (justice), granda (carissaopaca), palosa (acacia), sperkay (trachyspermum), big bees honey, and small bees honey, were directly collected from the honeycomb. In addition, branded honey samples (Al-Hayat, langnese, marhaba, paksalman, qarshi, versatile, young’s) and unbranded honey samples such as sperkay (trachyspermum), bekkarr (justice), beera, (ziziphus), granda (Carissa opaca), palosa (acacia), big and small bees honey were purchased from the local market at KPK. The samples were kept at 4°C for further analysis.

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 organisms20. Fresh culture suspension (100 μL) of the tested microorganisms was spread on respective media Sabouraud dextrose agar (SDA) plates. The concentration of culture was 1×107cfu/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.

Table (1):
Antifungal activities of branded honey samples against C. albicans and Rhodotorula sp.

Zone Diameter Inhibition (mm) at different dilution of honey
Test species Code Concentration Marhaba Qarshi Versatile Al-Hayat Young’s Paksalman Langness
C. albicans ATCC 90028 Undiluted
10%
30%
50%
Rhodotorula PCSIR 001 Undiluted * 08±0.01 06±0.02 07±0.01 09±0.10 07±0.02 05±0.04 06±0.02
10% 07±0.02 06±0.02 08±0.02 05±0.03 04±0.01 06±0.04 04±0.01
30% 08±0.02 07±0.04 09±0.02 06±0.01 06±0.03 07±0.07 05±0.03
50% 11±0.07 12±0.10 09±0.03 13±0.13 08±0.05 11±0.12 07±0.03

(-): not observed; * Results of the average of three replicates ± Standard Deviation.

Table (2):
Antifungal activities of unbranded honey samples against C. albicans and Rhodotorula sp.

Zone Diameter Inhibition (mm) at different dilution of honey
Test species Code Concen. Big bees
honey
Small bees
honey
Beera
(Ziziphus)
Palosa
(Acacia)
Sperkay
(Trachyspermum)
Bekerr
(Justicia)
Granda
(Carissa opaca)
C. albicans ATCC 90028 Undiluted
10%
30%
50%
Rhodotorula PCSIR 001 Undiluted *10±0.01 12±0.10 09±0.13 11±0.14 08±0.01 06±0.02 09±0.11
10% 07±0.06 08±0.04 07±0.07 08±0.10 06±0.01 07±0.05 07±0.06
30% 09±0.10 08±0.03 10±0.09 06±0.12 06±0.03 05±0.01 06±0.01
50% 15±0.14 14±0.18 11±0.04 15±0.18 9±0.07 13±0.13 10±0.08

(-): not observed; * Results of the average of three replicates ± Standard Deviation.

Table (3):
Antifungal activities of natural honey samples against C.albicans and Rhodotorula sp.

 Zone Diameter Inhibition (mm) at different dilution of honey
Test species Code Concen. Big bees
honey
Small bees
honey
Beera
(Ziziphus)
Palosa
(Acacia)
Sperkay
(Trachyspermum)
Bekerr
(Justicia)
Granda
(Carissa opaca)
C. albicans ATCC 90028 Undiluted
10%
30%
50%
Rhodotrula PCSIR 001 Undiluted *12±0.08 10±0.04 10±0.03 13±0.08 11±0.01 14±0.13 12±0.02
10% 09±0.06 10±0.07 09±0.09 11±0.15 08±0.04 10±0.10 09±0.07
30% 08±0.11 09±0.04 12±0.02 13±0.01 10±0.16 ND 09±0.10
50% 17±0.12 13±0.10 14±0.14 15±0.03 12±0.11 13±0.09 16±0.12

(-): not observed; * Results of the average of three replicates ± Standard Deviation.

The MIC of the tested branded honey sample was 53.33%-88.12%, and 11.76%-90.22% against Candida albicans and Rhodotorula sp., respectively (Table 4). MIC of the unbranded honey was 61.39%-93.88% for Candida albicans, and 9.90%- 95.55% against Rhodotorula sp. (Table 5). Natural comb honey exhibiting 67.19%-96.83% and 6.39%-98.87% MIC range against the two species of fungus (Candida albicans and Rhodotorula sp.), correspondingly (Table 6).

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

Honey sample Candida albicans Rhodotorula sp.
Concentration of sample Concentration of sample
Undiluted 50% (v/v) 30% (v/v) 10% (v/v) Undiluted 50%(v/v) 30%(v/v) 10%(v/v)
Marhaba 88.12 73.07 ≥100 ≥100 90.22 86.33 45.43 ≥100
Qarshi 84.03 75.33 ≥100 ≥100 88.26 85.16 36.62 ≥100
Versatile 81.44 77.42 ≥100 ≥100 83.21 80.17 27.13 ≥100
Al-hayat 76.34 72.31 ≥100 ≥100 81.47 76.21 22.55 ≥100
Young’s honey 73.32 68.51 ≥100 ≥100 77.35 62.41 19.66 ≥100
Pak-salman 70.21 64.55 ≥100 ≥100 72.61 65.10 16.22 ≥100
Langness 69.42 53.33 ≥100 ≥100 69.46 56.44 11.76 ≥100

MIC: Minimum Inhibitory Concentration.

Table (5):
Percent of growth inhibition (MIC) of C. albicans and Rhodotorula sp. by unbranded honey samples.

Honey sample Candida albicans Rhodotorula sp.
Concentration of sample Concentration of sample
Undiluted 50% (v/v) 30% (v/v) 10% (v/v) Undiluted 50%(v/v) 30%(v/v) 10%(v/v)
Big bees honey 93.88 89.45 ≥100 ≥100 95.55 93.08 54.46 ≥100
Small bees honey 90.74 85.22 ≥100 ≥100 94.87 95.55 44.22 ≥100
Beera 86.33 82.42 ≥100 ≥100 90.21 88.23 37.99 ≥100
Palosa 83.75 77.58 ≥100 ≥100 85.44 87.64 25.58 ≥100
Sperkay 78.90 70.69 ≥100 ≥100 81.73 78.37 19.21 ≥100
Bekerr 75.43 67.60 ≥100 ≥100 73.42 67.74 15.66 ≥100
Granda 73.47 61.39 ≥100 ≥100 70.77 69.33 9.90 ≥100

MIC: Minimum Inhibitory Concentration.

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

Honey sample Candida albicans Rhodotorula sp.
Concentration of sample Concentration of sample
Undiluted 50% (v/v) 30% (v/v) 10% (v/v) Undiluted 50%(v/v) 30%(v/v) 10%(v/v)
Big bees honey 96.83 92.43 ≥100 ≥100 98.87 91.21 57.76 ≥100
Small bees honey 91.44 88.57 ≥100 ≥100 95.43 89.55 48.33 ≥100
Beera 89.31 85.22 ≥100 ≥100 92.79 86.99 42.27 ≥100
Palosa 85.56 79.95 ≥100 ≥100 88.36 82.44 37.76 ≥100
Sperkay 83.78 74.39 ≥100 ≥100 83.43 77.21 31.65 ≥100
Bekerr 77.48 70.28 ≥100 ≥100 76.27 71.55 25.44 ≥100
Granda 75.66 67.19 ≥100 ≥100 72.77 64.45 6.39 ≥100
DISCUSSION

The incidence of fungal infections is growing worldwide.12, 13 The 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 honey from KPK (Pakistan) efficacy against clinically isolated Candida albicans and Rhodotorula sp. The result showed considerable variations. Our study showed that the inhibitory actions of branded, unbranded, and natural comb big bee’s honey samples (50% dilution) against Rhodotorula sp. was 13 mm (Alhayat), 15 mm (Palosa), and 17 mm (Big bees honey). Honey from different phytogeographic regions varies to inhibit yeast growth, suggesting the importance of botanical origin in displaying antifungal activity.21 In different kinds of honey, biological activity was attributed to their phenolic compounds, as their ability to denature proteins.12, 13, 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.19 The 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.

Declarations

ACKNOWLEDGMENTS
The authors would like to thank Food Technology Lab, PCSIR, Peshawar, Pakistan for providing the research facilities.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
KUR and KU designed the study, collected the data and wrote the manuscript. AR, AH, SU and ZG analyzed the data. IU and MFR revised the study. All others read and approved the final manuscript.

FUNDING
None.

ETHICS STATEMENT
This article does not contain any studies with human participants or animals performed by any of the authors.

AVAILABILITY OF DATA
All datasets generated or analyzed during this study are included in the manuscript.

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