Biochemical analysis, antioxidant, and antibacterial e ﬃ cacy of the bee propolis extract ( Hymenoptera: Apis mellifera ) against Staphylococcus aureus - induced infection in BALB/c mice : In vitro and in vivo study

Background ‒ Antibiotic resistance to Staphylococcal infections has prompted the pharmaceutical and scienti ﬁ c community to consider alternate treatments. Propolis is a natural substance produced by honey bees ( Hymenoptera: Apis mellifera ) from the exudates of di ﬀ erent plants. The aim of the current study was to evaluate the antibacterial activity of ethanolic extracts of Pakistani bee propolis ( PBP ) against Staphylococcus aureus in both in vitro and in vivo modeling. Methods ‒ The propolis sample was collected from the Kohat district and dried in the dark until processing. The antibacterial activity of the propolis extract was exam - ined using the agar well di ﬀ usion method. The S. aureus culture was incubated on Mueller – Hinton agar media. Five di ﬀ erent concentrations of propolis, 100, 200, 350, 500, and 650 μ g/ml, were used. Gentamicin disc was used as a positive control. For in vivo assay, BALB/c mice with an average weight of 30 g were purchased. Bacteria were inoculated into mice by the tape stripping technique. After abscess formation, mice were treated with propolis extract. Results ‒ The mean zone of inhibition and standard devia - tion for each concentration were 17 ± 0.816 at 650 μ g/ml, 14.6 ± 0.471 at 500 μ g/ml, 12 ± 1.41 at 300 μ g/ml, 9.6 ± 0.942 at 200 μ g/ml, and 2.3 ± 0.471 at 100 μ g/ml of the pro - polis extract against S. aureus . It was observed that by increasing the concentration of the propolis extract, the antibacterial and antioxidant activities also increased. The extracts showed less antibacterial potential compared to gentamicin. The abscess size was also decreased in mice groups treated with the propolis extract topically and orally in comparison with the untreated mice group. Conclusions ‒ To the author ’ s best knowledge, this study is the ﬁ rst attempt to demonstrate that an ethanolic PBP extract has antibacterial potential against S. aureus - induced infections.


Introduction
The Greek words, staphyle meaning "bunch of grapes" and coccus meaning "spherical bacteria," were combined to form the word staphylococci.The Latin word aureus meaning "gold" was applied to these bacteria because of their yellow to a yellowish-white colonial appearance on an enriched medium [1].A pathogenic member of the micrococcus family and the genus Staphylococcus, Staphylococcus aureus is a Gram-positive, non-motile, facultative anaerobe, and about 1 µm in size [2].Due to the formation of carotenoids and hemolysin, it produces golden colonies on a rich medium and hemolysis on blood agar containing 5% sheep and horse blood.However, because cell division occurs at various planes, it appears as bluish-grape-like colonies when stained with Gram stain [2].Humans' gastrointestinal tracts, nares, and skin are all colonized by S. aureus [3].About 20% of the human population has stable colonization, whereas the other 30% has varying levels of colonization [3,4].S. aureus is a very resilient organism that can endure dry surfaces for an extended amount of time.It is also resistant to desiccation and can withstand highlevel concentrations of salt, which serves as a basis for its selection of growth media over other bacteria [5].It is the causative agent of a wide range of infections in humans and animals with a significant impact on public health [6].Significant public health implications were brought about by host specialization, the capacity to acquire and lose virulence and resistance genes, and the possibility of zoonotic spread [6][7][8].Invasive diseases caused by S. aureus include bacteremia, sepsis, pneumonia, osteomyelitis, endocarditis, and skin and soft tissue infections [3].The development of purulent abscess lesions surrounding a nidus of the pathogen, predominantly as a result of neutrophil infiltration, is the pathological hallmark of S. aureus infection [9].It causes dermatitis in dogs, septicemia and arthritis in chickens, botryomycosis in horses, mastitis in cows, and botryomycosis in horses [6,10].Antimicrobial resistance is becoming more common all around the world.Pathogens that cause nosocomial infections have shown an increase in resistance and so have organisms that cause community-acquired diseases.In addition to the well-known pathogens, opportunistic microorganisms have developed resistance.Increased illness, mortality, and medical costs due to antimicrobial resistance highlight the need for novel antimicrobial agents [11].
Propolis is a mixture of beeswax and resins collected by the honeybees (Hymenoptera: Apis mellifera) from plant buds, leaves, and exudates [12].Bees use propolis to protect their hives against assault by other insects.The word "propolis" is derived from the Greek letters "pro" and "polis," which together indicate "town" or "city" [13].In addition to using it as a building material, bees use propolis to keep bacterial and fungal concentrations in the hive at low levels [14].Propolis serves as a biocide in the hive, active against invasive bacteria, fungi, and even invading larvae [15].The treatment of infections with propolis has a long history in traditional oriental medicine [15].As an antiseptic and anti-inflammatory agent for treating wounds and burns, propolis has long been used in traditional Chinese medicine to treat infections as well as in ethnopharmacology in Europe [16].Propolis has been well recognized as a beneficial ingredient in medicine due to its numerous pharmacological qualities, such as antifungal [17], antiviral [18], antioxidant [19], and antibacterial [20].Propolis samples have been shown to contain more than 150 different substances, including polyphenols, phenolic aldehydes, sesquiterpene quinines, amino acids, steroids, and inorganic substances.Propolis is an antioxidant-rich natural material that acts as a bodily defense against free radicals [15].Propolis has different characteristics and a different chemical makeup depending on where it comes from [17], and these variances in makeup are mostly caused by variations in the bearing plants [21].
As a result, the number of resistant strains is quickly increasing worldwide [22].In particular, given the increase in microorganisms that have evolved resistance to modern medicines, novel therapies for infectious disorders are urgently needed [23].In Pakistan's industrial apiaries, propolis is made alongside honey.Although it is scraped off from beehive walls and frames, it is discarded since it is deemed useless.Due to a dearth of studies into its many benefits, propolis is regarded as useless in Pakistan.To the author's best knowledge, there have been no studies conducted on the antibacterial potential (particularly, S. aureus) of Pakistani bee propolis (PBP) obtained from the current study area.Therefore, the objective of the current study was to assess the in vitro and in vivo antibacterial activity of PBP obtained from beehives in the Kohat region, against S. aureus-induced infections.

Sample collectiont
A sample of PBP was taken from beehives in the apiary of Kohat University of Science and Technology.The sample was manually collected, cut into small pieces, and kept in the dark until processing (Figure 1).

Extraction of propolis
The dried PBP sample was crumpled in an electric grinder.The ground PBP extract was diluted in an ethanol solvent in a ratio of 1:10 (1 g PBP/10 ml ethanol solvent) and kept in airtight bottles at 25°C for 14 days.The bottles were shaken briefly every 5 h.After 14 days, the solution was filtered via paper using a filter.By employing a rotary vacuum evaporator to evaporate the solvent, the filtrated extract was concentrated to obtain a crude extract.For experiments, the final crude extract was maintained at 4°C (Figure 2).

Study design
The in vitro activity of PBP was determined in the Lab of Molecular Parasitology and Virology, Department of Zoology, KUST.A culture of S. aureus was provided by the Department of Microbiology, and the BALB/c mice used in the in vivo study were provided by the Veterinary Research Institute, Peshawar.

Bacterial culture
The nutrient broth was primarily used to create bacterial suspensions.About 0.39 g of the nutrient broth powder was dissolved in 30 ml of distilled water.Then, 10 ml of the nutrient broth solution was added to three test tubes that were each 10 ml in size.Following that, 30 μl of the bacterial suspension from the stock culture was poured into two test tubes, and a third test tube was maintained as a control to be compared with the other test tubes to determine whether or not bacterial growth had taken place (Figure 3).Analysis of PBP against S. aureus-induced infection in BALB/c mice  3

Stock solution and dilution of propolis extracts
The Stock solution was prepared using 10 mg of the crude extract of PBP dissolved in 10 ml of ethanol.From this stock solution, concentrations of 100, 200, 350, 500, and 650 μg/ml were used for in vitro study.

In vitro activity
The agar well diffusion method was followed to determine the antibacterial activity of PBP against S. aureus.About 20 ml of the Mueller-Hinton agar medium was used to make test plates with a diameter of 10 cm.The medium was autoclaved along with Petri plates, loop, cork borer, tips and cotton swab, etc. at 121°C for 20 min to sterilize.The sterilized medium was poured into Petri dishes on cooling and shifted to the laminar flow hood under sterilized conditions.After the medium had solidified, using sterile glass-made pipettes connected to a vacuum pump, 8 mm diameter wells were punched in the agar plates.Sterile swabs were dipped into the bacterial suspension inoculated onto plate surfaces by rubbing it at 90°; again, the cotton swab was dipped in the bacterial culture and rubbed at 180°to make the bacterial culture lawn.Each well was filled with 30 μl of the extract.These plates were incubated for 24 h at 37°C.After the incubation period, the zones of bacterial growth inhibition around the holes were measured and recorded.Each of the antibacterial assays was conducted in triplicate and the mean of results were noted.A gentamicin antibacterial disc was used as a control during this study.

Mice model for in vivo activity
Thirty healthy BALB/c mice with an average weight of 30 g were purchased.All attempts were made to minimize the suffering of the mice during the trial.All mice were kept in vented plastic cages at a managed animal care facility, with a humidity of 50-60% and 12 h light/12 h dark cycles, with ample food and distilled water.

Preparation of propolis ointment for topical application
The ethanol extract of PBP was prepared as an ointment using petroleum jelly (melting point: 60-65°C) at a concentration of 50%.The ointment was kept in a sterile glass container, properly sealed, and preserved at 4°C for application.

Antibacterial agents
The commercial topical antibiotics (Effigenta: Cipro) were purchased from a local drugstore and it was used as a standard antibacterial drug in in vivo study.

Bacteria inoculation
Bacteria were inoculated into mice using the tape-stripping technique (Figure 4).Using a sterilized razor, mice's backs were shaved; then, small strips of elastic bandage tape were applied and removed from a 2 × 2 cm 2 dorsal area, until the skin glistened and reddened without bleeding.Then, 70% ethanol was swabbed on the mouse's bare back.A sterilized blade was used to shave the backs of the mice.Small strips of the elastic bandage tape were put and removed from a 2 × 2 cm 2 dorsal region 10-20 times until the skin glistened and reddened without bleeding (Tatiya et al., [24]).

Treatment of mice
Abscess appeared after 3 days of inoculation.The mice were divided into groups and subgroups and given treatment for up to 14 days after infection.Group-1 had 18 infected mice of which 6 mice were treated with 650 μg/ ml/day of the PBP ethanolic extract (orally and topically), 6 were treated with 500 μg/ml/day of the PBP extract both topically and orally, and 6 mice were treated with 350 μg/ml/day.Group-2 containing 3 mice was infected but untreated, Group 3 containing 3 mice was treated with petroleum jelly, and Group-4 containing 3 mice was treated with standard antibiotic drugs.Group-5 containing 3 mice is the control group.Treatments were carried out two times daily for a further 14 days (Figure 5).

Measurement of the abscess size
During the treatment, the width and length of abscesses of the different mice groups were measured from day 1 to day 14 with the help of Vernier calipers (Figure 6).The abscess volume were calculated using the abscess length and width measurements (Bunce et al., [25]; Lukomski, et al., [26]).

Hematological analysis
Blood was taken from each mouse through the intracardiac channel using 1 ml sterile disposable syringes having 26 mm × 6 mm needles.Ethylenediaminetetraacetic acid (EDTA) tubes were then used to collect the blood.The absolute counts for white blood cells (WBCs), erythrocytes, lymphocytes, hematocrit (HCT), platelets, and hemoglobin (HGB) were collected using an Auto Hematology Analyzer by the conventional technique with certain changes (Hoff [27]).

Determination of vitamin C
A colorimetric approach was used to estimate the amount of vitamin C [28].The extract was incubated in a water bath at a temperature of 60 o C with the addition of 2.5 ml of oxalic acid (4%), 0.5 ml of sulfuric acid (5%), 2 ml of ammonium molybdate, and 3 ml of distilled water.
After cooling, the absorbance of the resultant solution was measured at 515 nm using a spectrometer.A fresh ascorbic acid solution made was used to plot the calibration curve.

Determination of total phenols
Total phenols were determined by the procedure based on the Folin-Ciocalteu method [29].In a test tube, 500 ml of sodium carbonate, 250 ml of the Folin-Ciocalteu reagent, and 1 ml of the sample extract were combined.After being homogenized and allowed to react for 30 min at room temperature, the absorbance was measured at 710 nm using a spectrophotometer.

Determination of antioxidant activity
The antioxidant activity of the propolis extract was determined using the free radical-scavenging compound 1-1diphenyl-2-picrylhydrazyl (DPPH) [30].About 3 ml of a 60 mM ethanolic solution of DPPH was mixed with 1 ml of propolis extracts at varied concentrations.After 30 min, absorbance readings were taken at 517 nm at room temperature.The negative control was the absorbance of a blank sample made up of an equal volume of methanol and DPPH solution.The IC50 values were calculated using the % inhibition against the gallic acid (phenol) and ascorbic acid (vitamin C) contents.

Statistical analysis
Statistical assessment of obtained data was performed in vitro and in vivo using Minitab to examine the results of antibacterial activity of PBP extracts and to determine the variance in several parameters, such as mean and standard deviation.3 Results

In vitro assay of the ethanolic extract of PBP against S. aureus
The in vitro activity of the ethanolic extract of PBP is shown in Table 1 and Figure 7. Five different concentrations of the ethanolic extract of PBP (100, 200, 350, 500, and 650 μg/ml) were used in determining the in vitro activity in triplicates.At 650 μg/ml, the extract had the greatest antibacterial activity, followed by 500 and 350 μg/ml, with 100 μg/ml having the least inhibition of the bacterial activity.The inhibition of S. aureus increased with the increase in the concentration of the PBP extract.

3.2
In vivo assay of the PBP extract against S. aureus in mice 3.2.1 Mean abscess size in the mice group treated with the ethanolic extract of PBP (350 μg/ml/day) topically and orally The daywise distribution of the mean abscess size in the mice group treated with 350 μg/ml/day of the PBP extract topically and orally is shown in Table 2.The mean abscess size (topically and orally) was measured for up to 14 days.The wound-healing capacity of 350 μg/ml/kg/ day PBP was much lesser than at the other two concentrations (500 and 650 μg/ml/day).When compared with the other two concentrations of PBP, the lowest woundhealing capacity was 350 μg/ml/day of the PBP extract.
The topically treated mice group with a concentration of 350 μg/ml/day of PBP reduced the abscess area from 2.17 cm 2 ± to 0.33 cm 2 ± and reduced the abscess volume from 2.45 cm 3 ± to 0.16 cm 3 .The mice group treated with a concentration of 350 μg/ml/day of the PBP extract orally reduced the abscess area from 2.04 cm 2 ± 0.158 to 0.4 cm 2 ± 0.035 and the volume was reduced from 2.22 cm 3 ± to 0.22 cm 3 ± in 14 days of treatment.

3.2.2
Mean abscess size in the mice group treated with the ethanolic extract of PBP (500 μg/ml/day) topically and orally The daywise distribution of the mean abscess size for each group treated with 500 μg/ml/day of the PBP extract is given in Table 3.In the mice group treated with 500 μg/ml/day, there was a decrease in the abscess size but it was less than that at a concentration of 650 μg/ml/day.In these 14 days of treatment, the area decreased from 2.21 cm 2 ± 0.061 to 0.2 cm 2 ± 0.021, and volume reduced from 2.59 cm 3 ± 0.065 to 0.06 cm 3 ± 0.004 in the mice treated with 500 μg/day of the PBP extract topically.
The mice group treated orally with the same concentration of PBP reduced the abscess size.However, the wound-healing capacity of the mice treated topically at the same concentration was lower compared to that given orally.Overall, in the 14 days of treatment, the area decreased from 1.95 cm 2 ± 0.103 to 0.32 cm 2 ± 0.037 and the volume decreases from 2.08 cm 3 ± 0.188 to 0.14 cm 3 ± 0.026 (Table 3).

3.2.3
Mean abscess size in the mice group treated with the ethanolic extract of PBP (650 μg/ml/kg/day) topically and orally The daywise distribution of the mean abscess size in the mice group treated with 650 μg/ml/day of the PBP extract topically and orally is shown in Table 4.The mean abscess size (topically, orally) was measured for up to 14 days.The mice group receiving 650 μg/ml/day of PBP topically showed a decrease in the mean abscess size from 12.04 cm 2 ± 0.117 to 0.13 cm 2 ± 0.018 in area and a decrease in the volume from 2.09 cm 3 ± 0.276 to 0.04 cm 3 ± 0.008 after 14 days of treatment.
The mice group treated with the same concentration of PBP extract orally showed a decrease in the mean abscess size from 2.09 cm 3 ± 0.276 to 0.04 cm 3 ± 0.008 area while it showed a decrease in the volume from 2.73 cm 3 ± 0.056 to 0.12 cm 3 ± 0.048.Overall, the highest antibacterial activity of the PBP extract was observed at a concentration of 650 μg/ml.

Mean abscess size in the untreated mice group and the mice group treated with petroleum jelly
The abscess size begins to grow in the mice group in the untreated and treated with petroleum jelly.Petroleum jelly showed no role in the healing of the abscess and the abscess size increases considerably day by day; similarly, the abscess size also increases in the untreated mice group.In these 14 days, the abscess area in the untreated mice group increased from 2.35 cm 2 ± 0.245 to 4.07 cm 2 ± 0.230 and the volume increased from 2.44 cm 3 ± 0.064 to 6.47 cm 3 ± 0.584 (Table 5).

Mean abscess size in the mice group treated with the topical antibiotic
The mice group treated with the topical antibiotic showed considerable response to abscess healing.It was observed that as compared to the PBP extract, the wound-healing ratio of the topical antibiotic was quite positive.The mice group treated with the topical antibiotic was properly cured within 14 days.After a thorough observation of the wound till healing, it was finally concluded that the wound area was found maculated.In these 14 days, the abscess area decreases from 2.05 cm 2 ± 0.094 to 0.06 cm 2 ± 0.024 in the topical antibiotic-treated mice group.Similarly, the volume decreased from 2.29 cm 3 ± 0.128 to 0.04 cm 3 ± 0.042 (Table 6).

Hematological analysis
The hematological analysis of mice groups was performed after 14 days of bacterial infection in the PBP extract-treated mice groups and untreated mice group and was compared with the uninfected control group.The blood parameters include WBCs, granulocytes (GRA), red blood cells (RBCs), HGB, HCT, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), the mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), and the mean platelet volume (MPV), as shown in Tables 7 and 8.

Mean count of WBCs and GRA in mice groups
The mean count of WBCs in the untreated mice group was higher than the normal level (11.56 × 10 9 /L ± 0.826) compared to the control group (2.06 × 10 9 /L ± 0.205); similarly, the mice group treated with petroleum jelly showed an increase in the mean count of WBCs (10.93 × 10 9 /L ± 0.895).The mice group treated with the PBP extract topically and orally showed a slight increase in the mean count of WBCs.The mean count of WBCs was 7.9 × 10 9 /L ± 0.163 in the mice group treated with 650 μg/ml topically and 8.5 × 10 9 /L ± 0.804 in the 500 μg/ml topically treated mice group.While the number of WBCs in the orally treated mice groups is 8.8 × 10 9 /L ± 0.616 in the 650 μg/ml mice group and 7.2 × 10 9 /L ± 0.535 in the 500 μg/ml mice group treated orally (Tables 7 and 8).
The mean value of GRA slightly increased in mice groups treated with PBP extract (1.23 × 10 9 /L ± 0.249) in the 650 μg/ml topically treated mice group, and was 3.26 × 10 9 /L ± 0.329 in the mice group treated with 500 μg/ml topically.Similarly, the mean count of GRA in the mice group treated with PBP extract orally was 2.36 × 10 9 /L ± 0.205 in the 650 μg/ml treated mice group and 4.03 × 10 9 /L ± 0.498 in the 500 μg/ml treated mice group.While the increase in the mean count of GRA was observed in negative control mice groups (8.4 × 10 9 /L ± 0.430in the untreated mice group, (9.03 × 10 9 /L ± 0.451) in the mice group treated with petroleum jelly in comparison with a control group of mice (1.23 × 10 9 /L ± 0.249) (Tables 7 and 8).

Mean values of RBCs and HGB in mice groups
The mean values of RBCs were decreased in the untreated and mice group treated with petroleum jelly compared to the control group.The mean count of RBCs in the untreated mice group was 10.46 × 10 12 ± 0.917 and 9.73 ± 0.980 × 10 12 in the mice group treated with petroleum jelly, while the mean count of RBCs in the mice group  7 and 8).The level of HGB was normal in all mice groups, but this ratio was high in mice groups treated with topical antibiotics and in the mice group treated with 650 μg/ml/ kg PBP extract.

Mean percentage of HCT in mice groups
The HCT percentage was decreased in the mice groups in which S. aureus was inoculated compared to the mice group that was uninfected.The percentage of HCT was above the normal level in the mice group treated with topical antibiotic and the percentage was hardly equal to the normal level in the mice group treated with 650 μg/ml/kg of PBP extract (Tables 7 and 8).

MCV and MCHC in mice groups
The level of MCV was also decreased in all groups except in the mice group treated with the topical antibiotic and in the mice group treated with 650 μg/ml/kg of PBP extract.Similarly, it was normal in the mice group treated with 650 and 500 μg/ml/kg of PBP extract (Tables 7 and 8).The level of MCHC was only normal in the mice group that was uninfected and in the mice group that was treated with the topical antibiotic.In the rest of the mice groups, the level of MCHC decreased.

Yield percent, vitamin C, and total phenols in the ethanol extract of propolis
Estimating total phenols and vitamin C is important since the antioxidant activity is represented by these measurements.It is an important primary component that is responsible for antioxidant activity and is related to the protection of several oxidative stress-related illnesses [31].

Antioxidant activity
DPPH is often used to assess the antioxidant potential of different sample extracts [32].The free radical scavenging abilities of the propolis extract concentrations were measured.As shown in Table 10, the free radical scavenging activity was present at all the concentrations.All the concentrations showed free radical scavenging activity.The PBP sample concentrations showed 328.21, 365.40, 347.20, 335.88, and 445.92 µg/ml antioxidant activity.A comparison of the percentage of inhibition in the antioxidant activity of the ethanol extract of PBP sample concentrations was evaluated with gallic acid (phenol) and ascorbic acid (vitamin C) as standards for scavenging DPPH free radicals, and the results are presented in Table 10.

Discussion
To the author's best knowledge, no in vivo studies have been conducted earlier on PBP against any bacteria, particularly S. aureus; however, a single study showed that propolis obtained from other regions showed efficacy against S. aureus [33].The current in vitro results indicated that the extract of PBP has antibacterial activity against S. aureus.The current study showed an increase in the antibacterial activity with an increase in the extract concentration.The findings of the current study are in agreement with those of Lu et al. [33], who also showed that propolis has antibacterial action against S. aureus and that the bactericidal ability of propolis improves as the extract concentration increases.The findings of an in vivo investigation revealed that topical therapy with an ethanolic extract of propolis was more efficient than orally treating the group.The highest antibacterial activity of the PBP extract in vivo was observed at 650 μg/ml; the mean abscess area of the topically treated group with ethanolic extract of PBP before treatment was 2.04 cm 2 ± 0.117 and after 14 days of treatment, it was decreased to 0.13 cm 2 ± 0.018.Similarly, the mean abscess volume before treatment was 2.09 cm 3 ± 0.276 and after the treatment, it decreased to 0.04 cm 3 ± 0.008.The abscess area and volume of the mice before receiving the same ethanolic extract orally were found to be2.31 cm 2 ± 0.127 and 2.73 cm 3 ± 0.056; after treatment, they were 0.16 cm 2 ± 0.030 and 0.12 cm 3 ± 0.048.After 14 days of therapy, the center of the treated wounds developed a scar.All PBP-treated groups had fewer scars than the negative control group and those treated with petroleum jelly.The propolis wound-healing properties are most likely owing to its anti-inflammatory and antioxidant properties.
The hematological parameters, including RBCs, WBCs, Hb, platelet, and HCT (uninfected, ethanolic extract topically orally treated mice, topical antibiotic, and petroleum jelly treated mice groups) were analyzed after treatment.The mean counts of RBCs, Hb, and HCT were found to be decreased in the infected untreated mice group and mice groups treated with petroleum jelly groups, compared to uninfected groups; however, a slighter increase was observed in WBCs.The presence of a high WBC count indicates emotional and physical stress, as well as the immune system's fight against infection [34].The count of GRA increases in untreated mice groups and mice groups treated with petroleum jelly.GRA are WBCs with tiny granules that help fight viral and bacterial infections.For infections, autoimmune disorders, and blood cell cancer, the number of GRA (granulocytosis) increases, and the organism suffers abnormal breeding, pale skin, heavy sweating when sleeping, weariness, and appetite loss [35].There are no studies previously conducted on the hematological parameters of infected mice after oral and topical treatment with honey bee propolis against induced S. aureus infection in BALB C mice.The amount of active compounds present in the ethanolic extract of propolis might have contributed toward the scavenging of free radicals.
Based on the findings of this study, it is possible to infer that PBP has promising potential in the fight against S. aureus infection.For the greatest understanding, further research should be focused on identifying the antibacterial bioactive components in PBP and analyzing antibacterial properties at the compound level.

Conclusions
The current study concluded that an ethanolic extract of PBP had both in vitro and in vivo antibacterial potential against S. aureus-induced infections in mice.The in vitro study showed that inhibition of S. aureus increased when the concentration of PBP extract was increased.After in vivo treatment, a decrease in the abscess size was observed with an increase in the concentration of ethanolic extracts of PBP in BALB/c mice infected with S. aureus.Moreover, total phenols and vitamin C content showed antioxidant activity at all concentrations.It is recommended further to assess its mechanism of action for use as a natural antimicrobial agent in a variety of applications.

Figure 1 :
Figure 1: (a) Beehives at the research site at Kohat University of Science and Technology, Kohat.(b) PBP in small pieces kept in the dark until processing.

Figure 2 :
Figure 2: (a) Filtration of the ethanolic diluted solution of PBP.(b) The evaporation of solvent from the diluted PBP using rotary vacuum evaporation.(c) Crude extract of PBP.

Figure 3 :
Figure 3: Bacterial culture of S. aureus used in the study.

Figure 4 :
Figure 4: Tape stripping technique used in the current study for bacterial inoculation in mice.

Figure 5 :
Figure 5: Abscess in mice after 3 days of inoculation of S. aureus.

Figure 6 :
Figure 6: Abscess measurement carried out in the current study.

Figure 7 :
Figure 7: In vitro activity of the PBP extract against S. aureus.

Table 1 :
In vitro assay of the PBP extract against S. aureus

Table 2 :
Mean abscess size in the mice group treated with the ethanolic extract of PBP (350 μg/ml/day) topically and orally

Table 3 :
Mean abscess size in the mice group treated with the ethanolic extract of PBP (500 μg/ml/day) topically and orally

Table 4 :
Mean abscess size in the mice group treated with the ethanolic extract of PBP (650 μg/ml/day) topically and orally

Table 5 :
Mean abscess size in the untreated mice group and in petroleum jelly-treated mice group

Table 6 :
Mean abscess size in the mice group treated with the topical antibiotic

Table 7 :
Hematological analysis of mice groups treated with PBP extract topically treated with PBP extract topically and orally was normal.The mean counts of RBCs in mice groups treated with PBP were 12.2 × 10 12 ± 0.216 in the 650 μg/ml topically treated mice group, 11.4 × 10 12 ± 0.432 in the 500 μg/ml topically treated mice group, 10.46 × 10 12 ± 0.612 in the 650 μg/ml orally treated mice group, and 10.26 × 10 12 ± 0.478 in the mice group treated with 500 μg/ml/kg of PBP extract orally.The mean count of RBCs in the mice group treated with topical antibiotic was 12.66 × 10 12 ± 0.124 and in the mice group that was uninfected was 13.1 × 10 12 ± 0.492 (Tables

Table 8 :
Hematological analysis of mice groups treated with PBP extract orally Parameters Almost all the concentrations indicated a considerable amount of vitamin C that ranged between 23.53 ± 4.15 and 25.92 ± 3.49 and total phenols ranged from 11.75 ± 2.03 to 11.47 ± 1.68.The percent yield of extractable compounds in the ethanol extract of PBP is presented in

Table 9 :
Yield percent, vitamin C, and total phenols in the ethanol extract of PBP

Table 10 :
IC 50 values of the ethanol extract of propolis sample concentrations with ascorbic acid (vitamin C) and gallic acid (phenol)