ANTIBACTERIAL AND ANTIOXIDANT PROPERTIES OF SOME PLANT EXTRACTS WITH PROPOLIS

 

Sinem Aydin ¹, Gülşah Kadioğlu ²

 

¹ Associate Professor Doctor Sinem Aydin, Department of Biology, Faculty of Science and Arts, Giresun University, Giresun, Turkey

₂ Department of Biology, Faculty of Science and Arts, Giresun University, Giresun, Turkey

 

1. INTRODUCTION

Plant are valuable sources that can be utilized in many industries. Products based on natural substances attract attention in many industries, such as cosmetics and pharmacy in recent years Stuper-Szablewska et al. (2022).

Antimicrobial agents have important roles against pathogens. On the other hand, using excessive antimicrobial in the world cause drug resistance and undesirable effects. Many bacteria gained resistant to current antibiotics.  The increase in untreatable infections require the discovery and development of brand antibacterials.

Plants have been utilized to cure bacterial infections thousands of years ago. Many people in developing countries utilize herbal drugs for antibacterial illnesses Liang et al. (2022).

Oxidative stress may lead Alzheimer’s, cancer, and cardiovascular illnesses. Free radicals can be harmful proteins, lipids, nucleic acids and carbohydrates. The utilization of antioxidants can delay or retard the oxidation of biomolecules. Medicinal plants have been utilized as natural antioxidant agents for centuries by people Guchu et al. (2020).

Synthetic antioxidants create negative health effects. Hence, studies about natural antioxidants is valuable. Polyphenolic compounds can behave as antioxidants and free radical scavengers. Studies about polyphenols from plants has now achieve considerable attention Shahinuzzaman et al. (2020).

Papaver somniferum is an annual plant which belons to the family Papaveraceae. P. somniferum use as a foodstuff in manufacturing of bakery products.  Moreover, it’s oil has various medicinally important metabolites Chmelová et al. (2018).

Syzygium aromaticum possess various medical features like antimutagenic, antibacterial, anti-inflammatory and radical scavenging activity Faujdar et al. (2020). Moreover, It has antioxidant action so it utilizes in preventing some degenerative diseases. Syzygium aromaticum bud oil heals wounds and burns Alanazi et al. (2022).

Foeniculum sp. belongs to family Apiaceae and cultivated in India, China and Egypt. The volatile oil of fennel possess anti-allergic, diuretic, anti-spasmodic, antimicrobial and antioxidant potencies Eliuz et al. (2016).

Bees gather resins from plants, stir them with their own salivary enzymes and beeswax which composes propolis. Propolis possess many important effects such as antibacterial, radical scavenging, antiparasitic, antifungal and antiproliferative Przybylek & Karpinski (2019).

This investigation aims the compare biological features of extracts of propolis-Syzygium aromaticum mixture, propolis-Papaver somniferum mixture, propolis-Foeniculum sp. mixture.

 

2. MATERIALS AND METHODS

2.1. SUPPLYING OF PLANTS USED IN THE STUDY

Propolis, Syzygium aromaticum, Papaver somniferum and Foeniculum sp. were brought from a herbal shop in Giresun, Turkey.

 

2.2. TEST BACTERIA

Listeria monocytogenes ATCC 7644, Salmonella enterica serovar typhimirium ATCC 14028, Staphylococcus aureus subsp. aureus ATCC 25923, Bacillus cereus 702 ROMA, Yersinia pseudotuberculosis ATCC 911, Enterococcus faecalis ATCC 29212, Bacillus subtilis IMG 22, Enterobacter aerogenes CCM 2531, Gordonia rubripertincta (lab isolate) and Proteus vulgaris (lab isolate) were utilized in antimicrobial activity tests.

 

2.3. PREPARATION OF THE EXTRACTS

15 g of propolis-15 g of Syzygium aromaticum, 15 g of propolis-15 g of Papaver somniferum and 15 g of propolis-15 g of Foeniculum sp. were extracted in a shaker for 24 h utilizing 300 mL ethanol and hexane, separetely. The extracts were filtered through Whatman filter paper No. 1 and residues were evaporated (40 °C) with rotary evaporator Murugan & Parimelazhagan (2014).

 

2.4. ANTIBACTERIAL ACTIVITY

The discs (6 mm diameter) on the petri were impregnated with 20 μL of extracts, separetely. Gentamycine was used as standard antimicrobial agent. DMSO was used as negative control. Plates were incubated for 24 h at 37°C. Diameter of zones were measured with a ruler Murray et al. (1995), Šariš et al. (2009). The tests were carried out twice.

 

2.4.1.  THE DETERMINATION OF MINIMUM INHIBITION CONCENTRATION (MIC)

Minimum inhibition concentration of extracts which created ≥10 mm inhibition zones were determined. Method of Yiğit et al. (2009) were used to reveal MIC values of the tested extracts Yiğit et al. (2009).

 

2.5. ANTIOXIDANT ACTIVITY

All antioxidant tests were carried out three times. Results are expressed as the mean ± standard deviation (S.D.) of each triplicate test.

 

2.5.1.  TOTAL PHENOLIC CONTENT, TOTAL FLAVONOID CONTENT AND TOTAL ANTIOXIDANT CAPACITY

The total phenolic, flavonoid content and total antioxidant capacity were expressed as µg of gallic acid equivalent (GAE)/mL Slinkard & Singleton (1977), µg of cateschin equivalent (CE)/mL Zhishen et al. (1999) and µg of ascorbic acid equivalent (AAE)/mL Prieto et al. (1999), separetely.

 

2.5.2.  CUPRAC ACTIVITY

CUPRAC activity of the extracts were studied according to the method of Özyürek et al. (2009). Butylated hydroxytoluene (BHT) was used as a standard antioxidant agent. 

 

2.5.3.  DPPH RADICAL SCAVENGING ACTIVITY

Extracts were prepared at 250-1000 µL/mL concentrations by the method of Blois (1958). BHT and Rutin were used as standards.

The DPPH radical scavenging activity was calculated using the following equation:

                        

A0: Absorbance of control

A1: Absorbance of sample or standard

 

 

 

 

3. RESULTS AND DISCUSSION

In the current study it was tested antibacterial efficiencies of ethanol and hexane extracts of propolis-Syzygium aromaticum, propolis-Papaver somniferum and propolis-Foeniculum sp. against test bacteria. Table 1 demonstrates inhibition zones.

Ethanol extracts exhibited higher activity than hexane extracts. Antibacterial activities of mixtures were increased in the following order: propolis-Syzygium aromaticum> propolis-Papaver somniferum> propolis-Foeniculum sp. While inhibition zones was created by ethanol extracts ranges from 7±1.41 mm to 19.5±2.12 mm, inhibition zones was created by hexane extracts ranges from 7±0.00 mm to 14±1.41 mm. Gentamycine showed higher activity when compared with tested extracts. DMSO showed no activity against test bacteria.

Table 1

Table 1 Inhibition Zones, Which Was Created by Extracts, DMSO and Gentamycine (mm)
Bacteria	PSE	PFE	PPE	PSH	PFH	PPH	CN	DMSO
B. cereus 702 ROMA	8.5±0.70	NA	7.5±0.70	11±1.41	7±0.00	9±0.00	18±1.41	NA
K. pneumoniae (lab isolate)	11±0.00	11±1.41	15.5±0.70	7.5±0.70	7.5±0.70	11±1.41	21±1.41	NA
E. aerogenes CCM 2531	12±1.41	NA	11±0.00	7±1.41	7±1.41	NA	20±1.41	NA
G. rubripertincta (lab isolate)	NA	NA	NA	14±1.41	8±0.00	15±0.00	19±1.41	NA
E. faecalis ATCC 29212	14.5±0.70	10.5±0.70	17±1.41	12.5±0.70	10.5±0.70	13±2.82	20.5±2.12	NA
P. vulgaris (lab isolate)	18.5±2.12	10.5±0.70	12±1.41	9.5±2.12	8.5±0.70	8.5±2.12	21.5±2.12	NA
S. enterica serovar typhimirium ATCC 14028	11.5±0.70	9±0.00	10±0.00	9±0.00	8.5±0.70	7.5±0.70	22±2.82	NA
L. monocytogenes ATCC 7644	16±1.41	8±0.00	11.5±0.70	7.5±0.70	7±0.00	NA	19.5±0.0	NA
B. subtilis IMG 22	7.5±0.70	7.5±0.70	7±1.41	8±0.00	7±1.41	7.5±0.70	16.5±2.12	NA
S. aureus subsp. aureus ATCC 25923	19.5±2.12	8±0.00	15.5±2.12	9.5±0.70	7.5±0.70	11±1.41	18.5±2.12	NA
Y. pseudotuberculosis ATCC 911	12±0.00	12.5±0.70	12.5±2.12	9±0.00	8±0.00	10.5±0.70	21.5±2.12	NA
PSE: Ethanol extract of propolis-Syzygium aromaticum; PFE: Ethanol extract of propolis-Foeniculum sp.; PPE: Ethanol extract of propolis-Papaver somniferum; PSH: Hexane extract of propolis-Syzygium aromaticum; PFH: Hexane extract of propolis-Foeniculum sp.; PPH: Hexane extract of propolis-Papaver somniferum; CN: Gentamycine 10 µg/mL, NA: No Activity

 

MIC values of the extracts were given in Table 2. MIC describes as the minimum antimicrobial agent that inhibits the growth of microorganisms. Low MIC values means higher antibacterial effect. While the lowest MIC value was found in PSE as 0.0003 mg/mL against S. aureus, the highest MIC value was found in PSH as 1.5 mg/mL against B. cereus and in PPH as 1.5 mg/mL against Y. pseudotuberculosis.

Table 2

Table 2 MIC values of the extracts (mg/mL)
Bacteria	PSE	PFE	PPE	PSH	PFH	PPH
B. cereus 702 ROMA	-	-	-	1.5	-	-
K. pneumoniae (lab isolate)	0.0468	0.0468	0.1875	-	-	0.75
E. aerogenes CCM 2531	0.0468	-	0.0058	-	-	-
G. rubripertincta (lab isolate)	-	-	-	0.1875	-	0.1875
E. faecalis ATCC 29212	0.0117	0.0117	0.1875	0.1875	0.75	0.1875
P. vulgaris (lab isolate)	0.375	0.1875	0.1875	-	-	-
S. enterica serovar typhimirium ATCC 14028	0.1875	-	0.375	-	-	-
L. monocytogenes ATCC 7644	0.1875	-	0.1875	-	-	-
B. subtilis IMG 22	-	-	-	-	-	-
S. aureus subsp. aureus ATCC 25923	0.0003	-	0.1875	-	-	0.0234
Y. pseudotuberculosis ATCC 911	0.1875	0.1875	0.1875	-	-	1.5

 

·        Antioxidant activity

Table 3 summarizes total phenolic content of the extracts. When total phenolic content was compared between extracts, it was found that ethanol extracts have higher phenolic content than hexane extracts. The highest and the lowest phenolic contents were found as 389.81±0.001 µg GAE/mL and 100.57±0.012 µg GAE/mL in PSE and PFH, respectively.

Table 3

Table 3 Total Phenolic Contents of the Extracts (µg GAE/mL)
Extract	Total Phenolic Content (µg GAE/mL)
PSE	389.81±0.001
PFE	169.3±0.001
PPE	286.93±0.003
PSH	205.03±0.003
PFH	100.57±0.012
PPH	142.15±0.004

 

Total flavonoid content of the extracts were presented in Table 4. Ethanol extracts possess higher flavonoid contents than hexane extracts. Total flavonoid contents of the extracts as follows: PSE>PPE> PSH>PFE>PFH>PPH.

Table 4

Table 4 Total Flavonoid Content of the Extracts (µg QE/mL)
Extract	Total Flavonoid Content (µg QE/mL)
PSE	91.25±0.024
PFE	22.31±0.022
PPE	68.43±0.016
PSH	57.5±0.013
PFH	13.64±0.010
PPH	1.7±0.002

 

Total antioxidant capacity of the extracts were given in Table 5. The highest and the lowest values were found as 190.54±0.031 µg AAE/mL in PSH and 46.03±0.018 µg AAE/mL in PFH.

Table 5

Table 5 Total Antioxidant Capacity of the Extracts (µg AAE/mL)
Extract	Total antioxidant capacity  (µg AAE/mL)
PSE	119.33±0.021
PFE	65.44±0.012
PPE	147.11±0.009
PSH	190.54±0.031
PFH	46.03±0.018
PPH	65.85±0.005

 

Table 6 shows DPPH radical scavenging activity of the extracts. It was found no activity in PFH and PPH. PSE exhibits higher activity than Rutin and BHT which were used as standard antioxidant agents.

Table 6

Table 6 DPPH Radical Scavenging Activity of the Extracts and Standards
Extract	Concentration (µg/mL)	DPPH Radical Scavenging Activity (% inhibition)
PSE	250 500 750 1000	91.08±0.002 91.87±0.001 93.14±0.001 94.42±0.003
PFE	250 500 750 1000	86.62±0.002 87.26±0.001 89.35±0.005 89.72±0.004
PPE	250 500 750 1000	87.03±0.003 87.8±0.003 89.49±0.002 89.95±0.004
PSH	250 500 750 1000	85.01±0.007 86.94±0.004 88.74±0.002 89.41±0.005
PFH	250 500 750 1000	NA NA NA NA
PPH	250 500 750 1000	NA NA NA NA
BHT	250 500 750 1000	88.85±0.012 89.55±0.005 90.27±0.011 91.55±0.008
Rutin	250 500 750 1000	86.80±0.008 87.91±0.003 90.6±0.004 91.89±0.011
NA: No Activity

 

CUPRAC activity of the extracts were presented in Table 7. The highest activity was determined in PSE. All extracts were showed higher activity than BHT except for PPH and PFH.

Table 7

Table 7 CUPRAC Activity of the Extracts and Standard
Extract	Concentration (µg/mL)	CUPRAC Activity (nm)
PSE	250 500 750 1000	2.022±0.018 2.073±0.010 2.104±0.008 2.143±0.003
PFE	250 500 750 1000	2.007±0.003 2.029±0.004 2.066±0.005 2.081±0.056
PPE	250 500 750 1000	1.902±0.006 1.988±0.008 2.004±0.003 2.015±0.003
PSH	250 500 750 1000	2.009±0.008 2.062±0.009 2.091±0.005 2.136±0.004
PFH	250 500 750 1000	0.569±0.038 0.473±0.019 0.650±0.016 0.840±0.003
PPH	250 500 750 1000	0.352±0.027 0.427±0.035 0.620±0.008 0.812±0.012
BHT	250 500 750 1000	0.6945±0.023 0.7519±0.020 0.8509±0.029 1.0567±0.012

 

There is no study about antibacterial activity of propolis-Syzygium aromaticum, propolis-Papaver somniferum and propolis-Foeniculum sp. On the other hand, there are many studies about propolis, Syzygium aromaticum, Papaver somniferum and Foeniculum sp.

One of the most common and known most studied properties of propolis is its antimicrobial activity. Numerous studies have been conducted on its effects on fungi and viruses Albayrak & Albayrak (2008).

Veiga et al. (2017) reported that propolis inhibited gram posivite bacteria and gram negative bacteria such as methicilline resistant Staphylococcus aureus. Yildirim et al. (2016) investigated effect of propolis against tuberculosis and they found that propolis is efficient against many mycobacteria species.

It was recorded that propolis has antibacterial activity against many aerobic bacteria such as Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Micrococcus    luteus, Nocardia asteroids, Staphylococcus auricularis, Staphylococcus epidermidis, Staphylococcus  capitis,  Staphylococcus haemolyticus and  Staphylococcus warnerius Fokt et al. (2010).

In a study which was carried out by Nzeako et al. (2006) water extract and essential oil of Syzygium aromaticum showed antibacterial activity against Streptococcus pyogenes, Corynebacterium spp.. Salmonella spp. and Bacteroides fragilis.

Gupta & Prakash (2021) used extracts and essential oil of Syzygium aromaticum against Halobacteria sp., Lactobacillus sp., Pseudomonas sp., Micrococcus sp. and Streptococcus mutans which cause dental plaques and cavities. It was concluded that essential oil of Syzygium aromaticum had better activity than extracts of Syzygium aromaticum.

Masood et al. (2008) found aqueous infusions of Papaver somniferum seeds had no activity and aqueous decoction had very weak activity against 188 bacterial isolate.

Mishra and Pathak (2021) reported methanol and water extracts of Papaver somniferum seeds had activity against Salmonella and Escherichia coli but they were no activity against Pseudomonas.

Eliuz et al. (2016) revealed essential oil of Foeniculum sp.had antibacterial effect against E. coli, Klebsiella pneumoniae, Salmonella typhimurium, Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis.

Yıldırım et al. (2010) carried out a study about antibacterial activity of Foeniculum sp. It was reported that ethanol and aqueous extracts of Foeniculum sp. didn’t inhibit E. coli, S. aureus, B. cereus, K. pneumoniae and Enterobacter sp. but methanol extract of Foeniculum sp. inhibited S. aureus and Enterobacter sp.

Like antibacterial studies, there is no study about antioxidant activity of propolis-Syzygium aromaticum, propolis-Papaver somniferum and propolis-Foeniculum sp. On the other hand, there are many studies about propolis, Syzygium aromaticum, Papaver somniferum and Foeniculum sp.

Kocot et al. (2018) found that extracts of propolis had better DPPH and ABTS radicals scavenging activities than standard antioxidant agents such as BHT and ascorbic acid.

Can et al. (2016) investigated total phenolic content and antioxidant activity of propolis samples from Azerbaijan. It was stated that total phenolic contents of samples ranges from 10.94 to 79.23 mg GAE/g. Propolis samples which was obtained from Ismayilli. Zerdap and Qax had higher antioxidant activity when compared with other districts.

Muhson & Al-Mashkar (2015) investigated total phenolic content, total flavonoid content and DPPH radical scavenging activity and acetone extracts of stem and fruit parts of Syzygium aromaticum. DPPH radical scavenging activity was found %87.50 and %79.41 in fruit and stem, respectively.

Elaleem et al. (2017). studied DPPH radical scavenging activity of methanol, petroleum ether and chloroform extracts of seeds of Papaver somniferum. It was found that only methanol extract had activity.

 

4. CONCLUSION

The results revealed that these plant-propolis mixtures could play as a promising antibacterial and antioxidant agents, because of their high activities. Moreover, the current study suggests that these mixtures might be developed as pharmaceutical products. Hence, studies on the isolation and identification of substances responsible for biological activities in these mixtures should be expanded.

 

CONFLICT OF INTERESTS

None. 

 

 

 

 

ACKNOWLEDGMENTS

Current research was funded by Giresun University Scientific Research Projects (FEN-BAP-C-240222-17).

 

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