Study of The Antioxidant Activity and Inhibition Effect of Propolis Extract on Some Microorganisms
Mohammed A. Salman1,*, Nawal K. Zben2 , Lina S. Mohammed3 and Mariam A. Auribi4 College of Agriculture, University of Basrah, Basrah, Iraq
*Email: [email protected]
Abstract:
The study was conducted on types of spring and autumn propolis extracts and wax mixtures with propolis using two solvents, namely ethanol and diethyl ether, and their effect on the percentages of antioxidant activity. The results of the statistical analysis showed a significant effect (p<0.05) among the treatments, as spring propolis gave the highest activity when used with ethanol solvent, which reached 50.68%, followed by the mixture of wax and propolis, then the autumn propolis. However, using the solvent diethyl ether had the highest activity with the mixture of wax and propolis 74.65% at a concentration of 90 mg/ml and was less than the efficacy of Butylated Hydroxy Tolune (BHT). Whereas, the inhibitory activity of two types of propolis and a mixture of wax with propolis and between the comparison treatment (solvent) with addition rates of 0.1 ml and 0.2 ml by the agar well diffusion method was studied for four species of fungus, Penicillium sp, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and three types of yeasts: Candida albicans Candida glabrata and Candida parapsilosis. The statistical analysis results showed an inhibitory effect with a significant difference (P˂0.05) against the studied organisms and between the types of propolis and between the size of the addition and the types of solvents used.
Keywords: propolis, antioxidant activity, the inhibitory effect.
Introduction
Propolis is one of the products of honey bees, where this product has received the most studies in the last thirty years. It is a dark-colored sticky substance that honey bees collect from plants and use against pathogenic microorganisms, as well as use it as a building material in the hive. Propolis shows a variety of life activities including Antifungal, Antibacterial, and anti-inflammatory, as well as antioxidant activity (Bankova, 2005).
Propolis consists of various compounds, including resins, aromatic oils, minerals, flavonoids, as well as wax and bee secretions. It was used in folk natural remedies over time by containing active therapeutic compounds, including flavonoids, phenolic acids, and their esters, as well as volatile compounds (Bobis et al., 2017). Antioxidant compounds of plant origin can increase the shelf life of the food product by delaying the process of formation of peroxides in fats, which is one of the main causes of food spoilage during processing and storage. Furthermore, phenolic compounds have an important role in preserving human health because they reduce the risk of
diseases by reducing oxidative stress and inhibiting molecular oxidative within the body (Silva et al., 2004). Studies show that there are many bioactive compounds present in food materials or products of plant origin that can help protect against many factors causing food spoilage and their extracts can be used as a natural antifungal (Ansari et al., 2013). Propolis is an effective antifungal against a wide range of fungus that infects stored plants, and it has been applied to control fungus growing on various fruits during storage (Çandir et al., 2009). The efficiency of propolis as an antioxidant, antifungal, and antibacterial is due to its connection to its chemical composition, which varies according to the season and the plants from which it is collected. The high levels of flavonoids, phenols, and organic acids work to damage the structure of the germ cell and break down its walls and cytoplasm and the metabolic processes by preventing division and thus it shares with many antibiotics in the mechanism by which they work ((Kalogeropoulos et al., 2009).
Materials and methods of work
Autumn and spring propolis and a mixture of wax and propolis were obtained from the local apiaries of the Basra Specialist Society for Bees Husbandry and Production in Basra Governorate. Thus, 3 g of samples were taken, with 50 ml of solvent (ethanol, diethyl ether) was added to and placed in the Soxhlet device and left for 30 minutes at a temperature of 50 ℃. A centrifugation process was carried out at 5000 rpm. Then, the filtrate was taken and the precipitate was left, and the solution was concentrated by a rotary evaporator and then dried (Wijerante et al., 2006).
The antioxidant activity of the spring and autumn propolis extracts and the propolis extract with wax were tested according to the method mentioned in (Miguel et al., 2014).
Besides, the (DPPH) 2,2-diphenyl-1-picryhydrazyl reagent was prepared at a concentration of (60 μM) in methanol and mixed with (50 μl) of propolis extracts and propolis mixture with wax at concentrations of 30, 60, 90 mg/ml, and added to 2 ml of (DPPH), left for 20 minutes. The absorbance of each of them was measured over a wavelength (517 nm) using a Spectrophotometer, and both solvent diethyl ether and methanol (Blank) were used to reset the device. Results were compared with Butylated Hydroxytoluene (BHT), and the antioxidant activity was calculated according to the following equation:
Antioxidant activity% [(A0-A1 / A0) x 100] =
A0 = the absorbance reading of the standard sample (Blank) without the studied extract sample.
A1 = the absorbance reading of the studied extract sample.
The microorganisms used in the study: Aspergillus niger, A. flavus, A. fumigatus, and Penicillium sp , Candida albicans, C. glabrata, and C. Parapsilosis were obtained from the College of Education / Department of Biology. Moreover, the inhibitory efficacy of the extracts against the micro-organisms understudy carried by the agar well diffusion method presented in (Hanna, 2009; Al-salmani and Hassaan, 2011).
The yeasts and fungal isolates were activated on the PDA media and incubated at 25 ° C for 5-7 days. The plates containing the activated micro-organisms isolates and grown on PDA media were prepared. A hole was made in the center of the plate by the Cork Borer with a diameter of 7 mm, and the extracts were added in the hole by 0.1 ml and 0.2 ml with made a hole for comparison (solvent only). The plates were incubated at 25 ° C for a period of (5-7) days for the studied micro-organisms. Then the diameters of the inhibition area were measured in mm (Clear zone) the area free of micro-organisms growth.
Results and discussion
Antioxidant activity
The results in Figure 1 showed the antioxidant activity of spring and autumn propolis, a mixture of wax and propolis extracted with ethanol alcohol. The statistical analysis results showed significant differences (P˂0.05) between the percentages of antioxidant activity, as spring propolis gave the highest activity followed by a mixture of wax and propolis then autumn propolis compared to Butylated Hydroxy Tolune (BHT) .
Figure (1) the antioxidant activity of spring and autumn propolis and a mixture of wax and propolis extracted with ethanol alcohol
The statistical analysis results also showed that the antioxidant activity of spring and autumn propolis and the wax mixture with propolis increased significantly (P˂0.05) by increasing the concentration of samples from 30 mg/ml to 90 mg/ml. Since the activity of spring propolis reached 15.39% at concentration 30 mg/ml, also it increased to 40.49%
when increasing the concentration to 60 mg/ml, then to 50.68% at a concentration of 90
mg/ml. and so for all samples. Similarly, BHT increased from 10.04% to 48.24% for autumn propolis, and from 19.23% to 50.58% in a mixture of wax with propolis, and from 40.26% to 88.21% for BHT.
The results in Figure (2) showed the antioxidant activity of both spring and autumn propolis, and a mixture of wax and propolis extracted with a diethyl ether solvent. The results of the statistical analysis showed that there were significant differences (P˂0.05) between the percentages of antioxidant activity, as in the mixture of wax with propolis was the highest activity, followed by the spring propolis, then autumn propolis, comparing it with the (BHT) compound, it was lower. These results also indicated that the antioxidant activity of spring and autumn propolis and the mixture of wax with propolis increased significantly (P˂0.05) by increasing the sample concentration from 30 mg/ml to 90 mg/ml. Therefore, the activity of the wax mixture reached 45.18% at a concentration of 30 mg/ml, and it increased to 60.14% when increasing the concentration to 60 mg/ml, then to 74.65% at concentration 90 mg/ml, and so on for all samples.
Similarly, BHT increased from 42.33% to 73.22% in spring propolis, and from 47.18% to 65.25% for autumn propolis, and from 40.26% to 88.21. % for BHT.
Figure (2) Percentage of the antioxidant activity of spring and autumn propolis and the mixture of wax and propolis extracted with a solvent of diethyl ether
Identification and quantification of bioactive compounds in honey bee products, including the phenolic compounds in propolis, differ according to the different methods used in separation and extraction (Spulber et al., 2017). This result was consistent with Miguel et al., ( 2014) when studying the antioxidant activity of thirteen types of propolis from different regions of southern Portugal, as the antioxidant activity ranged between
these types 35.76 - 83.27%. The reason for the difference in activity was attributed to the difference in their content of phenols and flavonoids.
Popovaa et al., (2015) studied the chemical composition of three different extracts of propolis collected from three regions in Morocco, which are, Fez, Rabat, and al-Gharb.
The samples of Fez and Rabat showed a high content of flavonoids, and the Rabat sample contained a lower percentage of phenolic acid esters. Besides, it did not contain pinobanksin-3-O-acetate, as for the Fez sample, it contained a high percentage of diterpenes, 74.3%. The three samples showed high activity in inhibiting the enzymes of glucosidase, amylase, lipid peroxidation, and the ability to free radicals scavenging.
Therefore, 7 different samples of propolis were brought from different regions in Morocco, and they showed big differences in chemical composition and antioxidant and anti-microbial activity. Propolis with a high content of resin and wax contains a high percentage of antioxidant compounds and microbes, the samples of B, C, and G also contained caffeine, chrysin, pinocembrin, and galangin, which shows antioxidant activity and this depends on the predominant tree species in the sample collection area (Touzani et al., 2018). Some substances that contain a high amount of one or two compounds sometimes do not show high activity as antioxidants or antimicrobials. But, their synergistic effect is higher depending on the different proportions of their components, including the difference in the ratio of propolis to wax produced by bees (Burdock, 1998).
The inhibitory effect on yeasts
The results in Table (1) showed the relationship between the concentration of propolis extract in its autumn and spring types and the mixture of beeswax with propolis prepared with two types of concentrated solvents, which are ethanol and diethyl ether. As well as, the comparison treatment of concentrated solvents only (0 g / ml) in their effect on yeasts and for both types of solvents, with an addition of 0.1 ml and 0.2 ml. Accordingly, the results of the statistical analysis showed that there were significant differences (P˂0.05) between the concentrations used, as well as between the types of solvents in the experiment and between the types of yeasts.
Table (1) The inhibitory effect of autumn and spring propolis, a mixture of beeswax and propolis extracted with ethanol alcohol and diethyl ether against yeasts
Yeast type Candida albicans Candida glabrata Candida parapsilosis
Extract addition volume (ml)
0.1 0.2 0.1 0.2 0.1 0.2
Control (ethanol) Ethanolic extract of
autumn propolis
22
14.5
20 20
- -
25 19.5
14 11
19 30
Control (ethanol) 25 40 20 30 20 40
Ethanolic extract of a wax mixture with
propolis
15 16 - 17 - 30
Control (ethanol) Ethanolic extract of
spring propolis
20 18.5
15 20
12 17
25 23.5
13 14.5
35 22.5 Control (diethyl
ether) Extract of diethyl ether of spring propolis
20
21
17 20
13 15
25 20
15 16.5
30 24 Control (diethyl
ether) Extract of diethyl
ether of wax mixture with propolis
- -
- 20
- 15
17 15
- 20.5
- -
Control (diethyl ether) Extract of diethyl ether of Autumn propolis
- 20
- 20
- 22
- 25
- 20
- -
Table (1) showed that the highest inhibition of yeasts was found in the ethanolic extract of autumn propolis, ethanolic extract of wax mixture with propolis. Along with the extract of diethyl ether of spring propolis on genus C. parapsilosis with an additional volume of 0.2 ml and with an inhibition diameter of 30, 30, 24 mm compared with the control sample which reached 19, 40, and 30 mm, respectively, and also gave the most inhibitory effect on C. albicans. The inhibition diameter reached 20 mm for all treatments at a volume of 0.2 mm, except for the ethanolic extract treatment of the wax mixture with propolis, which gave an inhibition diameter reached 16 mm. Propolis is one of the most effective natural antibiotics that are characterized by their effect on a very broad spectrum of microbes (Lotfy, 2006). The ethanolic extract of propolis was used against the fungus Aspergillus ver. and Penicillium aur, it had a clear anti-effect, depending on the chemical composition and the concentration of propolis, as the concentration 10% gave the highest inhibition compared to concentrations 1% and 5%
(Temiz et al., 2013). The results were agreed with Herrera et al., (2010) when studied the antifungal activity of six types of propolis against three types of yeast, are Candida tropicalis C. albicans, C. glabrata, and that all of them were inhibited by the aqueous extract of propolis. Significant differences were observed between the concentration of phenols, which ranged between 9 ± 0.3 and 85 ± 2.1 mg/mL, and between the inhibitory activity of fungus, which is due to the presence of the following compounds: Kaempferol, quercetin, myricetin, caffeic acid, galangin, pinocembrin, apigenin, caffeic acid phenyl ester.
The inhibitory effect on fungus
Table (2) shows the effect of propolis extract in its autumn and spring types, and a mixture of beeswax with propolis prepared with solvents of ethanol and diethyl ether and its effect on fungus with an additional volume of 0.1 ml and 0.2 ml. Since the statistical analysis results showed significant differences (P˂0.05) between the concentrations used as well as between types of solvents in the experiment and between species of fungus.
Table (2) The inhibitory activity of spring and autumn propolis, and a mixture of beeswax and propolis extracted with ethanol alcohol and diethyl ether against fungus
Fungal type Penicillium sp. Aspergillus flavus Aspergillus niger Aspergillus fumigatus Extract addition
volume (ml)
0.1 0.2 0.1 0.2 0.1 0.2 0.1 0.2
Control (ethanol) Ethanolic extract
of autumn propolis
15 -
17 15.5
17 15.5
17 -
- -
15 -
-
17 16
Control (ethanol) Ethanolic extract of a wax mixture with propolis
17 13
13 20.5
13 20.5
20 -
-
- -
- -
20 17
Control (ethanol) Ethanolic extract of spring propolis
22 18.5
22 27.5
12 18
30 18.5
17 17.5
20 21
25 17
12 22.5 Control (diethyl
ether) Extract of diethyl ether of spring propolis
22 16.5
26 30
12 19.5
30 20
15 16.5
25 24
25 20.5
11 20.5 Control (diethyl
ether) Extract of diethyl
ether of wax mixture with propolis
- -
- 17.5
- -
- 27.5
- 15
- 27.5
- -
-
17.5
Control (diethyl ether) Extract of diethyl ether of Autumn propolis
22 19.5
15 22.5
15 20
- 23
- 15
20 18.5
- -
- -
The statistical analysis results showed that there were significant differences (P˂0.05) between the average concentrations used as well as between the species of fungus. It was found that the additional volume (0.2) ml was the best in inhibition of fungus, as the highest inhibition diameter of Penicillium sp. was 30 mm when treated with diethyl ether extract of spring propolis. Followed by fungus Aspergillus flavus and Aspergillus niger, with the inhibition diameter of 27.5 mm each when treated with diethyl ether extract of wax mixture with propolis. However, treatment with the two extracts of ethanol of spring
propolis and diethyl ether of spring propolis gave the highest inhibition diameter of Aspergillus fumigates amounted to 22.5 and 20.5 mm, respectively. The activity of propolis, pollen, and beeswax dissolved in methanol at a concentration of 70 and 99.9%
and ethanol at a concentration of 70 and 96% against fungi, yeasts, and bacteria in Slovakia against Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and yeasts Candida krusei, Candida, Candida. Candida glabrata Candida tropicalis. A. fumigatus was the most sensitive to propolis extract dissolved in ethanol 70%, and C. glabrata was the most sensitive to propolis extract dissolved in methanol at a concentration of 70%, and for beeswax extracts, the most sensitive were A. niger and C. Tropicalis (Miroslava et al., 2012). Finally, A study was conducted in Argentina by Quurioga et al., (2006) to determine the anti-fungal activity, as two compounds of propolis compounds, namely Pinocembrin and Galargin were compared and found that they have an effective anti-fungal effect. The antibacterial activity of propolis depends on its chemical composition, as inhibition is due to the presence of flavonoids, esters, acids, alcohols, aromatic compounds, terpenes, aliphatic carboxylic acids, and esters of aliphatic carboxylic acids (Temtz et al., 2011).
Conclusions
Spring and autumn propolis extract and wax mixture with propolis were used using ethanol and diethyl ether solvents. The best antioxidant activity was recorded at a concentration of 90 mg/ml and with varying proportions. The aforementioned extracts also recorded inhibitory efficacy for the studied yeasts and fungi with varying inhibition rates.
Acknowledgment
The researchers extend their sincere thanks to the Basra Specialist Society for Bees Husbandry and Production for providing samples of propolis, and to the Department of Biology at the Colleges of Science and Education / the University of Basra for providing samples of yeasts and fungi.
References
1. Al-salmani, A.k.m.; and Hassan ,I. MQ. (2011). Evaluation of the antibacterial activity of ethanol extract of Iraqi propolis in vitro on some pathogenic bacteria causing mastitis and pneumonia in cows. Journal of veterinary science, 4 (2): 97- 104.
2. Ansari, A. M.; Anurag, A. A.; Fatima, Z. and Hameed, S. (2013). Natural phenolic compounds: A potential antifungal agent. In A. Méndez-Vilas(Ed.), Microbial pathogens and strategies for combating them: Science, technology and education, 1189-1195.
3. Bankova,V.(2005).Chemical diversity of propolis and the problem of standardization. Journal of Ethnopharmacology,100:114-117.
4. Bobis, O.; Bonta, V.; Varadi, A.; Strant, M. and Dezmirean, D.(2017). Bee Products and Oxidative Stress: Bioavailability of Their Functional Constituents. Mod. Appl. Bioequiv. Availab., 1(3):P1-5 DOI:
10.19080/MABB.2017.01.555565
5. Boyanova L, Derejian S, Koumanova R (2003) Inhibition of Helicobacter pylori growth in vitro by Bulgarian propolis: Preliminary report. J Med Microbio.,l 52(5): 417-419.
6. Burdock, G.A.(1998). Review of the biological properties and toxicity of bee propolis. Food Chem Toxicol., 36(4): 347-363.
7. Çandir, E.E.; Özdemir, A.E.; Soylu, E.M.; fiahinler,N. and Gül, A. (2009).
Effects of propolis on storage of sweet cherry cv. Aksehir Napolyon. Asian. J Chem., 21: 2659-2666.
8. Hanna ,E. R. (2009). Antimicrobial activity of propolis agents on some pathogenic microbes. Journal of Baghdad science, 1(7):269-276.
9. Herrera , C., L. ; Alvear, M.; Barrientos, L. ; Montenegro, G.; and Salazar , L. A. (2010). The antifungal effect of six commercial extracts of Chilean propolis on Candida spp. Cien. Inv. Agr.,37(1):75-84.
10. Kalogeropoulos, N.; Konteles, S. J.; Troullidou, E.; Mourtzinos, I. and Karathanos, V. T. (2009). ‘Chemical composition, antioxidant activity and antimicrobial properties of propolis extracts from Greece and Cyprus. Food Chemistry, 116(2):452–461. doi: 10.1016/j.food chem.2009.02.060.
11. Lotfy, M. (2006). Biological activity of bee propolis in health and disease. Asian Pacific Journal of Cancer Prevention, 7(1):22-31. doi: 10.1007/s00114-011- 0770-7.
12. Miguel, M. G.; Susana, N.; Susana A.D.; Ana M, C. and Maria, D.A.(2014).
Phenols, flavonoids and antioxidant activity of aqueous and methanolic extracts of propolis (Apis mellifera L.) from Algarve, South Portugal. Food Sci. Technol, Campinas., 34(1): 16-23.
13. Miroslava K.; Vukovic, N.; Chlebo, R.; HaŠcík, P.; Katarína, R.; Cubon, J.;
Malgorzata, D. and Anna P.(2012). The Antimicrobial Activity of Honey, Bee Pollen Loads and Beeswax from Slovakia. Arch. Biol. Sci., Belgrade., 64 (3): 927- 934. DOI:10.2298/ABS1203927K of polyphenols from bee pollen and propolis.
Agro Life Scientific Journal, 6(2):183–194.
14. Popovaa, M.; Lyoussi, B.; Aazzac, S.; Antunesc, D.; Bankovaa, V. and Miguel G.(2015). Antioxidant and glucosidase inhibitory properties and chemical profiles of Moroccan propolis. Natur Prod Comm., 10(11): 1961-1964.
15. Quiroga, E. N.; Sampietro, D. A.; Soberón, ,J.R.; Sgariglia,M. A. and Vattuone, M. A. (2006). Propolis from the northwest of Argentina as a source of antifungal principles. Journal of Applied Microbiology, 101(1): 103-110.
16. Silva, B.M.; Andrade, P.B.; Valentao, P.; Ferreres, F.; Seabra, R.M. and Ferreira, M.A. (2004). Quince (Cydonia oblonga Miller) fruit (pulp, peel and seed) and jam: Antioxidant activity. J. Agric. Food Chem., 52:4705–4712.
17. Spulber, R.; Colța, T.; Băbeanu, N. and Popa, O. (2017). Chemical diversity standardization .Journal of Ethnopharmacology.,100:114-117.
18. Temiz , A . ; Sener,A. ; Özkök Tüylü , A. ; Sorkun, K. and Salih ,B. (2011).
Antibacterial activity of bee propolis samples from different geographical regions of Turkey against two foodborne pathogens, Salmonella Enteritidis and Listeria monocytogenes.Turk J.Biol. 35 ,503-511.
19. Temiz, A. ; Mumcu, A. S. ; Tüylü, A. Ö. ; Sorkun, K. and Salih, B. (2013).
Antifungal activity of propolis samples collected from different geographical regions of turkey against two food-related molds, Aspergillus versicolor and Penicillium aurantiogriseum . GIDA , 38 (3): 135-142.
20. Touzani, S.; Noori, A-W.; Nawal, E-M.; Bratko, F.; Adriana P.; Ilham, E-A.;
Wail, A-W. and Badiaa, L.(2018). Chemical analysis and antioxidant content of various propolis samples collected from different regions and their impact on antimicrobial activities. Asian Pacific Journal of Tropical Medicine , 11(7):436- 442.
21. Wijerante, S. S. K.; Abou-Zaid, M. M.; and Shahidi, F. (2006). Antioxidant polyphenols in almond and its co products. Journal of Agriculture and Food Chemistry, 54, 312–318.
