Biological Activity of Different Botanical Origin of Propolis

Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Microbiology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers related to the research across the whole spectrum of the subject. These including bacteriology, virology, mycology and parasitology. In addition, the journal promotes research on the impact of living organisms on their environment with emphasis on subjects such a resource, depletion, pollution, biodiversity, ecosystem.....etc www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Bees have been in existence for 125 million years and their evolutionary success has allowed them to become perennial species that can exploit virtually all habitats on Earth.This success is largely because of the application of the specific products that bees manufacture: honey, beeswax, venom, propolis (bee gum), pollen and royal jelly.Propolis regarded as the most important 'chemical weapon' of bees against pathogenic microorganisms (Wollenweber et al., 1990).Propolis (bee glue) is a natural products collected from beehives by worker bees from the bark of trees and leaves of plants.The color of propolis depend on the bees food, the common color is dark brown.This salivary and enzymatic secretions-enriched material is used by bees to cover hive walls to ensure a hospital-clean environment.propolis has a powerful effect on human health especially in fighting upper respiratory infections, such as common cold and influenza viruses (Focht et al., 1993).
The chemical composition of propolis is quite complicated.More than 300 compounds such as polyphenols, phenolic aldehydes, sequiterpene quinines, coumarins, amino acids, steroids and inorganic compounds have been identified in propolis samples.The contents depend on the collecting location, time and plant source.Consequently, biological activities of propolis gathered from different phytogeographical areas and time periods vary greatly (Lotfy, 2006).
The anterior nares are the major site of Staphylococcus aureus colonization in humans (Wertheim et al., 2005).S.aureus often cause β-hemolysis (Ryan and Ray, 2004).Common disease of S.aureus are pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome.Other Gram -positive that we used in this study is streptococcus pyogenues.S. pyogenus consider as the most actually life threating (Lamagni et al., 2009).S .pyogenes(betahemolytic; Lancefield group A) is a human pathogen that causes pharyngitis (strep throat), skin disease and many other infections.
The Pseudomonas aeruginosa can cause pneumonias, urinary tract infections as well as causing high morbidity and mortality in patients with cystic fibrosis due to chronic infections that eventually cause pulmonary damage and respiratory insufficiency (Ochoa et al., 2013).
Escherichia coli typically colonizes the gastrointestinal tract of human infants within a few hours after birth.E.coli that cause diarrhea, nonspecific gastroenteritis especially in children and the hemolytic uremic syndrome (Nguyen et al., 2006).
The objective of this work was to investigate antimicrobial properties of four ethanolic extract propolis samples obtained from different regions of the world against six microorganisms.

MATERIALS AND METHODS
The present investigation was carried out at the research laboratory, Biology section, faculty of Science, Taibah University, Al-medina to study the antibacterial activity of four samples of propolis on six types of bacteria.

Propolis samples:
The four samples of propolis were Saudi, Egyptian, Chinese and Turkish propolis.All these samples were obtained from commercial bee products market in Saudi and stored in the refrigerator until using.

Preparation of propolis extract solution:
Propolis samples were extracted by maceration at room temperature, with occasional shaking, in the proportion of 10 g of (Saudi, Egyptian, Chinese and Turkish) propolis to 100 ml of solvent (ethanol 80%v/v), extracts were obtained after 7 days of maceration and the ethanolic extracts were then filtered by Whatman (No.1) filter paper and incubated at room temperature until ethanol evaporated and the product obtained a honey-like consistence are referred to as ethanolic extract propolis, this method was reported by Ildenize et al. (2004).

Preparation of different concentrations of Saudi, Egyptian, Chinese and Turkish ethanolic extract propolis:
According to (Bauer et al., 1959), 1.0 g of each ethanolic extract propolis was dissolved in 10 ml ethanol (80%) by shaking, for 30 min protected from light.The resulting aqueous-ethanol extract was filtered through a Whatman (No.1) paper (Menezes et al., 1997).This final solution was (100mg/ml) employed for the antibacterial assay and Five serial concentrations were10.00,7.50, 5.00, 2.50 and 1.25 mg/ml of the ethanolic extract propolis to the assay.

Microorganisms:
Six bacterial strains were studied.Antimicrobial activity of propolis extracts was investigated using agar diffusion method.Test plates, were prepared of Muller-Hinton agar (MHA) according to (Muller and Hinton, 1941), and three wells were punched in the agar plates by using sterile glass-made pipettes attached to a vacuum pump.Sterile swabs were dipped into the bacterial suspension containing., and inculcated on to plate surfaces.Each well was filled with 100 µm of extracts (80 % ethanol and distilled water) the plates were kept for 2 hours at room temperature to allow the diffusion of the agents through the agar.Afterwards, the plates were incubated for 24 hours at 37° C. The control for all experiments was 80% ethanol; determination of the antimicrobial activity was done by measuring zones of inhibition using a ruler based on (Johnson and Case, 1995).

Statistical Analysis
Results were analysed using prism program and the probability p=0.05 was considered the critical value for all tests.

RESULTS
Antimicrobial activities of Saudi, Egyptian, Chinese and Turkish Ethanolic Extract Propolis against positive and negative bacteria test organisms are summarized in table 1 and illustrated in figure 1 in generally, all ethanol extracts of propolis samples showed inhibitory action on all tested bacteria, in addition antimicrobial activities of these extracts showed different inhibitory effect against all the tested bacteria, Gram-positive and Gram-negative bacteria.
Data recorded in table 1, and figure 1 showed that, Egyptian propolis had highly significant effect on different types of bacterial compared to control group which record 0.0 mm.The highest diameter of inhibition zone at 1.25and 2.5% concentration was (5.7and 6.7 mm) with Streptococcus pyogenes respectively, in addition.At 5% concentration the highest diameter inhibition zone was (7.0 mm) with Proteus mirabilis and The highest diameter inhibition zone was (8.7and 12.0 mm) At 7.5 and 10.0 % concentration with Proteus, respectively.in addition Saudi propolis recorded a highly significant effect against different types of bacteria compared to control group, the highest diameter inhibition zone at all concentration of study was on Proteus mirabilis was (6.4,9.0,9.1,9.4 and 14.0mm) at 1.25,2.5,5.0,7.5 and 10% concentration respectively . in the same line the Chinese propolis showed effect against different types of bacteria and Proteus showed the highest diameter inhibition zone at 1. 25, 2.50, 5, 7.50 and 10% concentrations (5, 7.3, 9.6, 11 ,13 mm) respectively.
While, Data recorded showed the effect of Turkish propolis against different types of bacteria, and its obvious that Turkish propolis showed the highest diameter inhibition zone on Klebsiella pneumoniae was (5.1, 6.3, 7.5, 10.5 and 10.5 mm) at 1.25, 2.5, 5.0, 7.5 and 10% concentration respectively.
From the previous results we noticed that, the lowest diameter inhibition zone among the four types of propolis under study were recorded in Proteus mirabilis by Turkish propolis and Streptococcus pyogenes by Chinese propolis but on other hand the highest diameter inhibition zone were recorded in Escherichia coli by Egyptian propolis , Pseudomonas aeruginosa by Chinese propolis and Klebsiella pneumoniae by Turkish propolis.Saudi propolis against.

DISCUSSION
As indicated from the results of Antimicrobial activities of these extracts which showed different inhibitory effect against all the tested bacteria, In addition, all ethanol extracts of propolis samples showed inhibitory action on all tested bacteria, on Gram-positive and Gram-negative bacteria, and the results of the high concentration of the propolis exerted a high antimicrobial activity, while the low concentrations revealed a low response.This activity may be due to the high concentration of each extract, such as flavonoid and total phenolic compounds because this compounds play an important role as bactericidal agent which reported by many researchers, so the bactericidal action of propolis was concentration dependant, more over The present findings indicate that the antibacterial activity of propolis, could not be correlated with their propolis concentration only but mostly to their chemical composition which can be variable according to the collection site and vegetal source and depends upon its botanical origin.
The studies of propolis against a wide variety of microorganisms all over the world show strong antibacterial activities (Koo, et al., 2000 andSteinberg, et al., 1996).
The present study showed a noticed effect of different types of propolis against the different types of tested bacteria, similar finding was reported by (Miyataka, et al., 1997 andBuono, et al., 2001).Propolis extracts also have antibacterial properties against some micro-organisms (Takaisi andSchilcher, 1994 andNieva, et al., 1999).Recent pharmacological studies stated that, propolis has a wide range of sophisticated composition including anti-inflammatory, anti-oxidative and anti-microbial (Burdock, 1998), immunomodulatory properties (Orsolic and Basic, 2003) also antitumor, anticancer, antiulcer, hepatoprotective, cardioprotective, and neuroprotective (Drago and Vecchi, 2007).Propolis also had polyphenols including flavonoids and phenolic acids (Cao, et al., 2004).The antibacterial effects of propolis in this study are parallel to (Soley, et al., 2011), whose found that, propolis had good in vitro antibacterial activity against E. faecalis in the root canals, suggesting that, it could be used as an alternative intra-canal medicament.Ferreira, et al., (2007) also found that, propolis had an antibacterial effect on selected endodontic anaerobic bacteria (Prevotella nigrescens, Fusobacterium nucleatum, Actinomyces israelii, Clostridium perfringens and E. faecalis).Bruschi, et al., (2006)  Tube dilution studies showed that, it was bactericidal for B. cereus at dilutions of 1: 160 to 1: 320 and that, growth of Mycobacterium tuberculosis was totally inhibited at 1: 320 and partially inhibited at 1: 640 (Grange and Davey, 1990).Propolis is mainly active against S. aureus and it is effective on P. aeruginosa at higher concentrations.That is sensitivity of S. aureus strains against antibacterial activity of ethanol extract of propolis with mean inhibitory diameter (17.66 ± 0.47-10 mm) followed by P. aeruginosa strains (7 mm) were recorded by (Zeighampour, et al., 2013).The chemical composition of propolis is quite complicated and its compounds and biological activities depend on many different factors such as the geographical region, collected time and plant source (Haggag, et al., 2006;Sforcin, et al., 2000 andBankova, et al., 2002).Among the numerous groups of substances identified in propolis samples from different localities, the most common are caffeic, p-Coumaric acid, aromatic acids, esters, chalcones, flavonoids, terpenoids, waxy acids, cinnamic acid derivatives, amino acid, fatty acids, vitamins like (B1, B2, B6, C and E), minerals like (Mg, Ca, I, Na, Cu, Zn, Mn and Fe) and enzymes like (adenosine triphosphatase, succinic dehydrogenase and glucose 6-phosphatase) (Marcucci and Bankova, 1999;Velikova, et al., 2000;Marcucci, et al., 2001 andZeighampour, et al., 2013).Flavonoids are thought to account for much of the biologic activity in propolis.The antimicrobial properties of this mixture of natural substances are mainly attributed to the flavonone pinocembrin, to the flavonol galangin and to the caffeic acid phenethyl ester (Takaisi andSchilcher, 1994 andBosio, et al., 2000).Active component of propolis, may be responsible for most of these biological activities (Hepsen, et al., 1977 andKoltuksuz, et al., 1999).
The mode of action is Propolis inhibits bacterial growth by preventing cell division, thus resulting in the formation of pseudomulticellular streptococci.In addition, propolis disorganized the cytoplasm, cytoplasmic membranes and cell wall, caused a partial bacteriolysis, inhibited protein synthesis also inhibit bacterial enzymes.The antibacterial mechanism also depends on the inhibition of bacterial RNApolymerase (Takaisi and Schilcher 1994).

Fig. 1 :
Fig. 1: the antibacterial effect of Saudi, Egyptian, Chinese and Turkish propolis against different types of bacteria.(Zones of Inhibition / mm).
Staphylococcus aurous and Streptococcus pyogenus, which are gram-positive bacteria,

Table 1 :
the antibacterial effect of Saudi, Egyptian, Chinese and Turkish propolis against different types of bacteria.(Zones of Inhibition / mm).