Egypt. J. Plant Prot. Res. Inst. (2019), 2 (4): 617 - 723

Egyptian Journal of Plant Protection Research Institute www.ejppri.eg.net

Potential antimicrobial activity of different types of Libyan honeys

Abouzeid, A.S. 1; Reham, A.M. El-Wassef2 ; Sarah, H. El-Dereny3 ; Khaled, M.A. Abdel- Hameed3 and Mohamed, S. Younis³ ¹Entomology Deparment, Faculty of Science, Ain Shams University. ² Economic Entomology Department, Faculty of Agriculture, Cairo University. ³ Plant Protection Research Institute, Agricultural Research Center, Dokki, Giza, Egypt. ARTICLE INFO Abstract: Article History Honey exhibits antimicrobial activity against a wide range Received: 21/ 10 / 2019 of bacteria. The aim of the present work was to evaluate the Accepted: 18 / 11 /2019 antimicrobial effects of the Libyan honeys harmal (Peganum ______harmala L.) , red camphor (Cinnamomum camphora), white Keywords camphor (Eucalyptus globule), sarou (Cupressus sempervirens), Honey, antimicrobial athl (Tamarix aphylla ) and kharoub (Ceratonia silique ) on activity, bacteria , Staphylococcus aureus, Bacillus subtilis, Klebsiella pathogens and Libya. pneumoniae, Enterococcus faecalis, Pseudomonas aeuroginosa, Escherichia coli, Bacteriods spp., Sarcina spp. and Candida albicans. Pathogens exhibited different sensitivities towards the honey samples. The results showed that C. camphora inhibited seven out of the nine tested microorganisms followed by T. aphylla honey, which inhibited six of them. The lowest effects were shown by P. harmala and C. semperviren honeys, where they only inhibited four different types of the tested microorganisms.

Introduction Honey is a complex natural food α-tocpherol (Ferreres et al.,1993). Honey produced from the honey bee Apis mellifera contains at least 181 components (White, L. (Hymenoptera: Apidae) feeding on plant 1975). nectar of blossoms, exudates of trees and The use of honey for the treatment of plants or from honey bees feeding on diseases and wounds has been mentioned honeydew produced by hymenoptran insects. since ancient time (2100-2000 BC), where Honey is a saturated solution of sugar of 31% Aristotle (384-322 BC) described pale honey glucose and 38% fructose and its colour and for sore eyes and wounds (Mandal and flavor vary considerably depending on it Mandal, 2011 and Vallianou et al. , 2014). botanical and geographical origin (Gheldof et Microorganisms such as Staphylococcus al., 2002) and of a moisture content of about aureus, Staphylococcus epidermis, 17.7% (Nagai et al., 2006). In addition to Micrococcus luteus, Streptococcus uberis, minor component of phenolic acids, Enterococcus faecalis, Pseudomonas flavonoids, glucose oxidase, catalase, aeruginosa, Escherichia coli and Klebsiella ascorbic acid, carotenoids, organic acids and pneumonia are frequently isolated from 717

Abouzeid et al. , 2019 human and animal skin wounds (Vuong and ulcers and the causative agent in many cases Otto, 2002; Nasser et al., 2003; Halcon and of dyspepsia has raised the possibility that the Milkus, 2004; Altoparlak et al., 2005 and therapeutic action of honey for symptoms of Basualdo et al., 2007). dyspepsia may be due to Manuka honey’s The healing effect of honey could be antibacterial properties. Somal et al. (1994) due to its physical and chemical properties demonstrated that after an incubation period (Rusell et al., 1990 and Snow and Manley- of 72 h.,5% Manuka honey completely Harris, 2004) and to its antioxidant and prevent the growth of H. pylori (the causative antimicrobial activity (Martos et al., 2000; organism of stomach ulcers). Similar invitro Escuredo et al., 2012; Vandamme et al., antimicrobial results of Manuka honey was 2013; Isidorov et al., 2015; Francine et al., reported against Campylobacter spp. (Lin et 2016; Almasaudi et al., 2017 and Leyva- al., 2009). Jimenez et al., 2019). Honey acts as an The aim of the present work was to effective thermal insulator and a protective evaluate the antimicrobial effects of the biofilm (Black and Costerton, 2010). Its Libyan honeys Harmal (Peganum harmala), antimicrobial activity is connected with its red camphor (Cinnamomum camphora), osmotic pressure which draws fluid from white camphor (Eucalyptus globule), sarou wounds, decreasing tissue edema (Molan, (Cupressus sempervirens), athl (Tamarix 2001). A possible reason for its activity aphylla) and Kharoub (Ceratoniasilique) on depends on its ability to generate hydrogen Staphylococcus aureus, Bacillus subtilis, peroxide by the bee derived enzyme glucose Klebsiella pneumoniae, Enterococcus dehydrogenase (Saleh et al., 2011). The faecalis, Pseudomonas aeuroginosa, strength of honey hydrogen peroxide is much Escherichia coli, Bacteriods spp., Sarcina lower than pharmacologic hydrogen spp. and Candida albicans. peroxide, causing no damaging to the healing Materials and Methods environment of a wound (Bang et al., 2003). The present investigation was carried Wound size is affected by pH value, Gethin out at the Beekeeping Research Section, et al. (2008) found that honey reduces the Plant Protection Research Institute, Giza, wound's pH and every 1 % reduction in pH is Egypt. associated with a 1 % reduction in wound 1.Honey samples: size. For wounds contaminated by Six types of Libyan honeys of mono methicillin-resistant Staphylococcus aureus, and multi-floral source were collected from Manuka honey supported better wound selected beekeepers during the harvesting healing than antibiotics (Gethin and periods and from local markets in Western Cowman, 2008). There are several studies Libya. The honeys of mono-floral source indicating the effectiveness of honey in were harmal (P. harmala, red camphor treating burns (Molan, 2001; Subrahmanyam, (C.camphora), white camphor (E. globule), 1991 and Subrahmanyam et al., 2001). sarou (C. sempervirens), athl (T. aphylla) and Sukur et al. (2011) studied the kharoub (C. silique). Honey samples were effectiveness of Tualang honey in healing kept in dark at room temperature prior to full-thickness burn wounds in rats. They analysis. The samples were investigated found that topical application of honey on microscopically to determine their containing burn wounds contaminated with of pollen grain types (Table, 1). Pseudomonas aeruginosa and Acinetobacter 2.Bacterial strains: baumannii give better results of healing Bacterial strains and C. albicans were compared with other treatments. kindly donated by the Microbial Genetic The finding that the bacterium, Department, Genetic Engineering and Helicobacter pylori is a cause of stomach

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Biotechnology Division, National Research control are shown in Table (2). It was Center, Giza, Egypt. observed that all honey samples inhibited the 3. Assay of antimicrobial activity: growth of C. albicans with different degrees, Antimicrobial activity of honey where P<0.001. Bacteroids spp. was the most samples was determined by the disc diffusion resistant bacteria, where it was only inhibited method (Collins et al., 1995). A by C.camphora honey with an inhibition concentration of 20% of each kind of honey zone of only 5.0+0.67 mm. Except C. silique in distilled water was prepared in clean sterile honey all honey samples affect the growth of test tube and kept in refrigerator at 4oC to be E. faecalis with different degrees. B. subtilis used for microbiological test. was moderately inhibited by T. aphylla and 4.Preparation of the microbial culture: C. silique honeys with inhibition zones of The tested organisms were inoculated 11.33+0.57 and 11.66+0.57 mm, in the appropriate liquid media and incubated respectively. C. camphora inhibited seven at 37 oC for 24 h. The microbial culture was out of the nine tested microorganisms used for the preparation of seed layer by followed by T. aphylla honey, which inoculating the agar medium with 2% (v/v) of inhibited six of them.The lowest effects were the microbial culture, thoroughly mixed and shown by P.harmala and C. semperviren immediately used as the seed layer of plates. honeys, where they only inhibited four 5.Preparation of plates: different types of the tested microorganisms. The appropriate agar medium was Escherichia coli, P. aeruginosa and distributed at the rate of 7 ml portion in petri Bacteriods spp. were found to be resistant to dishes. After solidification 5 ml of the seeded the antibiotic tetramycine (+ve control), agar was distributed over the surface of the while 20% sucrose sugar solution (-ve base layer and left for 15 min to solidify. The control) had no inhibitory effect on all previously prepared filter paper discs (each bacterial strains. disc was moistened with exactly 0.05 ml of The antimicrobial activity of honey is the diluted honey) placed side down on the mainly contributed to the high osmolarity and seeded agar and gently pressed with a tip of acidity. In addition, hydrogen peroxide, sterile forceps. Discs were placed volatiles, organic acids, flavonoids, phenolic symmetrically around the center of the dish. compounds, wax, pollen, propolis are Plates were incubated at 37 oC for 24 hours. important factors that provide antimicrobial for P. aeruginosa and M. leutus, plates were properties to honey. Shin and Ustunol (2005) incubated at 30 oC. Antimicrobial activity stated that the sugar composition of honeys was determined measuring the diameter of from different floral source are responsible inhibition zones around the discs to the for the inhibition of various intestinal nearest mm (Table, 2). bacteria. According to Moumbe et al. (2013) Three replicates were prepared for each the minor components of honey including honey sample. As a positive control method, proteins, minerals, phytochemicals and the antibiotic tetracycline (30 µg) was used, antioxidants are responsible for the while sucrose sugar solution (20%) was used antimicrobial activity of honey in the as a negative control method. treatment of infections, burns, wounds and 6. Statistical analysis: ulcers. Results are expressed as mean + Our results are in agreement with other standard deviation. ANOVA were applied at published studies, showing that some kinds a confidence level of 95%. of honey have an inhibitory effect against the Results and discussion fungus C. albicans and the bacteria S. aureus, The results of inhibition effects of B. subtilis, K. pneumoniae, E. faecalis, P. different honey samples in comparison to aeuroginosa, S. coli, Bacteriods spp. and

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Sarcina spp. (Basualdo et al., 2007; Mercan activities of different honeys. Our data are in et al., 2007; Al-Haj et al., 2009; Sherlock et agreement with the findings obtained by al,. 2010; Francine et al., 2016; Almasaudi et McCarthy (1995), who reported that, honey al., 2017 and Leyva-Jimenez et al., 2019). from different floralsources varies greatly in The results of this study are similar to their antibacterial activity. Rybak and the results obtained by Mohapatra et al. Szczęsna (1996) found that the minimum (2011), who reported that honey was concentrations of honey which inhibit the effective against gram-positive bacteria S. growth of B.subtilis were 5-10%. Molan et al. aureus, B. subtilis, E. faecalis and gram- (1988) reported significant differences negative bacteria E. coli and P. aeruginosa. between different kinds of floral honey in The inhibitory effect of honey against their activities on S. aureus at dilutions of S. aureus, E. coli and K. pneumonia is of 1/4, 1/8 and 1/16 original strength. Radwan et great importance due to the fact that al. (1984) reported that honey from Acacia Streptococcus species and coliforms are mellifera inhibits the growth of E.coli. Molan recognized pathogens. In this work the and Russell (1988) found that pollen present growth of Pseudomonas aeruginosa was in honey could be the source of the inhibited by 3 honey samples (C.camphora, antibacterial aromatic acids, which causes the E. globule and C. sempervirens). This type of component to act individually or synergically bacteria is always found in wounds, to prevent bacterial resistance (Cooper et al., especially those related to burns causing a 2010). In addition to pollen, propolis is also variety of systemic infections, particularly in found in honey. The antimicrobial and anti- victims with severe burns (Yau et al., 2001). inflammatory activity of European propolis is Irish et al. (2011) noted that temperature, the associated with the presence of flavonoids, time of storage, and the nature of flower's flavones, and phenolic acids and their nectar may explain the different antimicrobial derivates (Bankova, 2005).

Table (1):Types and floral sources of Libyan honeys. No. of samples Local name of honey Floral source Sample 1 Harmal Peganum harmala Sample 2 Red camphor Cinnamomum camphora Sample 3 White camphor Eucalyptus globulu Sample 4 Sarou Cupressus sempervirens Samples 5 Athl Tamarix aphylla Sample 6 Kharoub Ceratonia silique

Table (2): The diameter and standard deviation (in mm) of inhibition zones of different bacterial strains by honey samples compared to control. Bacterial strains Peganum Cinnamomum Eucalyptus Cupressus Tamarix Ceratonia Tetracycline Sucrose harmala Camphora globulu Sempervirens aphylla Silique solution Escherichia coli 10.33±0.57b 11.00±0.00b 11.66±0.57b 0.00 0.00 0.00 0.00 0.0 Enterococcus faecalis 21.0± 0.0c 23.66±1.52c 5.33±57a 24.0±0.00c 12.0±1.00b 0.00 23.66±1.52c 0.0 Staphylococcus aureus 0.00 0.00 0.00 21.33±1.15c 6.0±0.5a 5.66±1.15a 22.0±0.1.5c 0.0 Pseudomonas 0.0 10.33±0.57b 10.66±0.57b 6.66±0.57a 0.00 0.00 0.00 0.0 Bacillusaeruginosa subtilis 0.00 0.00 0.00 0.00 11.33±0.57b 11.66±0.57b 21.33±1.15c 0.0 Bacteroids spp. 0.00 5.0±0.67a 0.00 0.00 0.00 0.00 0.00 0.0 Sarcina spp. 22.0 ±0.67c 10.0± 1.12b 0.00 0.00 11.0±0.00b 11.0±0.00b 23.66±1.52c 0.0 Klebsiella pneumoniae 0.00 6.0±.00a 11.0.± 0.00b 0.00 6.0±0.50a 5.0.±0.00c 6.0±0.00a 0.0 Candida albicans 20.33±0.57c 20.66±0.57c 10.66±1.15b 11.0±0.00b 20.66±1.15c 21.33±0.57a 22.0±67c 0.0 Different letters indicate significant difference (P< ퟎ. ퟎퟎퟏ).

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