INTRODUCTION:

Except for few products like honey as food, silk for clothing and pollination of plants, mostly people think of insects as pests. So much so that even honey bees, which are in fact man’s best friends have been labelled as pests. The public perception of insects needs improvement and the profiles of beneficial species needs to be raised. This is especially important as more and more natural products are being extracted from insects and knowledge of their physiological roles is used for the benefit of man [1]. Honey bees are social insects and known as nature’s best architects and chemical engineersas their products have been used in home remedies of a variety of ailments since times immemorial. Apitherapy was only limited to the use of bee venom but nowadays Apitherapy is a much broader term covering the medicinal use of all the products of honey bees or bee hive like honey, pollen, propolis, royal jelly, bee venom and bee wax. Because of their natural occurrence and quality, bee products are also added as adjuvant in the formulation of different drugs and medicines.

Propolis is a basically a complex resinous material which the honey bees produced from plant exudates, bees wax and bee secretions [2]. It is used to seal the hive and it has anti microbial role in the hive itself. Propolis has a very complex composition containing around 300 constituents. According to different areas, seasons and vegetation of that particular area the composition of propolis varies. These constituents included wax, resins, balsams, essential oils, amino acids, sugars, flavonoids and derivatives of cinnamic acid [3,4]. Since the composition of propolis varies from area to area and also from season to season so is the case with its different activities. The abundance of one constituent increases its that particular activity and so on. The present study is focused to discuss the antibacterial role of bee propolis against Typhoid causing bacteria i.e. Salmonella.

Salmonella are Gram negative bacteria and have gained importance not only as a threat to worldwide public health but also as a model for studying of mechanisms of bacterial pathogenesis. Genus Salmonella has been divided into 2 major species viz. S. enterica and S. bongori [5] and which are further divided into various subspecies and serovars. Mainly two serotypes of Salmonella enteric i.e. Typhi and Paratyphi caused typhoid fever in India [6]. The identified risk factors include intake of street food, ice creams, contaminated water, poor sanitary conditions at home and excessive use of antimicrobial drugs [7,8]. The infecting dose of S. typhi ranged between 1000 and 1 million microorganisms for a healthy individual [9]. Every year around the world around 21 million cases of typhoid fever are reported [10,11,12]. The cases of this neglected illness are around 1600 per 100,000 individuals in some regions of South Asia. Due to increasing resistance to drugs in the endemic areas, typhoid fever is becoming more and more difficult to treat [13,14,15]. Since hematological indices provide very important information regarding the well being of an individual [16,17]. So, the present study discussed the effect of pretreatment of mice with different doses of propolis extract before infecting the mice with deadly Salmonella.

MATERIALS AND METHODS:

Collection of Propolis and preparation of extracts:

Propolis was obtained from honeybee hives kept in an apiary maintained by Department of Zoology, Panjab University, Chandigarh, India. The ethanolic extract of propolis (EEP) was prepared following the standard protocol [18].

Microorganisms:

The bacterial strain of Salmonella enterica serovar Typhimurium (MTCC 98) was procured from IMT E CH, Sector-39 Chandigarh and stored in the form of small aliquots at -20°C before subculturing. The strain was examined biochemically before storage and use.

Experimental Animals:

4-6-week-old BALB/c mice weighing 20-25g were used in the experiments. The animals were obtained from the Central Animal House, Panjab University, Chandigarh, India. The mice were fed standard pellet diet and water ad libitum. All the experiments were carried out strictly according to the guidelines and with the approval of the Animal Ethical Committee, Panjab University, Chandigarh (IAEC/411 dated 11/9/2013). Animals were checked regularly for any bacterial infection by streaking the tail vein blood directly on MacConkey agar.

Treatment regimens:

Animals were divided into six groups and each group consisting of six mice

Gp Normal: Normal mice given saline orally (negative control), Gp Infected: Salmonella enterica serovar Typhimurium infected: Mice given 2X10⁴CFU/mL (0.2 mL) once i.p. (positive control), Gp P1: Propolis at the dose of 300mg/kg b.w. for 10 days followed by S. Typhimurium infection, Gp P2: Propolis at the dose of 300mg/kg b.w. for 20 days followed by S. Typhimurium infection, Gp P3: Propolis at the dose of 500mg/kg b.w. for 10 days followed by S. Typhimurium infection, Gp P4: Propolis at the dose of 500mg/kg b.w. for 20 days followed by S. Typhimurium infection.

Sacrifices of preventive groups were made on the peak day of S. Typhimurium infection i.e. on 5^th day after infection.

Bacterial load:

Bacterial load in the blood and spleen of mice was assayed by plating the 10-fold serially diluted tissue homogenate on deoxycholate citrate agar (DCA) and maintaining overnight at 37°C [19].

Hematological studies:

Blood for hematology was collected in Ethylenediaminetetraacetic acid (EDTA) coated vials and examined for RBC count, WBC count, Hemoglobin, Packed cell volume (PCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Cell Hemoglobin Concentration (MCHC) etc by standard protocols [20,21].

Statistical analysis:

All values were expressed as mean ±standard deviation. Statistical differences between the various groups were evaluated by ANOVA (Tukey’s test). Values of p <0.05 were considered statistically significant.

RESULTS:

Two doses of propolis i.e. P300 and P500 were selected for analyzing the preventive activity of propolis against S. Typhimurium infection. Course of prophylactic medication was 10 and 20 days before giving infection. After giving infection sacrifice was made on the peak day of infection i.e. 5^th day to compare whether different doses of propolis had protective affect against the bacterial infection.

Survival percentage:

On the sacrifice day, 100% survival was observed in all the preventive groups with little or reduced signs of lethargy as compared to infected group. The animals of group P1 were very weak, lethargic and have hunched back.

Bacterial load in blood and different organs:

Bacterial load in Blood:

The bacterial count in the blood was observed to be 6.57±0.15 log CFU/mL at 120hrs. of infection. On pretreating with different doses of propolis, it was found that in case of P1 group, the bacterial count was 4.53±0.2 log CFU/mL by 120hrs. (p>0.05) which was not significantly less as compared to infected group. But in case P2, P3 and P4 the bacterial load was significantly (p<0.001) reduced. (Fig.1).

Fig.1. Graph representing bacterial load in blood of mice in preventive therapy.

Data is expressed as mean+SD.

#: p-value Infected vs P1, P2, P3 and P4 (#: p<0.001)

Bacterial Load in Spleen:

The bacterial count in infected group was 8.1±0.05 log CFU/g. In case of pretreated group (P1) the bacterial count was observed to be 7.55±0.04 log CFU/g which was almost the same as infected group. Whereas in case of P2, P3 and P4 groups significant reduction was observed (Fig. 2.).

Fig. 2. Histogram showing the bacterial load in spleen of mice on pretreatment with propolis and S. typhimurium infection.

Data is expressed as mean+SD.

*: p-value Infected spleen vs pretreated spleen.

(*: p<0.001)

Hematological studies:

Since the bacterial infection first comes in blood so the study of blood components is very important. The count of RBC, Hb, PCV, MCV, MCH, MCHC (Fig 3.), TLC and DLC (Fig.4) was significantly altered in infected group as compared to normal. In case of propolis pretreated groups specifically in P3 and P4 groups the levels of all the parameters were near normal range.

DISCUSSION:

Honey bees have been called Master alchemists since times immemorial because of the beneficial effects of the majority of bee products. The use of honey for the cure of common cough, colds, allergies and several types of weaknesses is well documented in our ancient epics. Today, however, the novel system of healing “Apitherapy” has been extended to the use of all bee products for the treatment of a variety of problems. It was in view of this background information that the present study originated. Earlier study showing the antibacterial effect of propolis on Salmonella infected mice confirmed that at a effective dosage of Propolis, a significant improvement was recorded in the survival percentage of experimental model [3]. Interestingly, Salmonella shows escaping behavior in the blood where suddenly the bacterial count decreases as Salmonella at that time leave the circulation and enters the reticuloendothelial system including liver and spleen. After that the survived bacteria multiplied in cells of these organs and were then released into blood which once again raised the bacterial count in blood causing secondary bacteremia, endotoxic shock and also rapid death [22,23]. Every drug weather natural or synthetic acts in dose dependent manner. Same is the case with propolis [24] as seen the above mentioned results. At low concentration the results were not significant but as the dosage was increased significant decline in the bacterial load both in blood as well as in spleen was recorded. Considering the effect of Salmonella on the hematological indices, it was very clear from the earlier studies that this bacteria causes anemia, leucopenia, eosinophilia [25,26,21] and this was may be due to haemophagocytosis, inhibition of haematopoeisis and bone marrow suppression [27,28]. And these effects were quite evident from the present study also. Previous studies have shown that if propolis could be added in diet, it helped in regenerating the lost concentration of Hb [29]. Propolis increases the permeability of membrane and thus inhibited bacterial motility and showed bactericidal activity thereby reducing the toxic effect of bacteria on blood cells and also the bacterial mobility. The phytochemicals especially polyphenolics and flavanoids present in propolis have protective effect for the blood cell membranes [30,31,32]. Due to increasing MDR cases in Salmonella treatment focus is shifting towards some alternative medicines evolved from natural products as these are showing significantly positive effects in treating Salmonella infection [33].

The overall results of the present work provides the baseline information that pretreatment of propolis can reduces the severity of Salmonella infection and opens new avenues for further research on the mode of action of this bee product.

ACKNOWLEDGMENTS:

The authors would like to thank Department of Science and Technology for their assistance at various stages of this research work through INSPIRE fellowship and DST FIST for infrastructural facilities.

REFERENCES:

1. Ratcliffea N A, Melloc C B, Garciaa E S, Buttb T M and Azambujaa P. Insect natural products and process: New treatment for human diseases. Insect Biochemistry and Molecular Biology, 2011; 41(10): 747-69.

2. Drago L, de Vecchi E, Nicola L and Gismondo M. In vitro antimicrobial activity of a novel propolis formulation actichelated propolis. Journal of Applied Microbiology, 2007; 103: 1914-1921.

3. Kalia P, Kumar N R and Harjai K. The therapeutic potential of propolis against the damage caused by Salmonella typhimurium in liver of mice: a biochemical and histological study. Archives of Biological Sciences,2015;67(3):807-16. doi:0354-46641500040K.

4. Al- Ani I, Zimmermann S, Reichling J and Wink M. Antimicrobial activities of European propolis collected from various geographic origins alone and in combination with antibiotics. Medicines (Basel), 2018; 5(1): 2.

5. Grimont P A D and Weill F X. Antigenic formulae of the Salmonella serovars, 9^th Edn. World Health Organization collaborating centre for reference and research on Salmonella. Institute Pasteur, Paris, France.2007.

6. Chandel D S, Chaudhry R, Dhawan B, Pandey A and Dey A B. Drug-resistant Salmonella enterica serotype Paratyphi A in India. Emerging Infectious Diseases, 2000; 6: 420-421.

7. Gasem M H, Dolmans W M, Keuter M M and Djokomoeljanto R R. Poor food hygiene and housing as risk factors for typhoid fever in Semarang, Indonesia. Tropical Medicine and International Health, 2001;6: 484-90.

8. Chidozie V N and Adoga G I. Potentiating effect of aqueous leaf extract of Anogeissus leiocarpuson Carica papaya aqueous leaf extract and Mangifera indica aqueous stem bark extract - A herbal product used against typhoid fever in Nigeria. International Journal of Current Microbiology and Applied Sciences, 2014; 3(10): 1046-1062.

9. Gillespie, S. H. (2003). Salmonella infections. In: Manson’s tropical diseases. 21^st Edn. (G. C. Cook and A. Zumla, eds.) W. B. Saunders/Elsevier, London publs. pp. 937-49.

10. Crump J A, Luby S P and Mintz E D. The global burden of typhoid fever. Bulletin of the World Health Organization, 2004;82: 346-353.

11. WHO, “Food Safety-Food borne diseases and value chain management for food safety,” in Proceedings of the Forging Links between Agriculture and Health, CGIAR on Agriculture and Health Meeting, 2007.

12. Crump J and Mintz E. Global trends in typhoid and paratyphoid Fever. Clinical Infectious Diseases, 2010;50: 241-246.

13. Clark T W, Daneshvar C, Pareek M, Perera N and Stephenson I. Enteric fever in a UK regional infectious diseases unit: A 10 year retrospective review. Journal of Infection, 2010; 60(2): 91-98.

14. Eng S K, Pusparajah P, Ab- Mutalib N S, Ser H, Chand K G and Lee L H. Salmonella: A review on pathogenesis, epidemiology and antibiotic resistance. Frontiers in Life Science, 2015; 8(3): 284–293.

15. Klemm E J, Shakoor S, Page A J, Qamar F N, Judge K, Saeed D K, Wong V K. et al. Emergence of an extensively drug-resistant Salmonella enterica Serovar Typhi clone harboring a promiscuous plasmid encoding resistance to fluoroquinolones and third-generation cephalosporins. mBIO, 2018;doi:10.1128/mBio.00105-18.

16. Adeneya A A, Ajagbonna O P, Adeleke T I and Bello S O. Preliminary toxicity and phytochemical studies of the stem bark aqueous extract of Musanga cecropioides in rats. Journal of Ethnopharmacology, 2006; 105: 374-379.

17. Promise N, Agomuo E N, Uloneme G C, Egwurugwu J N, Omeh Y N and Nwakwuo G C. Effect of Phyllanthus amarus leaf extract on alterations of haematological parameters in Salmonella typhi infected wistar albino rats. Scientific Research and Essays, 2014; 9(1): 7-12.

18. Kalia P, Kumar N R and Harjai K. Phytochemical screening and antibacterial activity of different extracts of propolis. International Journal of Pharmaceutical and Biological Research, 2013; 3(6): 219-22.

19. Kakkar K, Sharma S, Asnani P J, Banerjee C K and Sharma B K. Experimental haematogenous pyelonephritis in mice with uropathogenic, enteropathogenic and enterotoxigenic Escherichia coli. Antonie Van Leewenhoek, 1986;52(2): 153-61.

20. Jain N C. Hematologic technique. In: Schalm’s Veterinary hematology. 4^th Edn. (Lea and Febiger eds.) Philadelphia publs. pp. 20-86. 1986.

21. Kalia P, Kumar N R and Harjai K. Effect of propolis extract on the haematotoxi-city and histological changes induced by Salmonella enterica serovar Typhimurium in BALB/c mice. Archives of Biological Sciences, 2016;68(2):385-91 doi:10.2298/AB-S150902030K.

22. Makela P H and Hormaeche C E. Immunity to Salmonella. In: Host response to intracellular pathogens, (S. H. E. Kaufmann, ed.) Austin and Landes publs. pp. 143-66. 1997.

23. Zghair Z R. Histopathological study of Salmonella typhimurium infection in laboratory mice by using the light and electron microscope. Kufa. Journal of Veterinary Medical Sciences, 2012; 3(1): 124-31.

24. Boyanova L, Gergova G, Nikolov R, Derejian S, Lazarova E and Katsarov N. Activity of Bulgarian propolis against 94 Helicobacter pyroli strains in vitro by agar well diffusion, agar dilution and disc diffusion methods. Journal of Medical Microbiology, 2005; 54(5): 481-483.

25. Abro A H, Abdou A M, Gangwani J L, Ustadi A M, Younis N J and Hussaini H S. Hematological and biochemical changes in typhoid fever. Pakistan Journal of Medical Sciences, 2009; 25(2): 166-171.

26. Dangana A, Ajobiewe J and Nuhu A. Haematological changes associated with Salmonella typhi and Salmonella paratyphi in humans. International Journal of Biomedical and Healthcare Science, 2010; 6: 219-222.

27. Kumar K B H and Kuttan R. Chemopreventive activity of an extract of Phyllanthus amarus against cyclophosphamide induced toxicity in mice. Phytomedicine, 2005; 7: 494-500.

28. Lokhandwala A, Athar S and Turrin N P. Role of absolute eosinopenia as marker of enteric fever: experience from a tertiary care hospital in the United Arab Emirates. Ibnosina. Journal of Medical and Biomedical Sciences, 2012; 4(6): 249-53.

29. Haro A, Lopez-Aliaga I, Lisbona F, Barrionueva M, Alferez M J and Campos M S. Benefical effect of pollen and/or propolis on the metabolism of iron, calcium, phosphorus and magnesium in rats with nutritional ferropenic anemia. Journal of Agricultural and Food Chemistry, 2000; 48: 5715-22.

30. Youdim K A, Shukitt-Hale B, MacKinnon S, Kalt W and Joseph J A. Polyphenolics enhance red blood cell resistance to oxidative stress: In vitro and in vivo. Biochimica Et Biophysica Acta, 2000; 1523: 117-22.

31. Cushnie T T P and Lamb A J. Antimicrobial activity of flavonoids. International Journal of Antimicrobial Agents, 2005; 26: 343-356.

32. Kaur R, Kumar N R and Harjai K. Phytochemical analysis of different extracts of bee pollen. International Journal of Pharmaceutical and Biological Research, 2013; 4(3): 65-68.

33. Manore D, Pillai S, Joshi A and Punashiya R. Preliminary Phytochemical Screening and Antibacterial Activity of Ethyl Acetate Extract of Cuscuta reflexa Roxb. Research Journal of Pharmacy and Technology, 2012; 5(1).

Received on 25.09.2019 Modified on 23.11.2019

Research J. Pharm. and Tech. 2020; 13(7): 3389-3393.

DOI: 10.5958/0974-360X.2020.00602.2