THE THERAPEUTIC POTENTIAL OF PROPOLIS AGAINST DAMAGE CAUSED BY SALMONELLA TYPHIMURIUM IN MICE LIVER : A BIOCHEMICAL AND HISTOLOGICAL STUDY

Honeybee products are a rich source of nutritive supplements and traditional medication. The increasing resistance of bacteria towards various antibiotics and the limited scope of some vaccines makes it important to explore alternative therapies to combat bacterial diseases. The present study aimed to evaluate the antibacterial action of propolis using biochemical and histopathological methods in Salmonella typhimurium-infected BALB/c mice. Crude propolis was collected from an apiary and extracted with 70% ethanol. Hepatotoxicity was induced in mice by infecting them with Salmonella typhimurium (104 colony-forming units (CFU)), and the hepatoprotective activity of propolis was evaluated by administration of different doses of propolis (100, 300 and 500 mg/kg body weight) for 30 days. Biochemical and histopathological examinations were performed at regular intervals during the experimental period. Results obtained after treatment were compared with similar studies performed on normal control mice. Infected mice showed elevated liver marker enzymes and revealed the presence of characteristic typhoidal nodules in histological preparations. These results point to the therapeutic activity of propolis against Salmonella typhimurium.


INTRODUCTION
Typhoid is a major public health problem caused by Salmonella typhi.It is characterized by high fever, diarrhea, inflammation, colic pain, granulomatous inflammatory response (Bharadwaj et al., 2009), hepatic dysfunction and hepatic abscess (Soni et al., 1994).Around 21 million cases of typhoid are reported every year, out of which 1-4% proves fatal (WHO, 2008).Salmonella typhimurium causes an invasive disease in mice that has similarity with human typhoid.Transmission may occur by ingestion of contaminated food, mainly meat, or by fecal oral route from an infected individual.Children, the elderly, immunocompromised individuals and pregnant woman are at greater risk of typhoid infection (Klooster, 1997;Doffinger, 2005).Because of the limited scope of vac-cines against typhoid (Mastroeni and Menager, 2003) and increasing resistance to antibiotics (Hazir et al., 2002;Prabha et al., 2002), the efficacy of certain natural products that could be used against this disease needs to be considered.
Propolis is a complex resinous material produced by honeybees from plant exudates, beeswax and bee secretions (Drago et al., 2007).It is used as a sealant for unwanted open spaces in the hive and for scaffolding, reinforcing and protecting the hive from intruders and various pathogens.In traditional medicine, propolis is used as an emollient, immunomodulator, antioxidant and anti-inflammatory agent.Mirzova et al. (1997) confirmed its antibacterial effect on B. subtilis, E. coli and R. sphaeroides.Studies performed by Orsi et al. (2007) discussed the synergistic effect of Brazilian

THE THERAPEUTIC POTENTIAL OF PROPOLIS AGAINST DAMAGE CAUSED BY SALMONELLA TYPHIMURIUM IN MICE LIVER: A BIOCHEMICAL AND HISTOLOGICAL STUDY
and Bulgarian propolis and some antibiotics on Salmonella typhi.In the light of the biological properties of propolis, the present study was performed to assess whether propolis could ameliorate the damage caused by Salmonella typhimurium in the liver of infected mice.

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 (Kalia et al., 2013).

Preliminary phytochemical screening
Standard chemical tests were performed on EEP for the screening of phytochemicals (Misra et al., 2011;Vijayalakshmi et al., 2012).

GC-MS
Extraction and preparation of sample: 5 mg of dried EEP was mixed with 50 µl of dry pyridine and 75 µl of BSTFA (Bis-(trimethylsilyl)trifluoroacetamide) and heated for 20 min at 80°C (Papova et al., 2011).The silylated EEP was analyzed by gas chromatography-mass spectrometry (GC-MS) using Thermo Scientific Trace 1300 and Thermo Scientific TSQ 8000 (triple quadrupole) equipped with Thermo TG MS column (dimensions: 30 m X 0.25mm X 0.25µM).The temperature was programmed from 60°C for 2 min to 29°C at a rate of 10°C/min and maintained for 1 min.Helium was used as carrier gas at flow rate of 1 ml/min.The injector temperature was 250°C, transfer line temperature was 300°C and MS source temperature was 280°C.

Microorganisms
The bacterial strain of Salmonella 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.

Colony forming assay
Salmonella typhimurium (MTCC 98) was grown in nutrient broth overnight at 37°C.After overnight incubation the culture was centrifuged at 5 300 g for 10 min at 5°C and then rinsed twice with saline.Count was determined by plating 10 μl each of 10-fold serial dilutions of the culture on nutrient agar plates.The plates were incubated overnight at 37°C.Following incubation, bacterial colonies were counted and colony-forming units (CFU) were calculated (Shukla et al., 2009).

Experimental Animals
4-6-week-old BALB/c mice weighing 20-25 g 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.Animals were checked regularly for any bacterial infection by streaking the tail vein blood directly on MacConkey agar.

Treatment regimens
Animals were divided into seven groups and each group consisting of six mice: Group I: control (normal mice); Group II: Salmonella typhimurium infection at 2 ×10 4 CFU/ml intraperitoneally; Group III: Salmonella typhimurium infected + antibiotic (cefixime) (4 mg/kg body weight (bw) of mice); Group IV: Salmonella typhimurium + propolis (100 mg/kg bw) given orally for 30 days; Group V: Salmonella typhimurium infected + propolis (300 mg/kg bw) given orally for 30 days; Group VI: Salmonella typhimurium infected + propolis (500 mg/kg bw) given orally for 30 days; Group VII: only propolis (500 mg/kg bw) Animals in groups II were killed on the 5 th day post infection (Group II: 5 th day was the peak day of infection) Group III animals were killed after completion of the treatment (treatment regimen of cefixime for 5 days), whereas those in groups IV, V and VI were killed after the 5 th , 15 th and 30 th days by suffocating with diethyl ether; group VII was also killed after day 30.Propolis treatment lasted up to 30 days.Each experiment was conducted in triplicate.

Collection of blood and tissue
The animals were lightly anesthetized with diethyl ether.Blood was drawn from the jugular vein for biochemical investigations.After blood collection, the animal was sacrificed and the liver removed aseptically.The liver was weighed and a small portion was kept in 10% formalin for histological analysis while the rest was homogenized in saline in a glass homogenizer for quantitative bacterial culture.

Bacterial load
Bacterial load in the liver of mice was assayed by plating the 10-fold serially diluted tissue homogenate on deoxycholate citrate agar (DCA) and maintaining overnight at 37°C ( Kakar et al., 1986).

Assay of liver marker enzymes
The collected blood was allowed to stand for 30 min and then centrifuged to obtain the serum.The serum was used for analysis of various liver function tests like serum glutamate oxaloacetic transaminase (SGOT), se- rum glutamate pyruvic transaminase (SGPT), alkaline phosphatase and bilirubin using standard kits (Avecon).

Histopathological studies
Tissue was fixed in 10% formalin.Histological processing included dehydrating, paraffin embedding, block making, section cutting, staining with hematoxylin and eosin (H&E) and finally mounting according to the method of Baker (1945).

Statistical analysis
All values were expressed as mean ±standard deviation.Statistical differences between the various

Phytochemical analysis
Preliminary phytochemical studies were performed on EEP and results are summarized in Table 1.

Identification of compounds
The identification of peaks was performed by comparing the results with commercial reference libraries such as the NIST library.More than 200 compounds were identified as constituents of propolis.The chemical composition of propolis is highly variable due to the broad range of ecoflora visited by honeybees (Abozid and Ahmed 2013).The different biological properties of propolis are mainly contributed by pinocembrin, galangin and pinobanksin.The chemical composition of 70% EEP was assessed by GC-MS analysis.The compounds identified are listed in Table 2. Various flavonoids, esters, ketones and acids were identified.The major compounds in derivatized EEP were 4' ,5,7-Trihydroxy flavanone , 4H-1-benzopyran-4-one, isoquinoline, propanone and cinnamic acid.
In the surrounding area where the propolis was collected, Eucalyptus globulus, Azadirachta indica and Mangifera indica were found and were considered a probable source of resin for propolis synthesis.

Survival percentage
The total number of animals that survived treatment with different doses of propolis was calculated.
The results showed that in the case of the infected group no survival was observed by day 30, while at doses P-300 and P-500 survival was 77.53±9.98% and 83.3±0%, respectively, and in the case of P-100 the survival percentage was around 50% (Table 3).
From this data the therapeutic potential of propolis was analyzed, which was further authenticated by the results of different experiments discussed hereunder (Table 3).

Assessment of bacterial load
The bacterial count was quite high in case of infected animals by day 5 of infection and no survival was observed by the 15 th or 30 th days.Therefore, the Salmonella typhimurium-infected mice were killed on the peak day of infection, i.e. day 5.The count decreased significantly (p <0.050) in the propolis-treated groups by the end of 30 days of experiment (Table 4).

Assessment of liver marker enzymes
The levels of SGOT, SGPT, alkaline phosphatase and bilirubin were significantly high in the infected group as compared to that of the control group (p<0.05).The mice treated with propolis at doses of 300 and 500 mg/kg body weight showed results that matched the control values (Figs.1A-D).With the dose of 100 mg/kg bw results were

Histopathological examination
Histologically, liver architecture in the control mice showed hepatocytes arranged radially around the central vein (Fig. 2A).In the case of infected mice, the liver presented typhoid nodules, lymphocytic infiltration, localized clusters of lymphocytes, granulation and hepatocytic destruction (Figs.2B-D).The presence of vacuoles was prominent in infected mice.
Signs of recovery were quite noteworthy in the propolis (P-300)-treated group of mice.Infiltration of lymphocytes was reduced, there was less vacuolization and lymphocytic clusters were reduced (Figs.2E-H).

DISCUSSION
In the present study, preliminary phytochemical analysis and GC-MS analysis of EEP showed the presence of flavonoids and acids.Many honeybee products, such as honey, bee pollen and propolis are rich in phytochemicals (Kaur et al., 2013).The pres- ence of flavonoids, such as 4,5,7-trihydroxyflavone (galangin), 4 H-1-benzopyran-4-one (pinocembrin) was assessed in Spanish propolis (Viguera, 2013;Bosio et al., 2000).The flavonoids are manufactured by plants under stress conditions that may be environmental, microbial, etc. and have many properties, e.g.antimicrobial, antioxidant (Borelli et al., 2002).Moreover, along with the flavonoids, cinnamic acid was also found by GC-MS analysis.Similar results were reported by Markham et al. (1996).Cinnamic acid has various biological properties, e.g.antitumor, cell growth effect (Akao et al., 2003).The data obtained also confirmed the presence of sugars such as fructofuranose, fructopyranose and tagatofuranose in propolis from Canada (Viguerea et al., 1993).The most important plant sources for propolis in this region were Eucalyptus globulus, Azadirachta indica and Mangifera indica, which are rich in flavonoids and phenolic compounds (Lopes et al., 2003;Sultana et al., 2007;Thomas et al., 2002;Selles et al., 2002).
The rise in the level of liver marker enzymes observed during the present study is supported by the findings of previous studies that reported that Salmonella infection caused hepatic granulomas that led to the release of liver enzymes into serum, thus increasing the level of these enzymes when assayed in serum (Mert et al., 2004;Hasbun et al., 2006).It has been reported that the extent of hepatic dysfunction in typhoid fever depended upon various contributory factors such as endotoxins produced by Salmonella, damage to hepatocytes and invasion of hepatocytes by microorganisms (Calva, 1986).These might be responsible for the increase in liver function enzymes thus causing hepatic insult.yanpallewar et al. (2003) reported that the high serum concentration of liver markers indicated cellular leakage due to the disintegration of liver cell membranes.In the group treated only with propolis (Group VII), all the parameters were within control values.Earlier studies also confirmed that EEP showed no toxicological manifestations in different organs of BALB/c mice at different concentration (Kalia et al., 2014).
Previous studies have reported that mice infected with Salmonella typhimurium showed signs of histological damage (Everest et al., 1998).Umezawa et al. (1995) reported the presence of scattered, small nodular abscesses composed of degenerated hepatocytes and infiltrated polymorphonuclear (PMN) cells in mice infected with Salmonella typhimurium.Typhoidal nodules were formed by the infiltration of lymphocytes.The liver architecture in the control group revealed the characteristic radial arrangement of hepatocytes around the central vein.In the case of infected mice, liver injury was evident as indicated by lymphocytic infiltration, the presence of vacuoles, localized clusters of lymphocytes, granulation and hepatic destruction.The propolis-treated group P-100 showed no significant change, whereas treatment with P-300 and P-500 showed definitive signs of recovery, manifested as reduced infiltration of lymphocytes, reduced vacuolization and lymphocytic cluster formation (Figs. 2E-H).Pepeljnjak et al. (1985) reported that the antibacterial activity of propolis was due to the presence of phytochemicals such as flavonoids.According to Burdock (1998), it was the combined and specific concentrations of various phytochemicals that were responsible for the pharmacological activities of propolis.Preliminary studies during the present investigations confirmed the presence of flavonoids and other constituents responsible for its biological action (Kalia et al., 2013).
The mode of action of propolis on Salmonella is unknown but can be explained by the fact that propolis and its constituents, such as flavonoids and cinnamic components, uncoupled the energy transducing the cell membrane (Mirzoeva et al., 1997) and hampered the motility of bacteria that were responsible for virulence (Tamura et al., 1995;Finlay and Falkow, 1989).It was suggested that the effect of propolis on the membrane potential could be responsible for the overall cytotoxic action of propolis (Mirzoeva et al., 1997).The data obtained from our study revealed a significant reduction in the Salmonella typhimurium bacterial count in the propolis-treated groups.These results point to the repair of tissue damage and functional improvement of cells by propolis.

Table 1 .
Results of phytochemical analysis of the ethanolic extract of bee propolis.
groups were evaluated by Student t-test and ANOVA (Tukey's test) using Sigma plot software.Values of p < 0.05 were considered statistically significant.

Table 2 .
Compounds identified in EEP by GC-MS.

Table 3 .
Percentage of animals surviving after infection with Salmonella typhimurium and treatment with propolis at the end of experiment.

Table 4 .
Bacterial load in mice liver of different groups (log cfu/gm).Data are expressed as Mean± S.D.; *: p<0.050 , infected vs P100, P300 and P500 (Student's t test); $: p<0.050, day-wise comparison of different doses of propolis.At all doses by the 30 th day of the experiment, a significant reduction in bacterial load was observed at all doses of propolis (ANOVA: Tukey's Test).