Ameliorative Effects of Propolis Extract on Some Biochemical and Hematological Parameters of Burnt Skin of Male Guinea pigs

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. Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidate important biological, chemical, or physical mechanisms of broad physiological significance.

Propolis, a complex resinous material collected by honeybees from buds and exudates of certain plant.Propolis includes; fatty and phenolic acids and esters, substituted phenolic esters, flavonoids (flavones, flavanones, flavonols, dihydroflavonols, chalcones), terpenes, βsteroids, aromatic aldehydes and alcohols, and derivatives of sesquiterpenes, naphthalene and stilbenes (Marcucci et al., 1996).The use of propolis goes back to ancient times, at least to 300 BC, and it has been used as a medicine in local and popular medicine in many parts of the world, both internally and externally.In recent years, propolis has attracted researcher's interest because of its many beneficial biological effects, such as hepatoprotective, antitumour, antioxidant, antimicrobial, anti-inflammatory, antiviral, antifungal and antiparasite activities (Sforcin, 2007).The chemical composition of propolis depends on the specificity of the local flora at the site of collection.
Silver sulfadiazine (SSD) is the topical agent of choice in severe burns and is used almost universally today in preference to compounds such as silver nitrate and mafenide acetate.SSD cream, while being effective, causes some systemic complications including neutropenia, erythema multiforme, crystalluria and methaemoglobinemia (Hosnuter et al., 2004).
In this study, we examined the effects of propolis treatment on skin after thermal injury in an animal model compared to Silver sulfadiazine treated group.CBC counts, MDA levels, superoxide dismutase, catalase, Nitric oxide and lipid profile (cholesterol, HDL, LDL, vLDL and triglycerides) were measured 15 and 30 days post-treatment.

Propolis extraction:
Crude Propolis was obtained from an Egyptian honey bee keeper, Qena province, Egypt.Propolis was kept dry and freezed (-40°C) until used.Propolis samples were mixed with distilled water, heated gently and filtered through Whatman No:1 filter paper.Propolis was freshly prepared and the filtrate administered to animals by gavage at dose of 100 mg/kg b.wt.(El-Khayat et al. 2009).Dermazine ® cream (silver sulfadiazine) was used as a standard wound healing drug, manufactured by MUP company, EGYPT.Animals: Adult Guinea pigs of approximate (550 g body weight each) were selected.The animals were housed in the Animal House of the Faculty of science, South Valley University, Qena, Egypt, for two weeks under natural day and night periods and supplied with a balanced diet and water ad libitum.

Induction of burns in Guinea Pigs skin:
Standard burns were induced on Guinea Pigs according to (salman, 1995).as the following: At the time of experiment, the animal was shaved in a circle at the back of thoracic dorsum and a standard burn was induced under light diethyl ether anesthesia.At the center of the shaved area an opened glass tube with a diameter of 1.5 mm was fixed up right by hand in a vertical position.Five ml of continuously boiling water were poured in to the tube using a glass syringe within a very short time.The elapsed period of the hot water on the skin was justly approached to 30 seconds (the time was controlled by a stop watch) after which the tube was removed by tilting it away from the body.

Experimental Protocol:
Animals were divided in to 5 groups of 20 animals each.Animals were sacrificed every 15 or 30 days as the following: Group1 (normal group): Each of 20 animals were orally administered with 0.9% isotonic saline solution at a dose (10 ml/kg body weight) once a day along the experimental period and served as a normal group.Group 2 (Positive control group): Standard burns of Guinea pig skin are obtained wounds were treated orally with 0.9% isotonic saline solution at a dose (10 ml/kg body weight) once a day to observe the healing process occurring without treatment.Then10 guinea pigs were sacrificed after 15 days and the other 10 after 30 days.Group 3 (Propolis topically treated group): Standard burns of skin are obtained wounds were treated by topical application of Propolis (100 mg/kg b.wt).10 animals were sacrificed after 15days (i.e., after 15 doses of topical treatment).the other 10 animals were sacrificed after 30 days (i.e., after 30 doses of topical treatment).Group 4 (SSD topically treated group): Standard burns of skin are obtained wounds were treated by topical application of silver sulfadiazine (layer thickness of about 3-5 mm). 10 animals were sacrificed after 15 days (i.e., after 15 doses of topical treatment).The other 10 animals were sacrificed after 30 days (i.e., after 30 doses of topical treatment).Group 5 (Propolis top.and oral treated group): Standard burns of skin are obtained wounds were treated by orally and topically applications of Propolis (100 mg/kg b.wt).10 animals were sacrificed after 15 days (i.e., after 15 doses of topical and oral treatment).The other 10 animals were sacrificed after 30 days (i.e., after 30 doses of topical and oral treatment).

Collection of samples:
After 12 hrs Fasting the animals were sacrificed.Peripheral blood was divided into two portions (the first portion was used as whole blood for complete blood count (CBC), the second was used for separation of serum) also a part of the skin tissue was taken to prepare tissue homogenate as the following: Skin Tissue Homogenate preparation: Skin tissue was perfused with PBS (phosphate buffered saline) solution, pH7.4 containing 0.16 mg/ml heparin to remove any red blood cells and clots.Tissue then was homogenized in 5-10 ml cold buffer (i.e.50 Mm potassium phosphate, pH 7.4, 1 mM EDTA and 1 ml/L Triton X-100) per gram tissue.After this the sample was centrifuged at 4,000 rpm for 15 minutes at 4 °C.Finally the supernatant was removed for assay and freeze at -80 °C (Nishikimi et al., 1972).

Biochemical parameter of whole blood, serum and skin tissue:
Biochemical parameters; Lipid profile was estimated according to method of (Trivedi et al., 2004) by enzymatic method.Malondialdehyde (MDA) according to (Ohkawa, et al., 1979), Catalase CAT (Fossati et al., 1980) and super oxide dismutase SOD (Nishikimi et al., 1972), were analyzed also using available kits according the reported methods.Blood cell counts, hematocrit or packed cell volume (PCV) and hemoglobin (Hb) content were determined using Celltac α apparatus from NIHON KOHDEN (MEK-6410K).

Statistical analysis:
The variability degree of results was expressed as means ± standard deviation of means (Mean ± S.D).The significance of the difference between samples was determined using Graph Pad Prism 03n software, where appropriate.The difference was regarded significant at P<0.05.

Effect of treatment with propolis or Silver sulfadiazine (SSD) on serum lipid profile in burned Guinea Pigs:
The recorded results in Table (2) showed that TGs, T. Cholesterol, LDL and vLDL levels in burned male Guinea pigs (positive control group) after 15 days increased significantly (P<0.05) when compared with normal group.While after 30 days a highly significant (P<0.01)increase in their levels were recorded.In contrast, HDL level was significantly decreased after 15 days and was highly significant (P<0.01)decreased after 30 days.
Daily topical treatment with propolis or Silver sulfadiazine (SSD) for 15 days had a non-significant change in serum TGs, T. Cholesterol, LDL and vLDL levels in comparing with the positive control group.While after 30 days, a significant decrease was recorded (P< 0.05) in comparing with the corresponding positive control group.
Also the increase of HDL level was non-significant while the increase became significant (P<0.05) after 30 days.Treatment topically and orally with propolis for 15 days had a significant decrease (P<0.05) in serum TGs, T.Cholesterol, LDL and vLDL levels in comparing with the positive control group.While after 30 days, the decrease became highly significant (P<0.01) in comparing also with the corresponding positive control group.Also the increase of HDL level after 15 days was significant (P<0.05)while, the increase became highly significant (P<0.01)after 30 days, as shown in Table (

Effect of treatment with propolis or silver sulfadiazine (SSD) on antioxidant parameters catalase (CAT), superoxide dismutase (SOD) and nitric oxide (NO):
The activity of CAT and SOD in burned skin tissue of the positive control group after 15 days significantly decreased (P<0.05) and highly significant decreased (P<0.01) after 30 days as recorded in Table (3) and illustrated in fig ( 3).In addition the concentration of serum NO in the positive control group was significantly decreased (P<0.05) after 15 days and a highly significant decreased (P<0.01) after 30 days as shown in Table (3) and Fig ( 3).
There was a significant increase (P<0.05) in CAT, SOD and serum NO of the positive control group after 30 days of treatment with propolis or silver sulfadiazine (SSD) topically.The treatment with propolis topically and orally showed a significant increase (P<0.05) in NO, SOD activity and CAT activity after 15 days as recorded in table 3 and highly significant increase (P<0.01) after 30 days, as shown in Table (3) and fig ( 3).

Effect of treatment with propolis or silver sulfadiazine (SSD) on malondialdehyde level (MDA):
The level of MDA in burned skin tissue of the positive control group was significantly increased (P<0.05) after 15 days and a highly significant increased (P<0.01) after 30 days as recorded in Table (3) and illustrated in Fig ( 3).
In contrast, the treatment with propolis or silver sulfadiazine (SSD) topically recorded a significant decrease (P< 0.05) after 30 days as shown in table (3) and fig ( 3).While treatment with propolis topically and orally showed a significant decrease (P<0.05) after 15 days and highly significant decrease (P< 0.01) after 30 days, as recorded in Table (3) and illustrated in Fig. (3).

DISCUSSION
Early, adequate and repeated investigation of the circulating blood parameters is therefore an essential tool in the treatment of severe burns (Kramer, 2002).Muir, (1961) has shown that a general relationship exists between the extent of deep burn and the amount of red cell destruction.While Baxter, (1978) has observed a shorter life span of RBCs after burns.The progressive deficit in red cell mass may be accompanied by the appearance of multiple abnormal red cells contributing to early post burn anemia.In the present study, erythrocyte count and blood hemoglobin content significantly decreased in the burn group in comparison with the normal group.The improvement of RBCs, hemoglobin and hematocrit after treatment with propolis, are in accordance with other research.Propolis administration in female albino rats indicated an increase in RBCs, hematocrit and hemoglobin (Cetin et al., 2010).The administration of water soluble propolis derivatives in rabbits clarified a significant increase in RBCs count, hemoglobin and packed cell volume (Hager, 2010).
burned guinea pigs In the present study show decrease in platelet count and rise in WBCs in the initial post-burn days This finding coincides with similar observation in the studies by other authors (Maduli et al., 1999;Sarda et al., 2005;Pavic andMilevoj, 2007 andBelba et al., 2015).The results of present study clearly demonstrate that propolis treatment exerts a protective effect on platelets and WBCs by increasing platelet count and decreasing WBCs to the normal levels on subsequent postburn days.The effects of propolis on the platelets and WBCs may occur through its anti-bacterial properties.It has been reported that propolis displays antibacterial action against different pathogenic bacteria ( Kujumgiev et al., 1999).
Dyslipidemia after burn injury is one of the important alterations that resulted from many factors like hypermetabolic state in burn, release of hormones and inflammatory mediators and organ dysfunction (Birke et al., 1972 andCoombes et al., 1980).Furthermore, data obtained by others, showed that the plasma TGs level increase in burned patients due to mainly increased availability of free fatty acids released by stimulated lipolysis in adipose stores due to catecholamines, cytokines, tumor necrosis factor alpha, interleukin-1, interferon-alpha, beta and gamma; and recently interleukin-6, growth factors such as platelet derived growth factor, transforming and colony-stimulating factors; all modulate the lipid metabolism and may be the cause of these changes (Cree and Wolfe, 2008).In the current study, a marked increase in the concentrations of serum total cholesterol, triglycerides, LDL-C with a decrease in the level of HDL-C was found in the burned groups compared with normal group.Different studies indicated that propolis alleviated the high blood lipids, high total cholesterol and arteriosclerosis (Castaldo and Capasso, 2002).This result is in agreement to the present study with animals treated with propolis and this represents the powerful influence of propolis to reduce the risk of hyperlipidemia.Several studies are in agreement with the present study (Nirala et al., 2008) which proved the modulating effect of propolis on total cholesterol and triglycerides levels with a significant increase in total proteins content after beryllium toxicity and the improvement of serum level of HDL-C by propolis in a dose-dependent manner.
Oxygen radical formed and inflammation produced by heat burn causes lipid peroxidation (Hiramatsu et al., 1942).Cetinkale et al., (1997) reported that lipid peroxidation leads to serious damage of the endothelium, resulting to increased capillary permeability, damage of skin tissue, and necrosis.Furthermore, lipid peroxidation is essential for wound and shock caused by burns.Our results showed that lipid peroxidation was increased following burn injury.Propolis treatment, however, decreased MDA activity levels indicating inhibition of lipid peroxidation.
The antioxidant defense system is known to inhibit lipid peroxidation in mammalian tissues by destroying some of ROS that has an important role in initiation of the lipid peroxidation process.The antioxidant defense system operates through enzymatic and nonenzymatic components.The system is affected by burns.It has been reported that nonenzymatic antioxidants, such as glutathione, α-tocopherol and selenium, are decreased in the serum and tissues after thermal injury (Bekyarova and Yankova, 1998).Some authors have reported that SOD and CAT activities gradually decrease after burns (La Londe et al., 1996 andYoun et al., 1998).The tissue antioxidant enzyme activities were only decreased in the positive control group when compared to the normal group.This decrease may be related to the consumption of activated enzymes against oxidative stress.Propolis treatment resulted in improved enzyme activities.Shinohara et al., (2002) demonstrated that propolis was found to modulate antioxidant enzymes and decrease lipid peroxidation processes in plasma, liver, lungs, and brain of mice in a dose and tissue dependent manner.
Nitric oxide (NO) is involved in several processes in the skin including wound healing and pigmentation (Weller, 2003).In recent years, NO has emerged as a critical molecule in wound healing, where it increases collagen content in experimental wounds (Thornton et al., 1998 andShi et al., 2000).It is worth to mention that, in other study the wound healing in diabetic guinea pigs are improved by injection of bradykinin potentiating factor (BPF) which is the stimulator of NO (Elshater et al., 2011).So in this study, the burned skin healing in guinea pigs are improved by propolis.Propolis stimulation of the NO may be connected with the ability of the apitherapeutic agent to enhance the expression of transforming growth factor- (TGF-β) (Ansorge et al., 2003).Propolis, NO and TGF-β1 play central roles in collagen synthesis and can crossregulate each other (Khorasgan et al., 2010 andOlczyk et al., 2013).
Fig. 1: Effect of treatment with Top.Propolis, SSD alone and (oral administration of Propolis +Top.Propolis) on (RBCs, Hb, HCT, WBCs and Platelets) of male guinea pigs.
Fig. 2: Effect of treatment with Top.Propolis, SSD alone and (Oral administration of Propolis +Top.Propolis) on (Chol., TGs, HDL-C, LDL-C and vLDL) of male guinea pigs.
Fig, 3: Effect of treatment with Top.Propolis, SSD alone and (Oral administration of Propolis +Top.Propolis) on (MDA, SOD CAT and NO) of male guinea pigs.

Table 1 :
Effect of treatment with Top.Propolis, SSD alone and (Oral administration of Propolis +Top.Propolis) on (RBCs, Hb, HCT, WBCs, and Platelets) of male guinea pigs.

Table 2 :
Effect treatment with Top.Propolis, SSD alone and (Oral administration of Propolis +Top.Propolis) on (Chol., TGs, HDL-C, LDL-C and vLDL) of male guinea pigs.

Effect of treatment with propolis or silver sulfadiazine (SSD) on antioxidant status and MDA concentration in burned skin tissue of Guinea pigs:
2) and fig(2).

Table 3 :
Effect of treatment with Top.Propolis, SSD alone and (Oral administration of Propolis +Top.Propolis) on (MDA, SOD CAT and NO) of male guinea pigs.