Biochemical and Histological Study of Royal Jelly in male rats induced by Oxidative Stress

 

Sarah Abbas Obaid, Haider Salih Jaffat, Suhad Hamed Shabaa

 

ABSTRACT:

The study was divided into six groups (5 rats in each group) for 8 weeks. Group 1: control animals give water and pellet kept it as negative group. Group 2: administrated CCl₄ as in equal volume with olive oil (1/1) at 3 days in weeks. Group 3 and Group 4 were administrated royal jelly (RJ) at two concentrations 150,300 mg/kg of body weight. Group 5 and Group 6 were administrated CCl₄ along with royal jelly at two concentrations 150,300 mg/kg of body weight respectively. At the end of experiment (60 days), rats was scarified, serum sample obtained for assessment of laboratory analysis the results show significant decrease (p<0.05) in liver function (AST, ALT and ALP) in groups treated with RJ at both doses 150,300 mg/kg of body weight in compared with group treated with CCl₄ only that has significant raise in AST, ALT and ALP levels and histological filed the result showed histological change include hepatocyte necrosis, sinusoidal vessels congestion and cytoplasmic vacuolization but, normal liver section n groups treated with RJ at both doses 150,300 mg/kg of b.w.

 

 

 

INTRODUCTION:

Hepatotoxicity refers to liver dysfunction or liver damage that is associated with an overload of drug or xenobiotics¹. Hepatotoxic agents can react with the basic cellular components and consequently induced almost all types of liver lesions. Toxins and drugs are among the basic etiopathogenetic agents of acute liver failure². Antioxidant is "any substance that delays, prevents or removes oxidative damage to a target molecule''³. Antioxidants (AOX) have long been known to protect the biological system through inhibition or deterrence of oxidation stress tempted by reactive oxygen substances produced during usual metabolic activities or environmental factors⁴.

 

The royal jelly (RJ) is one of the products of honeybee workers that secreted by the salivary glands and is devoted mainly as food for the queen throughout the larval period, while another nurse honeybee larvae are fed royal jelly for only three days⁵.

 

MATERIALS AND METHODS:

Preparation of royal jelly:

The Royal jelly (RJ) sample was obtained from beekeeper in Najaf province(160 km South Baghdad) during the year 2016. Prepared by dissolving (5) g of RJ in (10) ml of distill water to prepare a stock solution, which is used to prepare the concentrations 150, 300mg/kg of RJ solution.

 

Experimental animals:

Thirty healthy adult albino male rats, (Rattusnovigicus) weighing between 205-290 g, were obtained from animal house in Faculty of Pharmacy, University of Karbala, Iraq, are used in the study. The animals were housed in the animal house of Science Faculty, University of Kufa, inatypical environment includes (degree of temperature 22-28°C) and controlled conditions to standard laboratory nourishment with commercial food (pellets) and water were provided to animals through the period of experimental that last 60 days. Rats were left to acclimatize for at least three weeks before the start of the experiment.

 

Laboratory analysis:

Determination of serum transaminase activity:

Colorimetric determination of ALT and AST activity, according to the Reitman and Frankel method (Reitman and Frankel, 1957), by using biomerieux kit.

Determination of Alkaline phosphatase Activity:

Colorimetric determination of APT according to Kind and King method (1954) by using biomerieux kit.

 

Histological study:

The ordinary histological technique was prepared for liver section. The liver fixed immediately in 10% formalin solution for later histological section according to (Bancroft and Gamble, 2002).

 

Biostatistical analysis:

The results were expressed as (mean = standard deviation). Pooled t-test was used for the comparison between control and other groups, in the measured parameters. One-way analysis of variance (ANOVA) followed by least significant difference (LSD) analyses at 0.05% probability of levels. All statistical analysis was performed using Excel program (2010) from Microsoft Office. USA, and Magastat. The difference will be significant when P<0.05 value.

 

RESULTS:

The results showed that serum activity of alanine amino transferase ALT, AST and ALP were elevated (p<0.05) in CCl₄-intoxicated animals for 8 weeks in contrast with control group. Treatment of the male rats with RJ at concentrations 150 and 300 mg/kg of body weight along with CCl₄ showed a marked decline in the serum ALT, AST and ALP activity compared to CCl₄-intoxicated animals. Other groups treated with RJ only at concentrations 150 and 300 mg/kg of b.w. for 8 weeks showed no significant difference (p<0.05) in ALT, AST and ALP levels. In contrast with control group but, significant decline when compared with CCl₄ group.

 

Activity of Royal Jelly and CCl₄ on ALP, AST and ALP levels

Groups	ALP U/L	ALT U/L	AST U/L
Control	52.20±0.92	40.40±0.24	80.80±0.49
CCl4	*98.00±0.55	*118.00±1.22	*144.80±0.73
RJ 150	*60.00±0.55	41.80±0.49	81.20±0.73
RJ 300	*58.00±0.55	*45.80±0.49	*84.20±0.49
CCl4 + RJ 150	*79.20±0.92	*78.80±0.73	*108.80±0.49
CCl4 + RJ 300	*77.20±0.97	*91.20±0.73	*118.80±0.73
L.S.D. 0.05	0.71	0.73	0.64

 

Histological of liver:

The results in figure (1) showed normal liver sections of control group. In figures (2) that, the CCl₄ could induce histological changes, including increased fatty degeneration, hepatocyte necrosis, inflammatory cells infiltration fatty liver disease (FLD) and marked sinusoidal vessels congestion with mild distribution histological configuration but, when the animals treated with RJ 300mg/kg showed normal hepatocytes and some cell in state of division cell and the bleeding decrease in group treated with RJ along with CCl₄ and showed necrosis, inflammatory cell and present of normal hepatocytes showed figure 4.

 

Figure 1: liver section of control showing normal hepatic section

(HandE 400X)

 

 

Figure 2: liver section of male rats treated with CCl₄ showed hepatocytenecrosis, inflammatory cells, degeneration and bleeding (HandE 400X).

 

 

Figure 3:liver section of male rate treatedwith RJ 300normal hepatocyte, some of hepatocytes cell in the state of division(HandE 400X)

 

 

Figure 4:Liver section of treated rats by CCl₄+RJ300 showed less bleeding, infusion in some liver cells, inflammatory, normal hepatocyte cells and nerosis, (HandE 400X)

 

DISCUSSION:

AST and ALT were found in serum and various body tissues but are mostly associated with liver parenchymal cells. The elevated level of AST and ALT will be observed in acute liver damage condition. In addition, the level of ALT will rise with intra hepatic cholestasis and infiltrative diseases of the liver⁶.  

 

Administration of CCl₄ causes deterioration of  liver function tests such as AST, ALT and ALP revealed hepatic dysfunction, which be a secondary events following CCl₄-induced liver damage, with the consequent leakage from hepatocyte⁷. AST is localized in the mitochondria, whereas ALT is distributed throughout the cytoplasm. In the case of hepatic stress, mitochondria damage with ROS accumulation tends to increase the level of AST rather than ALT^8,9.  In the present study also showed significant increase p<0.05 in AST, ALT and ALP levels in group treated with CCl₄ for 8weeks.

 

The study of Cemek et al. demonstrates that the administration of CCl₄ leads to severe acute liver affection in rats, revealed by significant increase of AST and ALT serum levels. The same study reveals that the treatment with RJ remarkably counteracts the severe acute hepatic affection induced by CCl₄, which can be seen in the decrease of the serum activity of AST and ALT. Furthermore, the histopathological evaluation shows that the hepatic lesions induced by CCl₄ improve after treatment with RJ¹⁰.

 

The royal jelly significantly restores the changes of ALP and AST due to its antioxidant effect and its ability to act as a free radical scavenger, thereby protecting membrane permeability¹¹. The group treated only with RJ at concentration 300 mg/kg of body weight for 8 weeks show increased in the hepatic marker enzymes.

 

The results showed the histological changes, the liver damage produced by CCl₄ revealed massive centrilobular necrosis, cell degeneration, fiilteraition¹². In only CCl4 given group, hydropic degeneration, occasional coagulation necrosis and sinusoidal dilatation were observed. Formation of fibrosis starting from the portal regions through the parenchyma with presence of occasional necrotic hepatocytes and mononuclear cellular infiltration was evident. Interlobular areas were widened due to development of fibrosis. Pseudolobule formations were also recognizable in severely affected regions¹³.

 

In the present study, RJ in concentration 300mg/kg of body weight and after both periods of experiment 8weeks have beneficial effect on liver structure and some of hepatocytes cell in state of cell division this study, agree to a study showed the liver of the RJ-only groups showed normal histological structures¹⁴.

 

Treatment with RJ along with CCl₄, show increased in number of apoptotic cells, and ordinary histological, these due to antioxidant action of RJ that act as scavenge of free radicals. RJ enhance recovery from CCl₄-induced liver damage in a manner partially dependent on their antioxidant properties and bioavailability, the RJ products can therefore be used for the prevention and treatment of various liver diseases, RJ are rich in natural antioxidant products¹⁵.

 

REFERENCES:

1.     Navarro VJ, Senior JR (2006). Drug-related hepatotoxicity. N Engl J Med 354:731-739.

2.     Grattagliano, I.; Bonfrate, L.; Catia, V. D.; Wang, H. H.; Wang, D. Q. H. and Portincasa, P. (2009). Biochemical mechanisms in drug-induced liver injury. World J. Gastro-enterol., 15: 76-4865.

3.     Halliwell, B. (2007). Biochemistry of Oxidative Stress. Bioche-mical Society Transactions, 35 (5): 1147-1150.

4.     Poljsak B, Dusan S, Irina M. Achieving the balance between ROS and antioxidants: when to use the synththetic antioxidants. Oxid Med Cell Longev. 2013; 2013:9556792.

5.     Ramadan, F.R. and Al-Ghamdi, A. (2012). Bioactive compounds and health-promoting properties of royal jelly: A review. Journal of functional food, 4 :39-52.

6.     Gaze,D. C.  ''The role of existing and novel cardiac biomarkers for cardioprotection,'' Current Opinion in Investigational Drugs, vol. 8, no. 9, pp. 711-717, 2007.

7.     Xu, J.W., Gong, J.; Chang, X.M.; Luo, J. Y, Dong, L.; Hao, Z.M. and Xu, G. P. (2002). Estrogen reduces CCl₄-induced liver fibrosis in rats. World J. Gastroenterol, 8:883.

8.     Hu Z, Lausted C, Yoo H, Yan X, Brightman A, Chen J, Wang W, Bu X, Hood L. Quantitative liver-specific protein fingerprint in blood: a signature for hepatotoxicity. Theranostics. 2014;4(2): 215-228.dol: 10.7150/ thno. 7868.

9.     Shen J, Zhang J, Wen J, Ming Q, Zhang J, Xu, Y. Correlation of serum alanine aminotransferase and aspartate aminotransferase with coronary heart disease. Int J Clin Exp Med. 2015;8(3):4399-4404.

10.  Cemek M, Aymelek F, Büyükokuroĝlu ME, et al. Protective potential of Royal Jelly against carbon tetrachloride induced-toxicity and changes in the serum sialic acid levels. Food and Chemical Toxicology. 2010; 48(10): 2827-2832.

11.  Ashry, K., and Elkady, A. (2014). Royal Jelly Modulates Hepatotoxic Effect of Rats Treated with Cisplatin.

12.  Gowri Shankar, GNL.; Manavalan, R.; Venkappayya, D. and Raji, CD. (2008). Hepatoprotective and antioxidant effects of Commiphoraberryi (Arn) Engl bark extract against CCl₄-induced oxidative damage in rats. Food Chemtoxicol. 46: 3182-3185.

13.  Musa Karaman, Hasan Zen, Serpil Dağ, Onur Atakişi, Gülşen çiğşar, Oktay Kaya. Ameliorative Effect of Omega-3 in Carbon Tetrachloride Toxicity. Kafkas Univ. Vet. Fak. Derg. 23 (1): 77-85, 2017 DOl: 10.9775/kvfd.. 2016.15862.

14.  Ali Karadeniz, Nejdet Simsek, Emre Karakus, Serap Yildirim, Adem Kara, Ismail Can, Fikrullah Kisa, Habib Emre and Mehmet Turkeli. Royal Jelly Modulates Oxidative Stress and Apoptosis in Liver and Kidneys of Rats Treated with Cisplatin. Hindawi Publishing Corporation, Oxidative Medicine and Cellular Longevity. Volume 2011, Article ID 981793, 10 pages doi: 10.1155/2011/981793.

15. Zlem Saral, Oktay Yildiz, Rezzan Aliyazicioğlu, Esin Yuluğ, Sinan Canpolat, Ferhat Ztürk and Sevgi Kolayli. Apitherapy products enhance the recovery of CCL₄-induced hepatic damages in rats. Tuek J Med Sci (2016) 46:194-202 doi:10.3906/sag-1411-35.

 

 

 

 

Accepted on 19.06.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2017;10(10):3426-3428.