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Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats



Sherif R. Mohamed, Lamiaa E. El-Sedeek, Mohamed M. Sief,, Sherif S. Mohamed, May M. Amer and M.H.M. Abdel-Aziz
 
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ABSTRACT

Background: Lead is potential environmental contaminants with the capability of causing human health problems and occurred in Egyptian food stuffs. Rosemary is known medically for its powerful antioxidant activity. So, the rosemary extract have been carried out to use as an antioxidant agent against lead hazards in rats. Materials and Methods: The rats divided into six groups including the control group, group 2 received lead solution, group 3 and 4 received rosemary at two doses are 100 and 200 μL, group 5 and 6 received lead plus rosemary at two mentioned doses. At the end of the experiment blood, liver and kidney tissues were taken for biochemical blood analysis and histological examination. Results: The obtained results indicated that lead induced negative effect on all biochemical tested parameters to be 78, 68 and 129 U L–1, 0.55, 3.8, 86, 135, 27, 32, 53, 0.41 and 2.9 mg dL–1 for AST, ALT, ALP, Bil., Alb, Ch., TG, HDL, LDL, urea, Cr and UA, respectively while, the same parameters were in normal value for control or groups received rosemary at two doses. It is worthy to report that the biochemical parameters of groups received lead plus rosemary extract at two mentioned doses were in normal value and close to control value. Histological confirmation was carried out and cleared almost the safety of liver and kidney tissues for all groups except the lead group. Conclusion: It could be concluded that the rosemary extract has the ability to protect against a lead hazard in rats.

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Sherif R. Mohamed, Lamiaa E. El-Sedeek, Mohamed M. Sief,, Sherif S. Mohamed, May M. Amer and M.H.M. Abdel-Aziz, 2017. Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats. Research Journal of Medicinal Plants, 11: 1-7.

DOI: 10.3923/rjmp.2017.1.7

URL: https://scialert.net/abstract/?doi=rjmp.2017.1.7
 
Received: July 15, 2016; Accepted: November 30, 2016; Published: February 23, 2019



INTRODUCTION

Lead is the most common environmental pollutant1, possess many undesired effects, including neurological, renal and hepatic2 immunological3 cardiovascular system and hematological dysfunctions4. Lead induced oxidative stress has been identified as the primary contributory agent in the pathogenesis5. Reactive Oxygen Species (ROS) generated as a result of lead exposure. Lead causes oxidative stress by inducing the generation of ROS, reducing the antioxidant defense system2. The levels of lead in the atmosphere of Egyptian industrial and urban areas were higher than the levels in other countries6. The annual mean concentration of lead exceeded both the Egyptian Standard7 and WHO air quality standard8.

Antioxidants acting as reducing agents, hydrogen donators and singlet oxygen quenchers that suppress the naturally produced free radicals and delaying oxidative reactiion9. Most pharmacological effects of rosemary are the consequence of high antioxidant activity of its main chemical constituents, which include carnosol, carnosic acid, ursolic acid, rosmarinic acid and caffeic acid. The potent antioxidant properties of rosemary have been mainly attributed to its major diterpenes, carnosol and carnosic acid, as well as to the essential oil components10. Based on the scientific reports aqueous rosemary extract was prepared to eliminate the lead hazard in rats.

MATERIALS AND METHODS

Materials: Rosemary was purchased from the Egyptian herbal Market, Dokki, Giza, Kits Biochemical analyses AST, ALT, ALP, Bil., Alb, Ch, TG, HDL, LDL, urea, Cr and UA were purchased from Biomeieux, Laboratory of Reagents and Products (France).

Experimental animals: The female rats were purchased from the Animal House Colony, National Research Centre, Giza, Egypt.

Methods
Preparation of rosemary extract:
One hundred grams of rosemary added to excessive distilled water:ethanol (2:8 v/v) and incubated at room temperature for 24 h, then the slurry was filtered through filter paper. The water extract was concentrated using rotary evaporator under reduced pressure and the residues were dissolved in 50 mL of distilled water.

Total phenol content: The total polyphenols content of rosemary extract was determined colourimetrically using the Folin-Ciocalteu reagent according to the modified method described by Gutfinger11.

Antioxidant activity
DPPH radical scavenging method:
The antioxidant activity of the phenol extracts was evaluated using the stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) according to a modification method of Bandoniene et al.12.

Experimental design: The experimental rats divided into six groups are: Control group fed on standard died only according13 to AIN-1993, group 2 received orally lead acetate solution at dose (4 mg kg–1), group 3 and 4 orally treated with rosemary extract at different two doses 100 and 200 μL and group 5 and 6 received orally lead acetate solution at dose (4 mg kg–1) plus rosemary extract at mentioned doses.

The animals were observed daily for signs of toxicity and weighted as well. At the end of experimentation period (i.e., day 30), blood samples were collected from all animals from retro-orbital venous plexus for biochemical analysis. Then all animals were killed and samples of the liver and kidney tissues of each animal were removed and hydrated in ascending grades of ethanol, cleaned in xylene and embedded in paraffin.

Histopathological examination: All histological analyses were performed in routinely processed formalin-fixed, paraffin embedded tissue sections of 5 mm thickness. They were stained with hematoxylin-eosin stain and the slides were examined with light microscope. Randomly selected fields were evaluated for cellular and tubular structures. Degeneration in epithelium and interstitial spaces were also noted.

RESULTS AND DISCUSSION

Table 1 shows the polyphenol and antioxidant activity of rosemary extract and found that the extract containing 9.98 mg mL–1 and 89.1%, respectively. Rosemary leaf extracts differed in the content of total phenolics and also in the amount of carnosic acid.

Table 1:Polyphenol and antioxidant activity of rosemary extract
Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 1:Effect of lead solution and rosemary extract at two dose as a protective agent on liver functions in female rats

Table 2:Effect of lead solution and rosemary extract at two dose as a protective agent on liver functions in female rats
Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Means superscripted with different letters are significantly different (p<0.05), R: Rosemary

Table 3:Effects of lead solution and rosemary extract at two dose on kidney functions in female rats
Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Means superscripted with different letters are significantly different (p<0.05), R: Rosemary

The content of total phenolics in rosemary leaf extracts ranged from 99-318 mg g–1 described by Helena et al.14. The antioxidant activity of rosemary extracts is primarily related to the presence of the two phenolic diterpenes carnosic acid and its derivative carnosol15.

The effect of lead solution and rosemary extract at different doses alone or in combination with lead solution were studied to explore of the effects on the biochemical parameters and histological picture in female rats because the female is also exposing to lead at many sites.

The tabulated data in Table 2 and Fig. 1 showed the effect of lead on the liver functions (AST, ALT and ALP U L–1), bilirubin (mg dL–1) and cleared that the lead induced negative effect and increased the mentioned parameters to be 78.6, 68.6, 129 and 0.55, respectively except albumin which decreased to be 3.40 mg dL–1. The same parameters were in normal range for control and the groups received rosemary at two tested doses and were in range between 29.3-29.6, 27-29, 96.6-102.6, 0.49-0.51 and 3.8-4.1, respectively. It is worthy to mention that treatment of rats with lead plus rosemary at two tested doses improved all biochemical parameters towards the safe limit for control group and gave the values in range between 25.2-28.2, 21.6-30.6, 86.3-119.6, 0.52-0.58 and 4.0-4.1, respectively. From the results cleared the safety of rosemary extract at two tested doses on the liver function and could be used as safe drink to eliminate the lead hazard, Doyl and Younger16 reported that lead is stored in almost all soft tissues and the liver is the largest repository of soft tissue lead. Reactive Oxygen Species (ROS) generated as a result of lead exposure has been identified in liver, kidney, brain, lung, endothelial tissue, testes and sperm. Lead causes oxidative stress by inducing the generation of ROS, reducing the antioxidant defense system of cells via depleting glutathione, interfering with some essential metal, inhibiting sulfhydryl dependent enzymes or antioxidant enzymes activities or increasing susceptibility of cells to oxidative attack by altering membrane integrity and fatty acid composition2 rosemary has been used empirically as a choleretic and hepatoprotective agent in folk medicine17. Most pharmacological effects of rosemary are the consequence of high antioxidant activity10.

Table 3 and Fig. 2 and 3 showed the effect of lead solution and rosemary extract at two tested doses alone or in combination with lead solution and the results showed that the normal values of urea, creatinin, uric acid for control and rosemary groups and their values were in range between 50.6-55.3, 0.46-0.51 and 2.6-2.7 mg dL–1, respectively. At the contrary the same kidney function parameters abnormality interrupted for the group treated with lead solution alone and gave the values 73.6, 0.41 and 2.9, respectively.

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 2:Effects of lead solution and rosemary extract at two dose on kidney functions in female rats

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 3:Effects of lead acetate solution and rosemary extract at two dose on kidney functions in female rats

Table 4:Effect of lead solution and rosemary extract at two tested doses as a protective agent on lipid profile of female rats
Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Within each column, means superscripted with different letters are significantly different (p<0.05), R: Rosemary, HDL: High density lipids, LDL: Low density Lipids

It is worthy to mention that the treatment of rats groups with lead plus rosemary at two tested doses improved the kidney function towards the normal control group and the same parameters were in range between 58.3-56, 0.48-0.49 and 2.4-2.5, respectively. Lead is stored in liver followed by kidney cortex and medulla2. Kidney function was interrupted by CCl4 and significantly restored after rosemary essential oil administration18.

Table 4 showing the effect of lead and rosemary at tested doses alone or in combination with lead on the lipid profile i.e., cholesterol, TG, HDL and LDL from the obtained results cleared that the control group possess the values 96.6, 115, 30.3 and 36.3 mg dL–1 for cholesterol, TG, HDL and LDL respectively. Slightly increase for the same parameters when rats received rosemary alone at two tested doses to be in the range between 103.3-116.6, 99.6-111.6, 30.0-33.3 and 53.6-64.3 mg dL–1, respectively., while the sharp decrease noted for cholesterol, HDL, LDL when rats received lead alone to record 86.6, 27 and 32.6 mg dL–1, respectively with increase of TG to be 155 mg dL–1.

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 4:Control untreated liver showing normal hepatocytes and blood sinusoids (H and E X200)

The current study was confirmed by histopathological examination for liver and kidney tissues to confirm the safety of rosemary extract on these tissues as shown in the Fig. 4-9.

Figure 4 control untreated rats liver showing normal hepatocytes and blood sinusoids, at the contrary Fig. 5 rats liver treated with lead 4 mg kg–1 b.wt., showing vacuolated hepatocytes and narrow blood sinusoids, while Fig. 6 rats liver treated with rosemary 100 mg kg–1 b.wt., showing degenerated hepatocytes and Fig. 7 rats liver treated with rosemary 200 mg kg–1 b.wt., showing focal area of hepatocytes necrosis infiltrated with mononuclear cells infiltrations.

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 5:
Liver treated with lead 4 mg kg–1 b.wt., showing vacuolated hepatocytes (black arrow) and narrow blood sinusoids (H and E X200)

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 6:Liver treated with rosemary 100 mg kg–1 b.wt., showing degenerated hepatocytes (H and E X200)

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 7:
Liver treated with rosemary 200 mg kg–1 b.wt., showing focal area of hepatocytes necrosis infiltrated with mononuclear cells infiltrations (H and E X200)

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 8:
Liver treated with lead 4 mg kg–1 b.wt.+rosemary 100 mg kg–1 b.wt., showing apparently healthy hepatocytes and blood sinusoids (H and E X200)

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 9:Liver treated with lead 4 mg kg–1 b.wt.+rosemary 200 mg kg–1 b.wt., showing vacuolated hepatocytes (H and E X400)

Figure 8 rat’s liver treated with lead 4 mg kg–1 b.wt.+rosemary 100 mg kg–1 b.wt., showing apparently healthy hepatocytes and blood sinusoids as well as Fig. 9 rats liver treated with lead 4 mg kg–1 b.wt.+rosemary 200 mg kg–1 b.wt., showing vacuolated hepatocytes.

The kidney histopathological pictures are as follow Fig. 10 control untreated rats kidney showing normal renal glomeruli and renal tubules, while Fig. 11 rats kidney treated with lead 4 mg kg–1 b.wt., showing degenerated renal tubules and interstitial mononuclear cells infiltrations.

Figure 12 shows that rats kidney treated with rosemary 100 mg kg–1 b.wt., showing slight degeneration in the glomerular tuft and renal tubular, as well as Fig. 13 shows rats kidney treated with rosemary 200 mg kg–1 b.wt., showing crystal ppt. in the renal cortex, together with degenerated renal tubules in Fig. 14, rats kidney treated with lead 4 mg kg–1 b.wt.+rosemary 100 mg kg–1 b.wt., showing apparently healthy renal glomeruli and renal tubules and in Fig. 15 shows rats kidney treated with lead 4 mg kg–1 b.wt.+rosemary 200 mg kg–1 b.wt., showing vacuolated glomerular tuft and degenerated renal tubules.

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 10:Control untreated kidneys showing normal renal glomeruli and renal tubules (H and E X100)

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 11:
Kidneys treated with lead 4 mg kg–1 b.wt., showing degenerated renal tubules and interstitial mononuclear cells infiltrations (H and E X200)

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 12:
Kidneys treated with rosemary 100 mg kg–1 b.wt., showing slight degeneration in the glomerular tuft and renal tubules (H and E X200)

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 13:
Kidneys treated with Rosemary 200 mg kg–1 b.wt., showing crystal ppt. in the renal cortex, together with degenerated renal tubules (H and E X200)

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 14:
Kidneys treated with lead 4 mg kg–1 b.wt.+rosemary 100 mg kg–1 b.wt., showing apparently healthy renal glomeruli and renal tubules (H and E X200)

Image for - Antioxidant Effect of Nutritive Extract from Rosemary Against Lead Hazards in Female Rats
Fig. 15:
Kidneys treated with Lead 4 mg kg–1 b.wt.+rosemary 200 mg kg–1 b.wt., showing vacuolated glomerular tuft and degenerated renal tubules (H and E X200)

CONCLUSION

It could be concluded that the lead should be evaluated in our foods and try to decrease its sources. The rosemary extract is very rich of polyphnol content as well as antioxidant activity which have the ability to protect against the lead hazard.

ACKNOWLEDGMENT

The study was supported by the Food Toxicology and Contaminants Department, National Research Centr, Dokki, Giza, Egypt.

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