Hepatoprotective and Reno-protective Effects of Artichoke Leaf Extract and Rosemary Extract against Paracetamol Induced Toxicity in Albino Rats

Background: Paracetamol overdose is a predominant cause of hepatotoxicity and nephrotoxicity in both humans and experimental animals. There is an emerging focus on plant products to find a highly effective and reliable drug for the prevention of paracetamol –induced toxicity. Objective: In this study, we investigated the Hepatoprotective and Reno-protective Effects of artichoke (Cynara scolymus L.) Leaf extract and rosemary (Rosmarinus officinalis L.) extract against paracetamol Induced toxicity in Albino Rats. Materials and Methods: Rats were divided into five groups: Negative control, paracetamol (1000 mg/kg dose) PCT, artichoke leaf extract “ALE” (1.5  g/kg, orally + paracetamol for 30 d), rosemary extract “RE” (125  mg/kg + paracetamol for 30 days) and the last group was treated with PCT+ ALE+ RE for 30 days. Results: Paracetamol caused marked liver damage as noted by significant increased activities of serum aminotransferases, alkaline phosphatase, gamma-glutamyl transferase and lactate dehydrogenase. Paracetamol also raised serum levels of urea, creatinine, and Cystatin-C. In addition, there was a significant decrease in serum total protein and albumin. Paracetamol caused Original Research Article Fadlalla and Galal; JPRI, 32(32): 67-81, 2020; Article no.JPRI.62462 68 an elevation in lipid peroxidation paralleled with significant decline in reduced glutathione (GSH) level and activities of glutathione-Stransferase (GST), glutathione (GPX) peroxidase, and superoxide dismutase (SOD) in the liver and kidney. These results are confirmed in the histological examination of the liver and kidney. Conclusion: Treatment with artichoke leaf extract (ALE) and rosemary extract (RE) produced a potential protection of the liver and kidney against biochemical and histological alterations and oxidative stress induced by paracetamol.

an elevation in lipid peroxidation paralleled with significant decline in reduced glutathione (GSH) level and activities of glutathione-S-transferase (GST), glutathione (GPX) peroxidase, and superoxide dismutase (SOD) in the liver and kidney. These results are confirmed in the histological examination of the liver and kidney. Conclusion: Treatment with artichoke leaf extract (ALE) and rosemary extract (RE) produced a potential protection of the liver and kidney against biochemical and histological alterations and oxidative stress induced by paracetamol.

INTRODUCTION
Paracetamol (PCT) used as a pain relief analgesic for arthritis, muscle aches, headache, fever and cold etc [1]. PCT is safe in therapeutic doses; however, overdose increase reactive oxygen species (ROS) production and worsen antioxidant defense [2,3].
Paracetamol has been repotted to cause acute kidney and liver injuries in experimental animal and humans [4,5].
Plant products are remarkably effective and reliable natural remedy for the prevention and cure of PCT -induced renal and hepatic-toxicity.
The researchers performed this study to investigate the hepatoprotective and Renoprotective effects of artichoke leaf extract and rosemary extract against PCT-induced toxicity in albino rats.

Reagents and Chemicals
Chemicals and paracetamol were purchased from Sigma Chemical Co. (St. Louis, MO). 40

Extraction of Plant Material
The Artichoke and Rosemary were collected on April, the identification and authentication of both plants were confirmed by the Botany Department of Ain Shams University.

Preparation of Artichoke leaves extract
2000 g of Fresh Artichoke leaves were blended mechanically with 2000 ml distilled water and filtered. The residue was re-dissolved in 1000 ml distilled water. The first aqueous extract was added to the later one, then condensed in rotary evaporator under vacuum. The condensed extract stored at 4°C. The method of extraction was carried out according to [15].

Rosemary leaves extract preparation
The rosemary (R. officinalis L.) leaves were dried, powdered. The extract was prepared by refluxing leaves with distilled water for thirty-six hours. The liquid extract was evaporated until transformed to powder. The powder was redissolved in distilled water before use [16].
At the end of the experiment, serum and organs samples were collected, properly handeled and stored at -20°C for biochemical parameters.

Total Flavonoids and Total Phenols Content in ALE and RE
Total phenolic (TP) content in the extract of both plants was assessed according to the assay of Folin-Ciocalteu [18], where Folin-Ciocalteu was mixed with the extracts. Then Na 2 CO 3 was added. The mixture left in dark for two hours. Then we eventually measured the optical density (O.D) against blank. (TP) content were expressed as mg GAE) /gm of the plant extract sample.
Flavonoid levels were calculated using a calibration curve prepared in parallel and in the same conditions as the samples obtained from a standard solution of quercetin.

Handling Liver and Kidney Specimens for Histopathological Examination
Liver and kidney sections were prepared for histological examination according to Luna [19] as follows: Tissue sections fixed in 10% formalin were washed in distilled water for 5 minutes, followed by dehydration using ethanol, clearing using xylene, infiltration, embedding by paraffin wax blocks, sectioning to thin 6-7 micrometer then fixed in slides for staining using the (Hematoxlin/Eosine stain). Liver and kidney tissue sections were subjected to histopathological examination using hematoxylin and eosin stains according to [20,21].

Parameters of kidney function
Serum creatinine and urea were determined according [24] and Kaplan [25] respectively. Serum cystatin C was determined according to Pergande and Jung [26].
Total protein was evaluated according to Henry, Cannon, and Winkelman [27]. Serum Albumin was assessed according to Doumas et al. [28].

Parameters of Oxidative Stress
Hepatic and renal Lipid Peroxidation (LPO) were determined in terms of malondialdehyde (MDA) production according to the method described by Rehman [29].
Reduced Glutathione (GSH) in the kidney and liver were estimated by estimating free-SH groups using the method defined by Sedlak and Lindsay [30]. Standard diet and served as control negative group Group 2 (PCT) Paracetamol was given orally by gastric tube at a dose level of 1000 mg/kg/ every other day for 30 days and served as control positive group. Group 3 (ALE) (Administered Artichoke and paracetamol) was given artichoke at a dose level of 1.5 g/kg body weight and paracetamol for for 30 days. [17] Group 4 (RE) (Administered rosemary and paracetamol) was given rosemary at a dose level of 125 mg/kg body weight and paracetamol for for 30 days. Group 5 (ALE+RE) (Administered was given artichoke at a dose level of 1.5 g/kg body weight + rosemary at a dose level of 125 mg/kg body weight and paracetamol for 30 days.
Hepatic and renal Superoxide dismutase activities were determined as described by Madesh and Balasubramanian [31].
Glutathione peroxidase activities were estimated as described by Paglia and Valentine [32].

Statistical Analysis
Values are expressed as mean ± S.E. The difference in mean between the different experimental groups was evaluated by one-way analysis of variance (ANOVA), by SPSS software statistical package (version 27; SPSS, Chicago, IL) according to [33] was used for statistical analysis. Table 2 showed that ALE has more phenolic content than RE, however the total flavonoids of RE was higher than ALE.

Effects of Artichoke Leaf Extract and Rosemary Extract on Hepatic Biomarkers
Paracetamol caused marked liver damage as noted by significant increased activities of serum aminotransferases (ALT, AST) and alkaline phosphatase (Table 3). Treatment with ALE and RE diminished PCT-induced elevation in these parameters PCT significantly increased, gammaglutamyl transferase (GGT) and lactate dehydrogenase (LDH) activities compared to the control. However, administration ALE and RE reversed paracetamol-induced elevation in GGT and LDH ( Fig. 1 and Fig. 2).
The ALT, AST and ALP activities are used to assess liver function [34]. Paracetamol administration significantly increases hepatic transaminases (ALT), (AST) and ALP.
The observe significant increase in serum transaminases in PCT-intoxicated rats revealed diminished hepatocytes functional integrity, this elevation might attributed to leak of the hepatocytes plasma membrane which eventually led to elevation of AST and ALT.
(GGT) and (ALP) are membrane bound enzymes. GGT activity is considered one of the best delicate indicators of hepatic function [35].
LDH has a vital role in the formation of pyruvate from lactate via NAD+ as coenzyme of NAD [36]. The elevated LDH activity could be due to drip of LDH into the blood stream due to cellular damage because of PCT -intoxication.
Paracetamol catalyzes formation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) via cytochrome P450. NAPQI directly induces oxidative stress, hepatocellular injury, and mitochondrial damage. Paracetamol has been reported to have traumatic impact on hepatic tissue [38,39].   However artichoke extract significantly reduced the elevated serum ALT, AST, ALP, LD and GGT levels which might be attributed to its precious phenolic content.
Artichoke extract remarkably reduced the elevated liver enzymes because of its capability to decrease free radical-induced oxidative damage in the liver (24), [40].
Domitrovic et al. [41] pointed out that Rosemary extract significantly inhibit CYP2E1 enzymatic activity in paracetamol-intoxicated rats. These results confirm our current findings of the inhibiting properties of RE.
Carnosol in the R. officinalis extract may contribute to RE hepatoprotective activity [42]. Treatment of paracetamol-intoxicated rats with either rosemary or artichoke extract can protect liver cells against damage, but the Co-treatment showed more ameliorating and promising effect as shown by the observed improvement in these biochemical parameters.
Cotreatment of ALE and RE improved hepatic biomarkers and showed obviously normal hepatocytes. This confirmed the protective effect of the ALE and RE or their combination and this effect might attribute to the diverse phytoconstituents and Flavonoids, which reveal protective influence on the liver. Furthermore, their precious antioxidant content may have a vital role in conserving hepatocellular membrane integrity.
These results are confirmed in the histological examination of the liver and kidney.

Effect of Artichoke Leaf Extract and Rosemary Extract on the Kidney Function
Paracetamol significantly raised serum urea, creatinine, and Cystatin-C as shown in (Table 4).
On the other hand treatment of PCT-intoxicated rats with ALE, RE or both together significantly improved renal function via the significant reduction in serum urea, creatinine and cystatinc.
The significant increase in serum urea in paracetamol -intoxicated rats might be attributed to the increased serum urea production, which exceeds urea clearance rate and, so tissue creatinine fragmentation which increases plasma creatinine levels [43,44].
High doses of paracetamol decrease glutathione levels and increase the production of toxic metabolites that are excreted through the kidney. These metabolites disrupt body homeostasis and may cause apoptosis and eventually resulted in renal dysfunction [45].
A systematic review about the relationship between paracetamol and renal impairment showed that PCT significantly increased the risk of renal impairment by (31%) in users without any history of renal disease [46].
The present study showed that the artichoke extracts attenuates induced elevation in serum creatinine and blood urea nitrogen (BUN) in rats.
Serum cystatin-C is an ideal marker of glomerular filtration [47,48]. It is considered one of the best markers for early diagnosis of renal dysfunction [49].
The improvement in renal functions upon ALE administration may be explained by the snoring content of artichoke, which accelerates urea metabolism and improves dieresis, which may initiate urea and creatinine excretion [50].
ALE may contribute in improving kidney functions via suppressing oxidative stress due to its phenolic content.
RE administration improved glomerular and renal function as shown by decreased Serum cystatin-C and serum creatinine which could be due to ROS-scavenging effect of RE.
Abd El-Ghany et al. [51] proved that aqueous extract of rosemary (RAE) inhibits free radical generation and lipid peroxidation and eventually recovers renal injury. The recovery effect of RE was due to its antioxidant constituents including rosmarinic acid, alpha tocopherol, carotenoids and diterpenoids.
The renal protective effect of Rosmarinus officinalis extract as manifested by the observed improvement in serum urea, creatinine and cystatin-C was proven by the normal appearance of kidney tissues.  Fig. 3 shows that paracetamol significantly decreased serum total protein and albumin, however coadministration of ALE and RE with paracetamol reversed these changes.
The decreased in serum total protein and albumin upon paracetamol administration might be due to defective protein synthesis.
Administration and co-treatment ALE and RE resulted in a significant improvement in protein metabolism parameters, which could be explained by enhancement in protein synthesis.

Effect of Artichoke Leaf Extract and Rosemary Extract on Hepatic and Kidney Antioxidant Enzymes
The impact of different treatments on oxidative stress parameters in liver and kidney are presented in Tables 5&6 and Fig. 4a,b. The results implied that liver and kidney MDA were significantly increased (Fig. 4), while there was a significant decline in reduced glutathione (GSH) level and activities of glutathione-S-transferase (GST), glutathione (GPX) peroxidase, in the liver and kidney paralleled with significant decline in superoxide dismutase (SOD) in the liver and kidney in the paracetamol group. ALE and RE treatment reversed the alterations in these parameters Tables 5&6.
Paracetamol caused an elevation in lipid peroxidation paralleled with significant decline in reduced glutathione (GSH) level and activities of glutathione-S-transferase (GST), glutathione (GPX) peroxidase and superoxide dismutase (SOD) in the liver and kidney.
Acetaminophen is converted to NAPQI via cytochrome P450 2E1 (CYP2E1). In case of PCT overdose, the extra NAPQI diminishes GSH, lead to attachment of extra NAPQI to sulf-hydryl groups in mitochondrial proteins which induce mitochondrial dysfunction. This elevated superoxide free radicals and oxidative stress [52,53].
Paracetamol led to depletion of liver GSH which increased lipid peroxidation and lead to liver damage [54].
One of the potential mechanisms for reversing the oxidative stress may be through CYP2E1 activity in paracetamol-intoxicated rats.
Furthermore, there were no significant differences in CYP2E1 enzyme activity between Rosmary and Artichock extract -treated groups (p<0.05).
GST is integral constituent of the detoxification system [39]. Our current research revealed that Paracetamol significantly (p<0.05) reduced hepatic GSH and GST, while rosemary and artichoke extracts reserved these effects.    Paracetamol administration increased lipid peroxidation as indicated by the significant increase (p<0.05) in liver and kidney MDA (Fig. 4).
In harmony with the results of the current study, increased hepatic lipid peroxidation during Paracetamol toxicity has been reported by other studies [55,56].
Our study showed that rosemary and artichoke extract decreased hepatic MDA which are in agreement with prior studies [57,58].
The decline in lipid peroxidation might be attributed to antioxidants constituents and cytoprotective properties of Rosemary extract [59]. RE owns ample biologically active antioxidants; include but not limited to rosmarinic acid, carnosic acid, betulinic acid, ursolic acid, rosmanol and rosmaridiphenol [60].
The role of ALE against oxidative stress are interrelated to its high flavonoids content which may express their effect through diminishing the ROS formation via inhibiting chelating trace elements or enzymes involved in the free radical production. These finding agreed with Mustafa et al., 2015 who reported that, ALE Flavonoids might exert these properties by scavenging ROS, and upregulate or protect the antioxidant defense [61].
ALE has been reported to increase glutathione peroxidase activity in liver and kidney.

Histopathology of Liver
Sections of livers in control group showed normal histological structure of hepatic lobule ( Fig. 6A), Meanwhile, liver sections of paracetamol -intoxicated rats showed apoptosis of hepatocytes (small arrow), portal infiltration with mononuclear cells and proliferated oval cells (large arrow) (Fig. 6B1), in addition fibroplasia in the portal triad (small arrow) associated with mononuclear cells infiltration and oval cells proliferation (large arrow) were reported (Fig. 6B2), while, liver tissue sections of Paracetamol -intoxicated rats treated with artichoke extract showed mild improvement in hepatic histopathology (Fig. 6C) as proved by slight vacuolation of centrilobular hepatocytes (small arrow) and activation of Kupffer cells (large arrow) in addition, slight improvement in liver Histopathology was observed in rosemary extract treated group as shown slight vacuolation of some hepatocytes (small arrow), activation of Kupffer cells (large arrow) and binucleation of hepatocytes (arrow head) (Fig. 6D) however, the joint effect ALE and RE significantly improved liver histopathology as proven by activation of Kupffer cells (small arrow) and binucleation of hepatocytes (large arrow) (Fig. 6E).
The observed progress in liver histopathology upon artichoke and rosemary treatments might be due to the coincide decrease in the elevated transaminases and ALP, which previously attributed to the antioxidant defense mechanism of ALE and RE due to their precious antioxidant content as shown in Table 2.

Histopathology of Kidney
Renal histopathological examination of the control negative group showed normal histological structure (Fig. 5A). and perivascular oedema (large arrow) in addition to congestion of glomerular tuft (small arrow) and focal necrosis of renal tubules (Fig. 5B1,2). However renal sections in experimental rats treated with ALE showing mild improvement in renal tissue with slight congestion of glomerular tuft (arrow) (Fig. 5C).
Treatment of paracetamol-intoxicated rats with RE moderately improved renal histologicay as confirmed by slight congestion of glomerular tuft (small arrow) and vacuolation of epithelial lining renal tubules (large arrow) (Fig. 5D).
The promising output from the current histopathological examination of kidney is the joint synergic effect of ALE & RE which observed clearly as improvement in kidney function as well as kidney histopathology where kidney of group 5 showed normal renal parenchyma (no histopathological changes) (Fig. 5E).
Current results are agreement with [59] who reported that rosemary prevented histopathological lesions and oxidative stress in liver, kidney.
The current improvement in renal structure might attributed to the simultaneous improvement in creatinine clearance, blood urea nitrogen and cystatin-c upon ALE and RE treatment and this improvement as mentioned earlier in the current research is due to the plentiful phenolic and flavonoid content of rosemary and artichoke extracts.

CONCLUSION
The current study proves that Artichoke Leaf Extract and Rosemary Extract are promising hepatoprotective and nephroprotective agents against Acetaminophen Induced toxicity in Albino Rats. ALE and RE restore urea, creatinine and Cystatin-C near to normal range, while coadministration of AE and RE has a significant antioxidant effect. Both Artichoke Leaf Extract and Rosemary Extract showed significant improvement in liver function. In conclusion Artichoke Leaf Extract and Rosemary Extract are highly advocated for paracetamol intoxication, to gain protection from liver and renal toxicity. Since each extract has its specific mechanisms in improving the tested parameters, a combination of both extracts may offer additional powerful effect for detoxification of liver and kidney.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
Authors declare that they follow the "Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985). The proper ethics committee has approved all experiments and protocol -Ain Shams University.