The liver is the primary organ for the metabolism of ingested alcohol 1. The liver is the largest and an important organ, and the site for essential biochemical reactions in the human body. It serves the function of detoxifying toxic substances and synthesizing useful biomolecules. Therefore, damage to the liver leads to adverse consequences. Alcohol induces oxidative stress, which is known to cause liver injury, and many biochemical metabolic reactions occur as a result of it. Some of these include redox state changes, production of reactive acetaldehyde, damage to the mitochondria of cells, cell membrane damages, hypoxia, effects on the immune system, altered cytokine production, induction of CYP2E1, and mobilization of iron 2. Alcoholic liver disease is a health problem that is prevalent worldwide and has three manifestations: fatty liver/steatosis, alcoholic hepatitis, and liver cirrhosis. At least 80% of chronic alcoholic consumers may develop steatosis, 10–35% may develop alcoholic hepatitis, and ∼10% may develop liver cirrhosis. Intake of alcohol leads to the accumulation of reactive oxygen species (ROS) such as superoxide, hydroxyl radical, and hydrogen peroxide in the hepatic cells, which oxidize glutathione (GSH), which in turn causes lipid peroxidation of cellular membranes, oxidation of protein, and DNA, resulting in hepatic damage 3. Prolonged consumption of alcohol increases the nitric oxide level, which leads to the formation of toxic oxidant peroxynitrite. Low capacity of antioxidants in this situation leads to damage of the cells of the hepatic cells and the cell organelles, with the release of reactive aldehydes and ROS 4. Treatment options available for common liver diseases such as cirrhosis, fatty liver, and chronic hepatitis are inadequate in modern medicine. Conventional drugs used in the treatment of liver diseases such as corticosteroids, antivirals, and immunosuppressants may lead to serious adverse effects; they may even cause hepatic damage on prolonged use. Therefore, alternative drugs in the form of herbal medicines that are now used for the treatment of liver diseases are sought instead of currently used drugs with unclear efficacy and safety 5. Protective actions of well-known antioxidants play a significant role in diseases – for example, vitamin C, vitamin E, β-carotene, and plant phenolics. Excessive use of synthetic antioxidants such as butylated hydroxyl toluene and butylated hydroxyanisole may cause hepatic damage and hence their use is restricted 6. A single drug in the form of herbal medicine cannot be effective for all types of liver diseases. It is therefore considered necessary to develop an effective formulation or combination using indigenous medicinal plants by pharmacological experiments and clinical trials based on the concept of polypharmacy 7.
Antioxidants are substances that, when present at a low concentration, significantly delay or reduce the oxidation of the substrate 8. Experimental studies in various models of liver and kidney injuries have been conducted on a wide variety of plants and their active principles. Recently, there has been an upsurge of interest in the therapeutic potential of medicinal plants as antioxidants. Current research trends are directed toward finding naturally occurring antioxidants particularly of plant origin in reducing such free radical-induced tissue injury 9–11. Many plant species have been investigated in the search for natural antioxidants 12 and novel antioxidants 13, but generally, there is still a need to obtain more information on the antioxidant potential of plant species. It has been reported that the antioxidant activity of plants might be because of their phenolic compounds 14.
Throughout history, herbs have been utilized as an important constituent of foods, industry, and folk medicine. One of the widely used vegetal species in various nations as medicine is parsley (Petroselinum crispum), which has remedial effects as a powerful diuretic agent 15,16, an abortifacient 17, and an expectorant 18,19. Parsley is a native herb of the central Mediterranean region (southern Italy, Algeria, and Tunisia). It is part of the Apiaceae family and is a species of Petroselinum20. The antioxidant activity of parsley essential oil has been confirmed in other investigations. Wong and Kitts 21 indicated that the phenolic compounds of parsley were responsible for its antibacterial and antioxidant activity. Zhang et al. 18 reported the antioxidant activity of this herb in terms of β-carotene bleaching capacity and free radical scavenging activity. This concept was then confirmed by further studies 22. Parsley possesses several flavonoids such as apiin and luteolin, and its essential oil contains apiol and myristicin. These components are believed to be responsible for the therapeutic effects of parsley 23. Kandaswami et al.24 reported the direct and indirect effects of flavonoids on tumor cells. Their studies showed that the hydroxylation pattern of the B-ring of the flavons and flavonols, such as luteolin and quercetin, seemed to affect their angiogenesis and anticancer activity, especially the inhibition of protein kinase activity and antiproliferation 24. Robak et al.25 believed that flavonoids are the superoxide anion scavengers of the media and this effect can also lead to their anti-inflammatory effects. Daly et al.19 observed bioactive phytochemicals, including carotenoids, in parsley. Carotenoids were shown to be associated with a low risk of several human chronic disorders including age-related macular degeneration and certain cancers. Kreydiyyeh and Usta 26 and De Campos et al. 27 reported that parsley is widely used in folk medicine which possess a diuretic, natriuretic and hypotensive effects. Further studies indicated more biological effects of parsley plants, such as provitamin A activity, and influence on cell signaling pathways. Parsley is a plant with various biological activities 24,25. In addition, it has immunomodulatory effects. Karimi et al.28 found that its inhibitory effect on phytohemagglutinin -stimulated splenocytes might be because of the production of cytokines such as interferon-γ and interleukin-2, which are vital for T-cell proliferation, or it may influence the signaling pathways. They also indicated that parsley essential oil can modulate the activity of macrophages without exerting a cytotoxic effect. The immunomodulatory effect of parsley essential oil and its modulatory effects on nitric oxide production and function of macrophages may point toward parsley as a useful natural candidate for the treatment of some autoimmune and allergic diseases.
On the basis of these previous studies, the present work aimed to assess the ameliorative effect of parsley oil against alcohol-induced hepatotoxicity and oxidative stress in rats.
Materials and methods
Adult male albino rats (Rattus norvegicus) weighing 120–150 g were used in the present study. The animals were obtained from the animal house in the Ophthalmology Research Center (Giza, Egypt). They were kept under observation for 2 weeks before the onset of the experiment to exclude any intercurrent infection. The animals were kept at room temperature and exposed to natural daily light–dark cycles. Rats were fed ad libitum and clean water was continuously available. All animal procedures were in accordance with the recommendations for the proper care and use of laboratory animals by the Canadian Council on Animal Care 29.
Parsley oil was purchased from the El Captin Pharmaceutical Company for medical oil extraction and cosmetics (Al Azhr, Cairo, Egypt). All other chemicals used for the investigation were of analytical grade.
Doses and treatment
Alcoholism was experimentally induced in animals by administration of absolute ethyl alcohol (10%) in drinking water for 15 days before the experiment 30. Parsley oil doses were adjusted to 50 mg/kg and were administered daily for 4 weeks 31.
Animals were divided into three groups of 10 animals each:
- Group 1 (normal control) was orally administered the equivalent volume of the vehicle 1 (distilled water) daily for 4 weeks.
- Group 2 (positive control) was administered absolute ethyl alcohol (10%) in drinking water for 4 weeks 30.
- Group 3 (treated with parsley oil and alcohol) was administered absolute ethyl alcohol (10%) in drinking water for 15 days before the experiment 30. The dose of parsley oil was adjusted to 50 mg/kg and was administered daily for 4 weeks 31.
Blood and organ sampling
Under diethyl ether anesthesia, 5 ml of blood sample was collected from the jugular vein of each animal in a centrifuge tube and left to clot at room temperature for 45 min. Sera were separated by centrifugation at 3000 rpm at 30°C for 15 min and kept frozen at −30°C for various physiological and biochemical analyses.
The liver from each animal was rapidly excised after dissection. Liver tissue of 0.5 g was homogenized in 5 ml 0.9% NaCl (10% w/v) using a Teflon homogenizer (Glas-Col, Terre Haute, Indiana, USA).
Serum total bilirubin concentration was determined by a colorimetric procedure using kits obtained from Biodiagnostics (Dokki, Giza, Egypt) according to Walter and Gerarde 32. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were determined using kits obtained from Biodiagnostics according to the methods of Reitman and Frankel 33. Serum alkaline phosphatase (ALP) and γ-GT were determined using kits obtained from Biodiagnostics according to the method of Belfield and Goldberg 34 and Szasz 35, respectively. Serum total protein was determined using kits obtained from Biodiagnostics according to the method of Young 36. Serum lactate dehydrogenase (LDH) was estimated according to the method described by Bühl and Jackson 37 using a reagent kit purchased from Stanbio Laboratories (Boerne, Texas, USA). Liver GSH was determined according to the method of Beutler et al.38. Liver lipid peroxidation was determined by measuring thiobarbituric acid-reactive substances according to the method of Preuss et al.39. Liver glutathione-S-transferase (GST) activity was assayed using the method of Matkovics et al.40 and Mannervik and Gutenberg 41. Liver catalase (CAT) activity was assayed following the method of Kar and Mishra 42.
Fixed liver tissue samples were embedded after dehydration in paraffin wax, sectioned into a thickness of 5 µm, and stained with hematoxylin and eosin for a general histopathological examination using a light microscope.
The data were analyzed using one-way analysis of variance (ANOVA) 43, followed by least significant difference (LSD) analysis to compare various groups. Results were expressed as mean±SE. F-probability, obtained from one-way ANOVA, showed the effect between groups.
The changes in different serum variables related to liver function are presented in Tables 1 and 2.
In the serum enzymes related to liver function, the alcohol-administered rats showed a significant increase (P<0.01; LSD) in ALT, AST, and ALP activities. Pretreatment with parsley oil successfully ameliorated the elevated activities of AST, ALT, ALP, and γ-GT.
Serum total bilirubin concentration was elevated in alcohol-administered rats, but a significant increase was observed in parsley oil-pretreated rats.
Serum total protein level increased as a result of alcohol administration, whereas it decreased significantly in animals pretreated with parsley oil. Serum LDH activity increased significantly in alcohol-administered rats, although parsley oil improved LDH activity.
Table 3 shows the effect of the tested parsley oil on the liver oxidative stress markers and the antioxidant defense system of normal and alcohol-administered rats. The pretreatment with parsley oil produced a potential increase (P<0.01; LSD) in the GSH level, GST, and CAT activities compared with the alcohol-treated group, which showed a significant decrease in the GSH level, GST, and CAT activities compared with the normal control group.
In contrast, liver lipid peroxidation showed a significant increase as a result of alcohol administration, whereas it decreased significantly in parsley oil-treated rats in the control group.
Figure 1 shows the histology of the liver of normal controls. No histopathological alteration was observed and the histological structure of the central vein and surrounding hepatocytes was normal.
The portal area in ethyl alcohol-administered rats (group 2) showed severe dilatation and congestion in the portal vein associated with periductal fibrosis surrounding the bile ducts (Fig. 2). Fatty change was observed in a diffuse manner in all the hepatocytes (Fig. 3).
The hepatocytes showed hydropic degeneration associated with severe congestion in the portal vein and periductal fibrosis surrounding the bile ducts at the portal area (Fig. 4) in the parsley oil-pretreated group.
One-way ANOVA showed that the significance level between groups was P<0.001.
The present study was carried out to assess the protective properties of parsley oil on hepatotoxicity and oxidative stress. In terms of serum enzymes related to liver function, pretreatment with parsley oil successfully ameliorated the activities of AST, ALT, ALP, and γ-GT, which increased with the administration of alcohol.
Pretreatment with parsley oil produced a potential increase in the GSH level, GST, and CAT activities compared with the alcohol-treated group, which showed a significant decrease in the above parameters. Inversely, the lipid peroxidation level increased significantly in alcohol-administered rats and decreased significantly in the group that received pretreatment with parsley oil.
In ethyl alcohol-administered rats, the portal area showed severe dilatation and congestion in the portal vein associated with periductal fibrosis surrounding the bile ducts. Also, fatty change was observed in a diffuse manner in all the hepatocytes. However, the hepatocytes of the parsley oil-pretreated group showed hydropic degeneration associated with severe congestion in the portal vein and periductal fibrosis surrounding the bile ducts at the portal area.
These results are in agreement with those of Al-Daraji et al.44, who found that the addition of fresh parsley leaves to the diet of geese resulted in a significant improvement in most hematological traits. This improvement may be attributed to the fact that parsley is a good source of iron, is required to produce RBC, and enhances the metabolism of nutrients, such as vitamin C, which is believed to improve general health and fight infections, and β-carotene, which is converted into vitamin A, which maintains the cell membrane status of all tissues 45,46. Parsley improves the digestion of proteins and fats, thus promoting intestinal absorption, liver assimilation, and storage. Because of its high enzyme content, parsley facilitates digestive activity and elimination 47. Osman et al.48 reported that the high vitamin C, β-carotene, B12, chlorophyll, and essential fatty acid contents render parsley an extraordinary immunity-enhancing food. Parsley is an immune-enhancing multivitamin and mineral complex in a green plant form and one of the most important herbs that provides vitamins for the body 49. However, it is known that parsley alleviates stress by enhancing general health and immunity 50. Zheng et al.51 reported that parsley is rich in myristicin, which has been shown to have high activity as an inducer of the detoxifying enzyme GST in the liver and small intestine mucosa of mice. Fejes et al.52 reported that parsley contains flavonoids (apiin, luteolin glycoside, pigenin glycoside), essential oil (apiol, myristicin), cumarines (bergapten and imperatorin), and vitamin C. The protective role of parsley may be attributed to the higher content of these flavonoids, which either scavenge free radicals or increase the production of GST. Ozsoy-Sacan et al.31 concluded that parsley extract, probably because of its antioxidant property, exerts protective effects against hepatotoxicity caused by diabetes and has free radical scavenging and membrane-protective effects 53. Chloro compounds in parsley have often shown significant biological activities – for example, antibiotic, antitumor, antiviral, antibacterial, anti-inflammatory, antihepatotoxic, pesticidal, and antioxidant activities, and dissolve cholesterol within the veins, which all promote general health 54–56. Parsley supports bladder, kidney, liver, lung, stomach, and thyroid function, it helps clear uric acid from the urinary tract, contains a substance that prevents the multiplication of tumor cells, expels worms, relieves gas, and stimulates normal activity of the digestive system. It is used to treat urinary tract infections, helps dissolve and expel gall stones and gravel, is used to prevent kidney stone formation, acts as a diuretic, increases urine volume, and is used to treat digestive weakness and bronchial and lung congestion. It may also be used to treat edema and high blood pressure and cholesterol 57. Al-Howiriny et al.56 reported that the efficacy of any hepatoprotective drug is essentially dependent on its capability to either reduce harmful effects or to maintain normal hepatic physiological mechanisms that have been unbalanced by the hepatotoxin. The ethanolic extract of parsley exerts significant hepatoprotective effects against experimental CCl4-induced liver damage in animals. The increase in the plasma levels of cytoplasmic and mitochondrial enzymes accurately reflects liver injury induced with CCl4. This increase in the levels of certain enzymes (GOT, GPT) under the influence of CCl4 has been attributed to the disturbed or the damaged structural integrity of the liver. Significant decreases of CCl4-induced increased GOT and GPT4 levels by the parsley extract definitely suggest protection of the structural integrity of the hepatocyte cell membrane by the extract. The increased level of the plasma ALP is another measure of liver damage occurring because of the de-novo synthesis by the liver cells. It was observed that on treatment with the parsley extract, there was a significant decrease in the plasma ALP level, indicating the ability of the extract to maintain the normal level. This effect might be because of either a decrease in de-novo synthesis or a feedback mechanism. However, the parsley extract could decrease the plasma bilirubin level, thereby indicating its effectiveness in maintaining the normal functional status of the liver. Parsley contains a number of chemical components, including flavonoids, tannins, sterols, and/or triterpenes, and all of these constituents are known to exert antioxidant activity and confer protection against cell damage 58, and exert free radical scavenging effects. Furthermore, CCl4 led to a significant reduction in the nonprotein sulfhydryl concentration. Parsley extract, however, replenished the nonprotein sulfhydryl concentration significantly. Thus, sulfhydryl groups seem to be involved in the hepatoprotective mechanism. Al-Howiriny et al.56 concluded that parsley showed significant anti-inflammatory activity and significant hepatoprotection against CCl4-induced toxicity.
Pandanaboina et al.59 found that alcohol treatment resulted in the depletion of superoxide dismutase, CAT, glutathione peroxidase, glutathione reductase, and GST activities, reduced the GSH content, increased the level of malondialdehyde, and decreased the levels of protein carbonyls, causing malfunction of hepatic and renal tissues.
Brown et al.60, Mallikarjuna et al.61, and Shanmugam et al.62 reported that GSH acts as an antioxidant and a powerful nucleophile, critical for cellular protection, such as detoxification of ROS, conjugation, and excretion of toxic molecules and control of the inflammatory cytokine cascade 60. The levels of GSH were significantly decreased in alcohol-treated rats, as reported in earlier published reports, which showed that the GSH concentration decreases during alcohol ingestion 61,62. The administration of parsley extract and glibornuride increased the content of GSH in the livers of diabetic rats. The elevated level of GSH protects cellular proteins against oxidation through the GSH redox cycle and also directly detoxifies ROS 63.
CAT acts as a preventive antioxidant and plays an important role in the protection against the deleterious effects of lipid hydroperoxide (LPO). Reports have shown that there is a significant decrease in the activities of CAT in alcoholic patients 64. The decreased activity of CAT was because of exhaustion of the enzyme as a result of oxidative stress induced by the alcohol. Presumably, a decrease in CAT activity could be attributed to cross-linking and inactivation of the enzyme protein in the lipid peroxides.
Liver lipid peroxidation was increased as a result of alcohol administration, whereas it was significantly decreased in the parsley oil group, corresponding to the control group. The current results were in agreement with those of Pandanaboina et al.59, who observed a significant increase in lipid peroxidation during alcohol consumption, as reported by earlier studies 65. The alcohol intoxication increases LPO production in various tissues, and is indicative of tissue oxidative stress. Nearly 60–80% ingested alcohol is metabolized in the liver, and this makes it more vulnerable than other organs to alcohol-induced oxidative stress 66. This concept is supported by the greater increase in LPO production in the liver compared with other organs.
Parsley extract may decrease the liver peroxides back to near-normal levels. This indicates that parsley extract may inhibit oxidative damage of hepatic tissue 31. Antioxidant effects have been reported for some plants that contain flavonoids, phenolic compounds, ascorbic acid, and tocopherol 67. Phytochemical results showed that parsley extracts are rich in flavonoids 53, phenolic compounds, ascorbic acid 68, and tocopherol 69. It is possible that the antioxidant effects of this herb are related to these components.
In conclusion, the present results showed that parsley oil plays an important role by exerting an ameliorating effect against alcohol-induced hepatotoxicity and oxidative stress. Further clinical studies are required to assess the benefits and safety of parsley oil before their use in humans and approval by the Food and Drug Administration.
Conflicts of interest
There are no conflicts of interest.
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