Hepatoprotective effects of walnut oil and Caralluma tuberculata against paracetamol in experimentally induced liver toxicity in mice

Walnut Oil and Caralluma are edible and form part of the traditional medicine system in many countries. These are frequently used in traditional medicine as remedies to relieve a wide range of illnesses and health prob- lems. Walnut Oil and Caralluma species have demon-strated anti-inflammatory, anti-nociceptive, antidiabetics, hepatoprotective, gastric mucosa protecting, antimalari-al, antioxidant, anti-trypanosomal, appetite suppressant and cytotoxic activities. The current study was planned to study the impacts of 21 days’ oral administration of walnut oil and methanolic extract of Caralluma tubercu- lata on the levels of some liver-associated parameters and hematological parameters in paracetamol intoxicated mice. It was observed that paracetamol intoxication resulted in a considerable rise in serum ALT, cholesterol, triglycerides, Creatinine, and urea levels while a decrease in HDL level in comparison to mice normal control group ( P <0.05). Serum ALT, cholesterol, triglycerides, creatinine, and urea levels of mice that were administered with walnut oil and methanolic extract of C. tuberculata at the doses of (1 ml/kg, 2 ml/kg and 3 ml/kg body weight) were significantly lower when compared to toxic control mice group ( P <0.05), While HDL level was significantly increased. The significant reduction had also been observed in the levels of serum parameters of mice group, which received standard hepato-protective drug i.e., vitamin C, at the dose of 8 mg/kg body weight ( P <0.05). Based on these results, it was evident that liver toxicity caused by the paracetamol administration has recovered toward the normal range by the walnut oil and C. tuberculata extract. There-fore, the present study revealed that (walnut oil and C. tuberculata ) exhibit hepatoprotective activities in paracetamol intoxicated mice.


INTRODUCTION:
The liver is the prime organ in the human body that helps in maintaining metabolic and physiologic homeostasis. It plays a central and important role in many metabolic pathways especially those related to growth and development, fighting off diseases, production and supply of nutrients and energy, metabolism of proteins, carbohydrates and fats, bile secretion and storage of vitamins (Ouassou et al., 2021;Sami et al., 2019, Tafere et al., 2019Ugwu & Suru 2021;Wahid et al., 2016). Furthermore, the liver is also a crucially important organ in drug metabolism and protecting humans against the harmful impact of many toxic agents. These toxic exogenous (toxins, alcohol, environmental pollutants, viruses, and xenobiotic substances) or endogenous (autoimmune diseases) agents are a major risk factor for hepatic injury leading to hepatitis fibrosis, cirrhosis, cancer, and other diseases of the liver (Maev et al., 2014;Real et al., 2019;El-Hadary & Ramadan Hassanien, 2016). Hepatic damage, due to any reason, greatly hampers the normal functioning of many metabolic processes. In the majority of the cases, the damage is obvious from alteration in alanine transaminase (ALT) and aspartate transaminase (AST); the enzymes found in different body tissues and serum but originating from parenchymal cells of the liver .
Only some conventional drug remedies are available to treat and improve hepatic functions, but they are also associated with hepatic injury when overdosed (Gagliano et al., 2007). These drugs can induce hepato-toxicity resulting in raised ALT and AST and associated liver damage. Of these, Acetaminophen (N-acetyl-p-aminophenol, paracetamol), an antipyretic and painkiller drug is the most common hepatotoxic agent. The drug act by selective suppression of the Cox-3 enzyme pathway in the spinal cord and brain thus n reducing pain and fever (Chandrasekharan et al., 2002). Paracetamol is metabolized by the liver and then excreted through the kidneys . Paracetamol is one of the most commonly available, over-the-counter drugs that is administered by oral, intravenous injection or rectal route to relieve pain and fever (Pathan et al., 2014). In normal dosage, the drug is safe, but an overdose can induce liver damage, nephrotoxicity, extrahepatic lesion and even death (Bhattacharyya et al., 2003;Darbar et al., 2011 evidence suggests that almost 40% of drug-associated liver disorders are associated with paracetamol overdose (Omotayo et al., 2015) for instance, chronic liver diseases and drug -induced hepatic injuries are among the top ten leading causes of death worldwide (Harsha et al., 2021;Saleem et al., 2010), thereby requiring remedies that are safe and effective. In this context, natural herbal preparations offer a promising alternative that is not only effective to treat and prevent liver damage but also safe and free of side effects.
Pakistan is home to a rich and diverse flora consisting of almost 5700 species. Among these, around 2000 species are believed to possess important pharmacologic activities against various illnesses and thus constitute a large group of medicinally important plants (Ullah, 2017). Walnut (Juglans, Juglandaceae) is a commonly grown tree in mountainous terrains of the world including Pakistan. Different parts of this have traditionally been used for medicinal purposes owing to its antioxidant, antimicrobial, anti-inflammatory, immune-modulatory and wound healing activities. The most important part of the tree is the walnut fruit, consisting of an outer green shell cover or husk, the middle shell which after cracking releases the kernel. Kernel, the nutrient-rich part of the fruit is commonly used as a food supplement and cosmetic products across the world (Pereira et al., 2007;Stampar et al., 2006;Britton et al., 2009). Similarly, walnut tree leaves are a rich source of different compounds possessing health promoting activities and therefore, commonly used in folk medicines for the curing of venous insufficiency, inflammation of skin and ulcers (Eidi et al., 2013). Recent research studies, both in human and animal models have also reported antidiabetic, antioxidant, and lipid-lowering effects of the leaf extracts from walnut tree (Mollica et al., 2017). Flavonoids PUFAs are the main bioactive compounds isolated from the leaves, with proven beneficial activities as reported previously (Panth et al., 2016;Carey et al., 2013).
Correspondingly, C. tuberculata, a family member of Asclepiadaceae (milkweed family), is an edible, juicy, leafless stiff plant that commonly grows in dry, undomesticated regions of Pakistan and other countries such as Saudi Arabia, Nigeria, and Iran (Mudrikah et al., 2015). C. tuberculata is extensively found in the mountainous areas of Khyber Pakhtunkhwa province of Pakistan, where it is called Pamankay in the local Pashto language. The plant has commonly known for its ethnomedicinal applications including the treatment of dysentery, jaundice, constipation, stomach pain, blood purification, liver ailments and hypertension (Baig et al., 2021;Delaviz et al., 2017;Adnan et al., 2014). The disease prevention and health-promoting effects of the plant are primarily attributed to its antioxidant, antibacterial (Mudrikah et al., 2015), hypolipidemic, anti-hyperglycemic (Abdel-Sattar et al., 2011;Essam et al., 2011), and in vitro anticancer potentials (Waheed et al., 2011). The plant has been shown to contain many bioactive secondary metabolites including steroids, reducing sugars, terpenoids, beta cyanin, tannins and amino acids. Taking the above medicinally important activities into account, the current study was designed to assess hepatoprotective effects of walnut oil and C. tuberculata extract in paracetamol-induced liver damage in mice.

Preparation of plant extracts and walnut oil
C. tuberculata plants and walnut fruits were purchased from a local market in Dir (Lower), Pakistan. The plants were allowed to dry in shade for one month followed by mechanical grinding to obtain a fine powder. The powder (800 g) was soaked in 90% methanol at room temperature and after 10 days, the extract was filtered using filter paper. The filtrate was collected and vaporized using Rotary Evaporator. The methanol was evaporated at a temperature of 70 o C in a water bath and a pressure of 50 Pa, the concentrated extract was obtained. The extract was then kept at room temperature. Walnuts were taken and the shells were removed, endosperm was collected and processed through the cold pressing method to extract the oil.

Experimental animals
One month-old, healthy, Albino mice were obtained from the National Institute of Health Islamabad and kept in the Bio Park of the Biotechnology Department, University of Malakand. Mice were housed in polypropylene cages, each containing 8-10 animals, sustained under standard conditions and provided the same dried food and water ad libitum until the day of experiments. Standard ethical guidelines regarding the care of the animal were followed. Ethical approval of the study was obtained from the ethics board of Islamia College University, Peshawar.

Grouping and extract administration
Mice were randomly divided into nine different groups each containing 10 mice. The positive control group was intoxicated with 200 mg/kg body weight paracetamol while the negative control group only received normal feed and water ad libitum. Group A, B and C mice were intoxicated with paracetamol (200 mg/kg body weight) and fed with walnut oil at a dose of 1, 2 or 3 mL/kg body weight, respectively. Group D, E and F also receive the same paracetamol dose and C. tuberculata extract at a dose of 1, 2, 3 mL/kg body weight, respectively.

Laboratory analysis
Complete blood count (CBC) was done by Automatic Digital Machine (Sysmex Kx-21) for the assessment of Leucocytes, Neutrophils, Lymphocytes, Platelets count (PLT), Monocytes and Eosinophils count. Serum biochemical parameters such as total cholesterol, triglyceride, uric acid, creatinine, HDL, LDL and ALT were analyzed using Enzyme-linked immunosorbent assay (ELI-SA) following manufacturer instructions.

Histopathological examination
Once the blood was collected, the mice were sacrificed. Mice livers were collected, cut into small pieces, and fixed in 10% formalin. After fixation, the specimens were dehydrated, then embedded in wax and sectioned into 5 µm thicknesses. The sections were then stained with eosin and Hemotoxillin. For slide preparation, the mice's liver was carried out in 10% formalin for 14 hours. For dehydration propanol was used in the percentage of 70% for 1 hour, 80% for 1 hour and 95% for 1 hour. After dehydration, de-alcoholization was done by xylene. Two types of xylenes were used namely xylene pure and xylene alcohol. This process was repeated three times. First, the specimen was taken in xylene alcohol for 1 hour, then in xylene pure for 1 hour and then again in xylene pure. This process removed all the alcohol from the specimen tissue. Paraflast wax was used to remove xylene. These are embedded wax melted at 55 o C. Then the L-Blocks which are made of alloy were taken and were cut into blocks with the help of a microtome (rotary microtome).

Evaluation of hepatoprotective activity
Based on the paracetamol-induced liver damage method as described in various studies (Hussain et al., 2014;Qadir et al., 2014;Saleem et al., 2014), the in-vivo hepatoprotective activity was assessed.

Slide formation and its microscopic study
Staining was done in two steps i.e., slides were put in xylene, dilute for 1 hour and then put in xylene pure for 1 hour for the removal of waxes from the slides. Then they were put in propanol concentrated for 1 hour and again in propanol pure for another 1 hour, then in water for 30 minutes and shaken the slide. After that, they were then put in hematoxylin which started staining in fifteen minutes. Hematoxylin in nature is nucleophilic. They were put in water and then in acid alcohol. The stain was taken by all slides. With the help of acid al-cohol, the stains were removed from those areas which were not needed. Then again, they were put into water to remove acid alcohol and then in ammonia water for 5 minutes which give a reddish color. Propanol was used again to attach with eosin to give reddish color easily, then again diluted propanol was used to remove eosin from slides except for staining areas. Xylene was used again to remove water and alcohol. Candabalsam and dpx (mixture of distyrene, plasticizer and xylene) were applied to the thin slice and then a cover slip was put to cover it. The slides were ready for microscopic examination. The slides were studied by an electric microscope model No. M 7000 D (SWIFT, Japan) and the pictures were captured by a digital camera of microscope DCM 130 (SWIFT, Japan) (USB 2.0) with a resolution of 1.3 M pixels which is commonly known as the CCT camera.

Statistical analysis
All samples were measured in triplicate. Statistical analysis was carried out using one-way analysis of vari- ance (ANOVA) and Tukey test using GraphPad Prism 5 for windows.

Effect on body weight
Paracetamol administration to the mice at the dose of 200 mg/kg body weight showed increased levels of total cholesterol, triglycerides and ALT while decreased levels were noticed HDL as shown in Fig. 1. Similar findings have been reported by (Oyagemi & Odetola, 2010); that paracetamol significantly increased the levels of total cholesterol, triglyceride and ALT, and reduced HDL level further confirmed by Zakaria and others (Zakaria et al, 2020). From Fig. 1 it can also be seen that walnut oil showed decreases in the level of cholesterol and ALT, while increasing the level of HDL. The cholesterol and Triglyceride levels were also decreased by vitamin C. It also increases the level of HDL. Furthermore, C. tuberculata decrease the levels of cholesterol, triglycerides and ALT and increase HDL level.

Serum biochemical parameters
Estimation of different serum enzymes and markers has always been a useful quantitative and validated marker to assess liver injury and toxicity. In the current study, we have used different biochemical parameters as markers of liver damage and paracetamol-induced toxicity. As shown in Fig. 1, the levels of cholesterol, triglycerides, ALT, creatinine, and urea were significantly elevated in paracetamol-fed mice (positive control) compared to the negative control, indicating hepatic damage. However, pre-treatment with low, medium, and high doses of walnut oil and C. tuberculata extracts have significantly reduced these enzymes, almost to the level of normal values, especially on day 21. Of all these markers, ALT is the most common biochemical marker of liver toxicity. Following acute paracetamol toxicity, the level of ALT rose significantly reaching as high as 101±2.6 IU\L on day 21 compared to the negative control (60±1.0 IU/L). Pretreatment with high doses of both walnut oil (WHD group) and C. tuberculata extract (EHD group) had a significant impact on ALT levels, especially on day 21 when their levels were almost similar to the negative control group (63±1.0 IU/L for WHD group and 62±1.0 IU/L for HD group).

Hematological parameters
Complete blood count including total leukocytes, neutrophils, lymphocytes, and platelets was also assessed on days 7, 14 and 21. Following acute paracetamol toxicity, the total leukocyte count remains nearly constant in negative controls (5.4×109 cells per liter) while in positive controls, a nearly 3-fold increase (14±5 109 cells per liter) was detected. Pretreatment with different doses of walnut oil and C. tuberculata extracts have attenuated the paracetamol toxic effects, bringing the leukocyte count to almost normal at day 21 as shown in Table 1. CBC also revealed that paracetamol toxicity results in elevated lymphocytes, but lower neutrophils count compared to the untreated negative control. Pretreatment has successfully reversed the effects of paracetamol toxicity in all doses, especially on day 21. The effect of different doses of Walnut Oil and C. tuberculata extracts on hematological parameters is summarized in Table 1.

Liver Histopathology
Figure 2 (A-I) demonstrates the histopathological examination of liver sections from all experimental groups of mice. In the normal control group of mice ( Fig. 2A), the liver parenchyma as well as endothelial linings of central veins had normal morphology with no evidence of pericentral fibrosis. Kupffer cells showed non-reactivity the orientation of the hepatic cord was well defined. Furthermore, the hepatic portal vein and artery showed normal structure with no signs of inflammation, necrosis, or fibrosis. In contrast, liver sections from paracetamol-treated mice (Fig. 2F) displayed visible signs of inflammation, necrosis, swelling of hepatocytes and mild steatosis despite no significant effect on liver parenchyma. Pretreatment with a low and medium dose of walnut oil had no significant impact on these parameters ( Fig. 2D and E) while those treated with high doses (Fig. 2F) have normal liver parenchyma and hepatocytes and signs of inflammation. However, liver sections from mice treated with C. tuberculata extract, in all doses, have displayed normal liver parenchyma and hepatocytes with only mild inflammatory changes and necrosis (Fig. 2G-I) just like vitamin C treated mice (Fig. 2C).

DISCUSSION
The current study aimed to investigate, the impacts of paracetamol on different biochemical and hematological parameters along with the histopathological study of the liver. Also, to study the roles of walnut oil and C. tuberculata against paracetamol-induced hepatotoxicity. In this study experimental animals (Mice) were divided into various groups. In these mice, hepato-toxicity was induced by paracetamol and then treated with walnut oil, C. tuberculata and vitamin C for 21 days.
In the present study, different biochemical parameters were studied. Paracetamol administration to the mice at the dose of 200 mg/kg body weight increased the levels of total cholesterol, triglycerides and ALT while decreased has occurred in the level of HDL. Similar findings have been reported; that paracetamol significantly increased the levels of total cholesterol, triglyceride and ALT, and reduced HDL level (Oyagbemi & Odetola, 2010). The same results have also been in agreement with (Zakaria et al., 2020). There was an increase in the lipid profile by Intoxication with paracetamol in tissues and serum. An increase in the total cholesterols level changes the function and structure of the membrane (Tatiya et al., 2012). In the current study, walnut oil decreases the level of cholesterol and ALT, while increasing the level of HDL. The cholesterol and triglyceride levels were also decreased by vitamin C. It also increases the level of HDL and C. tuberculata decreases the levels of cholesterol, triglycerides and ALT and increases HDL level.
Administration of paracetamol produced a notable increase in the level of ALT enzyme and can demonstrate damage to the structural integrity of the liver. After cellular damage, it discharges into circulation which shows the onset of liver toxicity (Tatiya et al., 2012). The serum ALT is the most important biochemical marker for the analysis of liver dysfunction (Mohammad et al., 2022;Islam et al., 2021;Mahmood et al., 2014). ALT is an enzyme found in liver cells (hepatocytes). In the blood, it is leaked out where it is measured. Its level increases in acute liver toxicity like an overdose of paracetamol and viral hepatitis. Liver damage due to paracetamol is assessed by the increased level of ALT, when the liver is damaged it leaks out into the blood circulation (Ukpabiugo et al., 2016). An elevated level of ALT after feeding the mice with paracetamol was also observed by (Gyawali et al., 2017) C. tuberculata is a succulent, angular, and leafless plant that flourishes in the wild (Sultan et al., 2014). Caralluma is used as a hepatoprotective, anti-inflammatory, antiulcerogenic, anti-nociceptive and antioxidant (Kumar et al., 2018). In the current study, C. tuberculata decrease the levels of total cholesterol, triglycerides and ALT and increase HDL level. This was also supported by Abdel-Sattar and others (Abdel-Sattar et al., 2011) who observed that C. tuberculata not only reduced the levels of cholesterol and triglycerides but also increase the level of HDL. This study has also been an agreement with Poodineh and others (Poodineh et al., 2016) that showed the protective effects of C. tuberculata that decrease the levels of ALT, TG and cholesterol while increasing the level of HDL. It also significantly decreases the levels of urea and creatinine in diabetic mice. The serum level of ALT is an indicator of liver function.
Walnut oil contains the largest concentration of polyunsaturated fatty acids (PUFA), which is up to 78% of the total fatty acids content as compared to other vegetable oils (Aye et al., 2019;Shah et al., 2014). In the present project, walnut oil decreases the level of cholesterol, TG and ALT, while increasing the level of HDL. Similar results have also been observed by Tariq and others (Tariq et al., 2010). Walnut oil significantly reduces the levels of cholesterol, TG and rise in the level of HDL as observed by Zibaeenezhad and others (Zibaeenezhad et al., 2017). The hepatoprotective effects of walnut against carbon tetrachloride (CCl 4 ) induced liver toxicity was studied and it was found that walnut reduces the level of plasma enzymes that were raised by (CCl 4 ). Damage to liver cells alters their membrane permeability and transport causing the leakage of enzymes from the cell (Eidi et al., 2013). Walnut oil reduced the level of cholesterol was reported by that observed that walnut oil decrease cholesterol and it may be due to the polyunsaturated fatty acids content of walnut oil that caused a reduction in the synthesis of cholesterol or a result of phytosterols of walnut oil, which have structural similarity with cholesterol and thus decrease the serum cholesterol by inhibition of cholesterol absorption (Fink et al., 2014).
The result from this study demonstrates that vitamin C reduced the level of total cholesterol, and tri-glycerides while increasing the level of HDL, which is in contrast to the results of Eteng and others (Eteng et al., 2006), that observed the effects of oral administration of vitamin C on lipid profile and serum of mice and it was found that vitamin C decreases total cholesterol but a non-significant increase in HDL. In the present study, vitamin C increases the level of HDL. The reason for this deviation might be the duration and dose of feeding and administration of walnut oil alone.
It was also noticed that there was a high level of creatinine in the paracetamol-fed group in comparison to the control group. It showed that paracetamol elevates the level of creatinine that was reduced by C. tuberculata and vitamin C as compared to walnut oil. The result was an agreement with previous studies which showed a rise in the level of serum creatinine after the administration of toxic doses of paracetamol (Mohammad et al., 2022;Islam et al., 2021;Mahmood et al., 2014). An increase in the level of serum creatinine was also supported, as there was a rise in the creatinine level of the paracetamol-intoxicated group (Rosita et al., 2018). The decrease in the level of creatinine by C. tuberculata was also observed by (Poodineh et al., 2016). There was a considerable rise occurred in the level of urea in the paracetamol-fed group as compared to the control; walnut oil and vitamin C were more effective in curing the level of urea as compared to C. tuberculata. The same findings have also been reported that the serum urea level was significantly increased in the paracetamol-fed group (Gupta et al., 2017). The increase in blood urea is associated with paracetamol-induced nephrotoxicity which has been confirmed that paracetamol elevates urea level and causes renal impairment (Hua et al., 2018).
There was also a significant rise in TLC and lymphocyte counts and a drop in neutrophils, monocytes, eosinophils, and platelets count in the paracetamol-fed group as compared to the normal control group. The same results have also been reported that paracetamol produces a considerable increase in lymphocyte count and a drop in neutrophils, monocytes as well as eosinophils counts in paracetamol-induced liver toxicity in mice (Juma et al., 2015). There was a significant deviation from the present results when hematological parameters were studied in paracetamol-fed mice and it was found that paracetamol caused no significant changes in lymphocytes, neutrophils, monocytes, eosinophils, and platelets count compared to their control group. The non-significant change of lymphocytes showed that the body's immune responses have not been compromised by paracetamol. The non-significant change in neutrophils showed that the ability of the body to destroy invading viruses, bacteria and other injurious agents has not been compromised by paracetamol. The non-significant change in monocytes indicates that the phagocytic function of the body has not been compromised, while the non-significant change in eosinophils count showed that the body's anti-allergic responses have not been compromised by paracetamol (Oyedeji et al., 2013). In another study it was observed that liver damage produces by paracetamol caused drops in leucocytes, lymphocytes, and platelets count (Senthilkumar et al., 2014). A previous study performed by Okokon and others (Okokon et al 2017), of it, was studied that treatment of mice with paracetamol did not significantly affect the percentage of neutrophils but decreased the monocytes, lymphocytes, and eosinophils percentages.

CONCLUSIONS
It was concluded from this study that there were positive effects of walnut oil and C. tuberculata extracts on the serum ALT values in all the treated groups. Elevation in serum ALT activities was found with paracetamol administration in mice. Walnut oil and C. tuberculata extract significantly reduced the serum ALT level when compared with normal and toxic control groups. In the present study lipid profile such as cholesterol, HDL, and TG levels, were also studied. Therefore, the curative effect of walnut oil and C. tuberculata extracts on serum lipid profile was investigated. A clear reduction was observed in mice administered with low and high doses of walnut oil and extract. Effects of walnut oil, C. tuberculata and vitamin C were similar as these normalized the serum lipid profile values. In the current study creatinine and urea levels were also studied. Paracetamol administration elevated the levels of creatinine and urea in experimental mice. Walnut oil and extract significantly reduced the levels of creatinine and urea when compared with a toxic positive control group.
In this some important hematological parameters like leucocytes, lymphocytes, neutrophils, monocytes, eosinophils, and platelets counts were investigated in paracetamol intoxicated mice. Oral administration of paracetamol caused a significant decrease in total neutrophils, monocytes, eosinophils, and platelets counts while increase in leucocytes and lymphocyte counts. Considerable changes were observed in hematological parameters with the administration of walnut oil, extract, and vitamin C. Based on the results obtained after the treatment of paracetamol intoxicated mice with walnut oil and C. tuberculata extracts, the following recommendation is given. Walnut oil and C. tuberculata extracts are hepatoprotective and renal-protective in nature. The walnut oil and extract are also beneficial and can be used as a remedy for cardiovascular diseases. The extract of walnut oil is also beneficial for some hematological parameters. C. tuberculata and walnut oil possess antioxidant properties, which could be used as the best source of advanced medication.

Declarations
Ethical Approval. Ethical approval of the study was obtained from the ethics board of Islamia College University, Peshawar.
Conflict of interest: All the authors declare no conflict of interest.