, Leyiga Justice Maagbo1, Mgbahurike Augustina Amaka*2
HEPATOTOXICITY EFFECTS OF PARACETAMOL-DENIK CLEANSERÒ CO-ADMINISTRATION
Cecilia Nwadiuto Obasi1, Leyiga Justice Maagbo1, Mgbahurike Augustina Amaka*2
1Department of Experimental Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Rivers State, Nigeria.
*2Department of Clinical Pharmacy and Management, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Rivers State, Nigeria.
Aim and Objective: The concurrent use of herbal products with orthodox medicine is on the rise with the risk of herb-drug interaction that could be beneficial or harmful to the body. Paracetamol (acetaminophen) is an anti-pyretic and analgesic drug metabolized by CYP2E1 to give the major hepatotoxin, N-acetyl-p-benzoquinone imine (NAPQI). Denik cleanserâ is an oral herbal preparation from plants part of Occimum grattissimum, Colocynthis citrullus, Khaya ivorensi. The aim of this study was to evaluate the hepatotoxic effects triggered by Denikâ and paracetamol co-administration in rats. The study sought to mimic conventional usage of Denik cleanserâ followed by ingestion of paracetamol, a likely scenario given the popularity of both compounds.
Method: Twenty animals were randomly assigned to four groups, the first group (control) received 0.3 mL distilled water, 2nd group received paracetamol 100 mg/kg, 3rd group received Denik cleanserâ2 mL/kg while the 4th group received both paracetamol and Denik cleanserâ at 100 mg/kg and 2 mL/kg daily for 3 days after which biochemical and histological analysis were carried out.
Results: From histological analysis revealed that rats that received Denikâ-only and Denikâ/paracetamol (concomitantly) showed markedly distorted liver architecture compared to control indicating toxicity. Similarly, the biochemical analysis results showed a significant (p<0.05) increase in AST and ALT for the Denikâ/paracetamol group compared to the control group indicating a hepatotoxic event. A non-significant increase in ALP and GGT were also observed in the Denik cleanserâ + paracetamol group. Enhanced metabolism of paracetamol by Denik cleanserâ to NAPQI (hepatotoxic metabolite) is indicated as possible mechanism.
Conclusion: The results of this study demonstrated the in vivo potential for a herb/drug interaction involving paracetamol and Denik cleanserâ resulting in liver injury.Therefore, caution is strongly advised against its casual, non-medically supervised usage.
Keywords: Denik cleanserâ, herb-drug interaction, hepatotoxicity, paracetamol.
INTRODUCTION
Evidences have shown that nutrients in foods, fruits, vegetables, and herbal supplements can affect the therapeutic activities of drugs by affecting their bioavailability through drug - food interactions. This can occur via interaction with the drug transporters, or drug-metabolizing enzyme systems, by formation of complexes, or modification in gastric emptying/pH1. Increased use of herbal products and wide spread poly-pharmacy have enhanced the chances of drug interactions and adverse drug reactions (ADR)2. Disease co-morbidity is one major reason for co-administration of certain drugs with herbs, leading to increased risk of hepatotoxic effects of drugs. For instance, pre-existing liver disease has been identified as an essential risk factor for hepatotoxicity of certain drugs3. This has been observed in patients with viral hepatitis and tuberculosis co-infections who develop liver injuries due to co-administration of antiviral and anti-tuberculosis drugs4. The presence of impurities and other hepatotoxins are also risk factors for hepatotoxicity of herbal preparations3,5. Some herbs may affect drugs metabolized by cytochrome P450 enzymes and thereby impair the activity of cellular drug transporters and glucuronidation pathways6.It has been shown that certain herbs have dose-dependent inhibition effect on CYP3A4 and reduces expression of P-glycoprotein, leading to drug accumulation and increased hepatotoxicity of co-administered drugs4,7,8. Often, co-medication with herbs and synthetic drugs occur, predisposing drug-herb interaction at the hepatic cytochrome P450 (CYP) system8,9,10.
Acetaminophen (Paracetamol) is a common antipyretic /analgesic drug which is easily obtained over-the-counter (OTC). It is metabolized by CPY3A4 to N-acetyl-p-benzoquinone imine (NAPQI) in the liver11. NAPQI is produced in little amount at therapeutic doses, but overdosing of paracetamol, leads to increased production of NAPQI11,12 with consequent formation of conjugates with glutathione. The depletion of glutathione is the major reason for severe liver damage as it conjugates with NAPQI13. This depletion is the main reason for hepatotoxicity and necrosis13,14. Paracetamol effectively relieves pain due to selective inhibition of cyclooxygenase-3 (COX3) in the central nervous system by interrupting the descending serotoninergic pathways and to some extent blocking the activity of pain mediators (bradykinin, substance P)15. Therapeutic doses are safe and well tolerated, but large intake of the drug may cause hepatotoxicity16. Paracetamol is essentially metabolized in the liver. It is mostly changed by glucuronidation (40-60%) and sulphation (20-46%). Pharmacological inactive metabolites are formed, while less than 10% is oxidized to a toxic metabolite, i.e. N-acetyl-p-modulate the P-gp activity11. Herb- drug interactions involving paracetamol has been identified in the study by Ewing et al., that investigated interaction between Cannabidiol-rich Cannabis (CBD) extract and paracetamol17. Data obtained demonstrated a potential for CBD extract/drug interactions resulting to hepatotoxicity17.
Denik cleanserÒis a herbal oral preparation made from a blend of various parts and fruits of medicinal plants such as; O. grattissimum, C. citrullus, Khaya ivorensis. This product is claimed by the producers to possess important therapeutic effects such as antioxidant, fertility, weight loss, and immune boosting effects. It is claimed to be effective against indigestion and also in the reduction of cancer risk, blood purification, enhancing eye and skin health. Given the wide spectrum of pharmacological use of Denik cleanserâ by the Nigerian populace, a possible co-administration of this herbal drug with paracetamol is inevitable. The aim of this study was to investigate the effect of co-administration of paracetamol and Denik cleanserâ on the histology and biochemical parameters of the liver.
MATERIALS AND METHODS
Materials
Distilled water, methylated spirit (JHD, China), Xylene, Diethyl ether, 10% formalin, Denik cleanserâ (Kayfahd herbaceuticals), Paracetamol 500mg tablets BP (Panadolâ from GlaxoSmithKline). All other solvents and chemicals were of analytical grade.
Apparatus/Equipment
Weighing balance (Ohaus, Advanturer), centrifuge (Techmel and Tecgmel USA), measuring cylinder, syringes, latex gloves, cotton wool, tissue paper, heparinized tubes, 5mL sample bottles, surgical blade, refrigerator, cannula.
Source of herbal formulation
The herbal formulation under study was obtained in August 2020, from a medicine store at Rumuokoro, East West Road, in Port Harcourt, Rivers State, Nigeria. The herbal formulation was manufactured by Kayfahd herbaceuticals. Exclusively for: Purity biz.com FCT Abuja, Nigeria.
Animals
This study was conducted using an experimental design previously described by Ewing et al.,17 with little modifications. Twenty (20) healthy adult male rats (140±0.25 g) were used for the experiment. They were acclimatized in the animal house of the Department of Experimental Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Port Harcourt, Nigeria for two weeks and fed ad libitum with standard feed (Broiler finisher- Guinea feeds) with free access to water before experiment. They were maintained under standard conditions of humidity and temperature. Animal ethics and proper handling methods were strictly adhered.
Method
Animal experiment
The dosage for Denik cleanserâherbal mixture was chosen using its prescription label. The drugs were administered to the rats in the test group orally using an oral cannula with rubber tubing, Animals were randomly assigned to four groups (1-4) of five animals each. Group I consisted of the control group; animals who were given 0.3 mL of distilled water orally daily for 3 days. The group II animals received paracetamol orally at a dosage of 100 mg/kg daily for 3 days. Group III animals received Denik cleanserâ orally at a dosage of 2 mL/kg daily for 3days. Group IV animals received both paracetamol Denik cleanserâ orally at a dosage of 100 mg/kg and 2 mL/kg respectively concurrently daily for 3 days. Animals were then fasted overnight on the third day of treatment and sacrificed under ether anesthesia on the fourth day. With this experimental setup, we sought to mimic conventional usage of Denik cleanserâ followed by ingestion of paracetamol, a likely scenario given the popularity of both compounds.
Blood sampling and biochemical analysis
Blood samples were collected via cardiac puncture. The blood samples were collected by cardiac puncture and kept at a temperature of 4°C for 6 hours. The blood samples were then centrifuged at 3000 rpm for 10 minutes and used for biochemical analysis. In the present study, the liver function was evaluated with serum levels of Glutamyl transferase (GGT), Alkaline phosphatase (ALP), Alanine amino transferase (ALT) and Aspartate amino transferase (AST) using comer-cial diagnostic kits (Randox laboratory kit, England).
Histopathology results
Liver sections were fixed in 10 % formalin for 6-12 hours. They were processed and examined for histological changes at the college of health sciences Pathology Facility. For light microscopy examination, the formalin fixed tissues (liver) were dehydrated through ascending grades of alcohol, cleared in three changes of xylene, and were embedded in paraffin. Serial sections, each of 4-micron thickness, were cut and stained with H and E as per standard protocol.
Ethical issues
The protocol of this study is designed in accordance with the ethical principles of the International Committees for the Protection of Animal Rights Laboratory. This project was approved by Ethics Committee of the University of Port Harcourt, Rivers State, Nigeria (Ref No. UPH/798EC/8/2020).
Histopathological Analysis
Liver sections were fixed in 10 % formalin for 6-12 hours. They were processed and examines for histological changes at the college of health sciences Pathology Facility. Samples of the liver tissue were cut, about 3mm by 3mm in size, with a sharp knife. Each tissue was then fixed with 10% formalin for about 6-12 hours and subsequently dehydrated by passing the tissue slowly through ascending grades of absolute alcohol (50%, 70%, 90% and 95%) and lastly in the absolute alcohol (100%), for a period of 1-2 hours in each grade of alcohol. The tissue is cleared using xylene for about 1-2 hours twice. It is then placed in the molten paraffin wax at a constant temperature of 56 – 60oC in an oven or paraffin bath (changing it twice keeping for about 2 hours each time) for infiltration. The tissue is embedded by placing it in special L-shaped metal blocks filled with molten paraffin wax and after solidification, the L-metal blocks are removed. For microscopic study, extremely thin (5-15 um) sections of the tissues were cut using a microtome. The tissues were then stained using haematoxylin-eosin, and the stained tissue on the slide is then mounted in Canada balsam under a cover slip and ready for examination. The microscopic examination was carried out on the livers of both the control and treated groups.
Statistical analysis
Statistical analysis involved use of the Microsoft Excel. Data are expressed as the Mean ± SD. Statistics were performed using one-way Anova and t-tests. p values less than 5% were considered statistically significant (p < 0.05).
RESULTS
The organ-body weight index is recorded in Table 1. There was little reduction in organ-body weight index at P<0.05. The microscopic structure of the liver depicted in Figures 1-4 shows no abnormalities in the control and paracetamol-only groups. For the Denikâ-only and paracetamol + Denikâ groups, there was markedly distorted liver tissue showing hepatocytes with microvesicular steartosis and councilman’s bodies (arrowed), patent central vein (CV) Results from biochemical analysis (Figure 1- Figure 4) revealed higher levels of liver function markers Aspartate aminotransferase (AST) Alanine aminotransferase (ALT), alkaline phosphatase (ALP) and Gamma-glutamyl transferase (GGT) enzymes in the paracetamol + Denik cleanserâ group compared to the control group indicating liver toxicity. The elevation in ALT, AST and GGT in the paracetamol+ Denikâ group was statistically significant at p< 0.05.
DISCUSSION
Emerging evidence indicates that certain herbs pose a significant risk for hepatotoxicity3,9,10. Liver damage is associated with cellular necrosis, increase in tissue lipid peroxidation and depletion in the tissue gluthathione (GSH) level10. In addition, serum levels of many biochemical markers like serum glutamic-oxaloaxetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), triglycerides, cholesterol, bilirubin, alkaline phosphate (ALP) are also elevated with liver damage18. Therefore, biochemical abnormalities and histological characteristics, in association with clinical presentation, help to define the pattern of a drug or substance-induced liver injury. Paracetamol is metabolized by a cytochrome P450 enzyme known as CYP2E117. Certain herbs have been shown to induce CYP2E1 resulting in enhanced production of N-acetyl-para-benzo- quinone imine (NAPQI) which is a hepatotoxin17,19. In a study by Zendulka et al., Cannabidiol from Cannabis sativa (commonly used for its anti-seizure activity) was shown to induce drug interactions involving the modulation of various cytochrome P450 enzymes responsible for paracetamol metabolism20. Paracetamol is a common analgesic medication. When paracetamol is administered using its indicated dosage, the risk of adverse effects is low. However, when the therapeutic dosage range is exceeded, paracetamol toxicities emanate17.
The most serious adverse reaction associated with paracetamol is hepatotoxicity attributable to a major metabolite known as N-acetyl-p-benzoquinone imine (NAPQI)21. Denik cleanserâ is an oral herbal preparation obtained from O. grattissimum, C. citrullus, Khaya ivorensi. It is claimed by the manufactures to boost immunity, aid digestion and to treat a wide array of ailments. Generally, with an increase in the utilization of herbal remedies for different conditions, the concomitant administration of herbal medicines with conventional drugs might be inevitable19 and this has created a need to consider possible herbal-drug interactions. From our study, data obtained revealed no histological changes in the paracetamol-only group (control). For Denik cleanserâ-only and paracetamol-Denikâ groups, there were marked distorted liver tissues indicative of hepatotoxicity. This was further supported by the results obtained from the biochemical analysis were elevations in the levels of the liver enzymes AST, ALT, ALP, and GGT were observed. The rise in AST and ALT levels in the paracetamol-Denikâgroup was statistically significant at p <0.05 indicative of toxic events in the liver on co-administration. For the Denikâ-only groups, there was increase in the levels of AST, ALT, ALP and GGT, however, this was not statistically significant. Paracetamol metabolism to the reactive metabolite (N-acetyl-p-benzoquinone imine; NAPQI) by the CYP P450s, especially CYP2E1, is well recognized for its role in the initiation of toxicity17. There was elevations in the levels of liver enzyme biomarkers and abnormalities in the liver architecture in the paracetamol ± Denikâ group, we hypothesize that phyto-constituents in Denik cleanserâ may have increased metabolism and bioactivation of paracetamol resulting to enhanced production of NAPQI by the co-administered herbal remedy.
Results obtained from the present study indicates a potential hepatotoxic effect of Denik cleanserâwhen administered alone and when co-administered with paracetamol.
Limitations of the study
The high cost of the animals and the cost of their maintenance limited the number of animals used for the study.
CONCLUSION
The results of this study demonstrated the potential for in vivo herb/drug interaction involving paracetamol and Denik cleanserÒ resulting in liver injury. Our data suggest that Denik cleanserÒ creates a significant drug interaction that could lead to serious adverse health such as hepatotoxicity. However, this is subject to validation via pharmacokinetic study. Nevertheless, caution is strongly advised against its casual, non-medically supervised usage.
AUTHORS’ CONTRIBUTIONS
The first author developed the research idea, the second author conducted and evaluated the literature search, the first and third authors designed and conducted the bench work. They also, articulated the results and typeset the manuscript and the third author served as the corresponding author.
ACKNOWLEDGEMENT
The authors acknowledged the assistance of the laboratory technician who prepared and processed the samples for histological studies
REFERENCES