Terminalia arjuna restores the levels of alkaline phosphatase and aspartate aminotransferase of acetaminophen intoxicated mice

Full Length Research Article

Terminalia arjuna restores the levels of alkaline phosphatase and aspartate aminotransferase of acetaminophen intoxicated mice

Nida Irshad, Hassan Hameed, Maryam Mumtaz, Zainab Aslam, Andleeb Batool, Imran Sohail*

Adv. life sci., vol. 10, no. 1, pp. 104-108, March 2023
*- Corresponding Author: Imran Sohail (Email: imransohail@gcu.edu.pk)
Authors' Affiliations

 Government College University, Lahore – Pakistan
 [Date Received: 04/11/2022; Date Revised: 28/02/2022; Date Published: 31/03/2023]

Abstractaa download_button
Introduction
Methods
Results
Discussion
References 

Abstract

Background: Herbal medicines are natural and comparatively safer than conventional treatments and are well-documented for carrying little potential for harm.  Therefore, phytonutrients have occupied a central stage in the therapeutics. Terminalia arjuna, a medicinal plant, has been reported to have homeostatic, laxative, diuretic, antidiabetic, anticancer and cardiotonic actions.

Methods: The current study was designed to investigate the protective role of T. arjuna leaf extract at three dose levels (100, 250, 500 mg/kg body weight) against acetaminophen (250 mg/kg body weight) induced liver damage.

Results: The administration of hepatotoxin (Acetaminophen) resulted in disturbance of hematological and serological profile including alkaline phosphatase (ALP) and aspartate aminotransferase (AST) which was assayed in control and drug treated experimental models. Treatment with T.  arjuna leaf extract for 7 days restored the normal levels of markers and response was dose dependent.

Conclusion: This study adds to the very limited existing literature regarding hepatoprotective effect of T. arjuna against acetaminophen toxicity. It is also important to get a step closer to development of accessible, authoritative, and independent information resources about herbal medicines and wide-ranging health disorders, which are currently lacking in Pakistan.

Keywords: Hepatoprotective; Terminalia arjuna; ALP; AST    

Introduction6th button-01

Liver is a major detoxifying and metabolizing organ and is continuously exposed to variety of environmental pollutants, drugs and other highly reactive substances [1-3]. Wide range of exogenous and endogenous stresses and inability of cellular defence to counteract these cellular insults predispose the cells to undergo pathogenesis [4]. Research over the years has refined the knowledge regarding phytonutrients and nutrigenomics [5]. It has been documented that the Terminalia species act as a potential medicinal plant against variety of diseases. The members of Terminalia species are widely used because of their homeostatic, laxative, cardiotonic, antidiabetic, anticancer, antioxidant, anticoagulant, antihypertensive, antithrombotic, antiviral, antifungal, antibacterial and diuretic activities [6-10]. Different parts of plant have been investigated for the presence of phytoconstituents and pharmacological actions [11].   Phytochemical extract have been attributed to possess antimicrobial and antioxidant activity [12]. The experiment showed that T. arjuna (200 mg/kg) significantly reversed the effect of cadmium induced toxicity and proved that it has hepatoprotective and antioxidant potential [13]. Terminalia arjuna extracts in both alcohol and water significantly reduced the activity of the CYP3A4, CYP2D6 and CYP2C9 enzymes. According to an enzyme kinetics investigation, treatments resulted in the fast, reversible, non-competitive inhibition of all three enzymes in human liver microsomes [14]. Aqueous and ethanol extracts of Terminalia arjuna bark were found to have hepatoprotective potential against the liver damage caused by paracetamol/CCl4 in Wistar albino rats [15]. Additionally, it was noted that the purified flavonoids in the methanolic extract of Terminalia arjuna stem bark have hepatoprotective effects against CCl4-induced hepatic damage [16]. Interestingly the hepatoprotective role of Terminalia arjuna leaf is still incompletely defined. Therefore, we tested whether or not Terminalia arjuna leaf extract had hepatocurative potential by taking into account the serological and hematological parameters.

Methods6th button-01

Preparation of plant extract:

Leaves of Terminalia arjuna were collected from Scholars Garden of Government College University Lahore, Pakistan and were identified by an expert  (Professor Dr. Zaheer-Ud-Din Khan, Ex-Chairperson Botany Department, Government College University, Lahore, Pakistan). Collected leaves were washed thoroughly with distilled water. Then they were shade dried for two weeks. Using electric blender the dried leaves were pulverized and 500 grams of powder was soaked in 4 liters of absolute ethanol (95%) for 15 days with sporadic shaking. After filtration, the filtrate was subjected to rotary evaporator at 42-45ºC to evaporate the solvent (ethanol). After the distillation process crude extract was obtained. The extract was then stored at 4ºC until further use.

Experimental animals

The albino mice weighing 25-27g were purchased from University of Veterinary and Animal Sciences Lahore. The animals were housed in Government College University animal house under standard conditions (22 ± 2°C; 12:12 hours light dark period) and fed with standard diet (poultry feed) and water. All the experimental work in animals such as administration of drug and plant extract, and blood collection were performed according to the national standards under appropriate regimes with veterinary services. The ethical permission to use animal has been taken from institutional ethical committee (GCU-IIB-114).

Assay kits and Chemical Reagents:

Acetaminophen (Paracetamol) was used for liver intoxication. Test kits for measuring AST, and ALP were supplied by synchron® systems (Ireland).     

Experimental Design:

In this experiment, twenty-five male albino mice were randomly allocated into five groups, each consisting of five animals (n=5).

Group I: Normal healthy control

The animals were fed on standard diet and water ad libitum.

Group II: Untreated Acetaminophen control

Animal was given orally 250mg/kg of acetaminophen, suspended in distilled water for seven days. Blood sampling was done on 8th day. The blood was analyzed for various serological and hematological parameters.

Group III: Acetaminophen+ 100mg/kg body weight of Plant Extract

The animals of this group were treated with 100mg/kg body weight plant extract as well as 250mg/kg acetaminophen per day for 7 days. The dose was given orally.

Group IV: Acetaminophen+ 250mg/kg body weight of Plant Extract

The animals of this group were treated orally with 250mg/kg body weight plant extract as well as 250mg/kg acetaminophen per day for 7 days.

Group V: Acetaminophen+ 500mg/kg body weight of Plant Extract

The animals of this group were treated orally with 500mg/kg body weight plant extract as well as 250mg/kg acetaminophen per day for 7 days.

Blood Sampling

On 8th day the mice were anesthetized and dissected. Approximately one milliliter blood was collected directly from the heart by cardiac puncture. Half of which was transferred into EDTA sample tubes for hematological studies, while the other half was transferred into 1.5 ml polypropylene tube-(Eppendorf SE, Hamburg, Germany) which was then allowed to clot by leaving it undisturbed for half an hour. The latter was centrifuged at 4000 rpm for 10 minutes to separate the serum for serological studies.

Data Analysis

Data was analyzed by using SPSS software version 25. The values represent mean ± SD. Statistical significance was determined by ANOVA followed by Tukey’s post-hoc. A P value ≤ 0.05 was considered as significant.

Results6th button-01

Hematological and Serological Parameters

The values of various hematological and serological parameters are shown in Table 1. The table shows the comparison of mean±SD of white blood cells (WBCs), granulocytes (GRA), lymphocytes (LYM), aspartate transaminase (AST) and alkaline phosphatase (ALP). The number of WBCs observed in normal healthy control was 2.7 ± 0.16 x103/µl. In untreated acetaminophen control the number of WBCs increases and the value observed was 3.61 ± 0.67 x103/µl. In the treatment groups III, IV and V, WBCs number decreases with administration of extract. The WBCs values observed in group III, IV and V were 2.51 ± 0.035 x103/µl, 2.37 ± 0.06 x103/µl and 2.29 ± 0.025 x103/µl, respectively.

The number of lymphocytes observed in the control group was 3.28 ± 0.07×103/µl. This number is elevated in group II in which only acetaminophen was administered, and the value observed was 4.50 ± 0.50 x103/µl. The number of lymphocytes observed in group III, IV,  and V which were treated with leaf extract was 1.67 ± 0.03 x103/µl, 1.58 ± 0.04 x103/µl and 1.53 ± 0.16 x103/µl, respectively (Table 1). The number of granulocytes in untreated healthy control was 1.32 ± 0.02 x103/µl. After acetaminophen was administered this value rises to 1.93 ± 0.07 x103/µl. The GRA count was found to be significantly decreasing in group III, IV, and V i.e., 0.66 ± 0.02 x103/µl, 0.32 ± 0.03 x103/µl and 0.29± 0.04 x103/µl.

The serum level of AST in untreated (Control) was 63.92 ± 3.2 U/l. After acetaminophen administration in group II, a significant elevation in serum AST level i-e 87.14 ± 3.15U/l was observed which indicate liver damage. In treatment groups III, IV, and V in which extract was given at the rate of 100, 250 and 500mg/kg of body weight along with acetaminophen the values observed  for serum AST level were 83.09 ± 6.5 U/l, 76.49  ± 4.9 U/l and 69.70 ± 3.40U/l, respectively.

The concentration of ALP in untreated healthy control was 24.47 ± 3.6 U/l. After the administration of acetaminophen (250mg/kg body weight) for 7 days in group II this value rises to 51.90 ± 4.42 U/l indicating liver damage. The values of serum ALP level observed in group III, IV, and V which were treated with extract along with acetaminophen was 46.67 ±5.47 U/l, 39.19 ± 4.72 U/l and 28.25 ± 5.37 U/l, respectively.

 

 

Figures & Tables

 

 

 

Discussion6th button-01

In this study the effect of T. arjuna leaf on serological and hematological parameters was evaluated. For this purpose, acetaminophen (paracetamol) was used to damage the liver in experimental model. Previous studies have proved that various parts of T. arjuna are effective against a diverse range of diseases. Fruits are used as tonic and deobstruent  [17]. Moreover studies have also revealed that T. arjuna fruit extract has hepatoprotective and hepatocurative role against acetaminophen intoxication[18].In our study the level of hepatic enzymes i.e. AST and ALP increased in blood after administration of acetaminophen. The treatment with Terminalia arjuna leaf extract at all doses (100, 250 and 500mg/kg) normalized enzyme activities which is in concordance with the work of [19]. Similarly our data is in accordance with other research works in which levels of AST and ALP are elevated after administration of acetaminophen, CCl4 (Carbon tetrachloride) or thioacetamide observed in mice[20]. Various other researchers also explored the likely correlation between pharmaceutical toxicology and phytonutrients and suggested that paracetamol induced pathology was reversed significantly by silymarin [21]. Our results are in line with [22], who demonstrated that hydroalcoholic extract of bark of T. arjuna possessed hepatoprotective effect

The blood related functions of plant extract and its product can be explained by investigating certain biochemical parameters [23]. The levels of WBCs, Lymphocytes and granulocytes increased dramatically with oral administration of acetaminophen which might be related to an immunological response upon liver damage when compared with control. However experimental mice treated with herbal extract displayed a decline in level of WBCs and other cell counts declined at all doses.This indicates that the extract might have some bioactive components that could harm or impair the production of white blood cells. WBCs and other cell counts were normalized especially at dose of 500 mg/kg body weight of extract along with paracetamol. This observation is in accordance with the work of  [24], who reported the effect of ethanolic extract of B. speatabilis leaves on haematological and serum lipid variables in rats. Studies have shown that T. arjuna contains flavonoids, tannins and minerals. Flavonoids have been reported to exert antioxidant effect. The hepatoprotective role of T. arjuna in our findings might be due to enhanced antioxidant activity [12].

In conclusion, T. arjuna leaf extract was found to demonstrate hepatoprotective efficiency and also improves hematological abnormal profile against acetaminophen toxicity. Although these studies partially describe the underlying mechanisms which are targeted by herbal extracts to safeguard cells against cellular insults. Yet detailed information is necessary for a better understanding of the cellular mechanisms which are nonfunctional or which are inactivated during pathogenesis. Additionally, a greater awareness of the bioactive components of the plant is vital for its utilization and likely translation to an effective drug that can be used in clinical trials.

Author Contributions

The study was planned by Imran Sohail. Imran Sohail, Maryam Mumtaz, Zainab Aslam conducted experiments. Initial draft of the manuscript was written byImran Sohail, Hassan Hameed, Nida Irshad.Imran Sohail, Andleeb Batool, Nida Irshad, and Hassan Hameed participated in revisions.

Conflict of Interest

The authors declare that there is no conflict of interest.

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References

  1. Yousafzai AM, Shakoori A. Hepatic Responses of A Freshwater Fish Against Aquatic Pollution. Pakistan Journal of Zoology, (2011);43(2).
  2. Montanari F, Pinto M, Khunweeraphong N, Wlcek K, Sohail MI, Noeske T, Boyer S, Chiba P, Stieger B, Kuchler K, Ecker GF. Flagging drugs that inhibit the bile salt export pump. Molecular Pharmaceutics, (2016); 13(1): 163-71.
  3. Sohail MI, Mughal MS, Arshad N, Arshad M. Incidence of hepatitis B and C in industrial areas of Sheikhupura and Kasur. Pakistan Journal of Zoology, (2010);42 (6).
  4. Kurutas EB. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutrition journal, (2015);15(1): 1-22.
  5. Prusti A. Antibacterial activity of some Indian medicinal plants. Ethnobotanical leaflets, (2008); 2008(1): 27.
  6. Saleem A, Husheem M, Härkönen P, Pihlaja K. Inhibition of cancer cell growth by crude extract and the phenolics of Terminalia chebula retz. fruit. Journal of Ethnopharmacology, (2002); 81(3): 327-36.
  7. Singh G, Singh AT, Abraham A, Bhat B, Mukherjee A, Verma R, et al. Protective effects of Terminalia arjuna against Doxorubicin-induced cardiotoxicity. Journal of ethnopharmacology, (2008); 117(1): 123-9.
  8. Sivalokanathan S, Ilayaraja M, Balasubramanian M. Efficacy of Terminalia arjuna (Roxb.) on N-nitrosodiethylamine induced hepatocellular carcinoma in rats. (2005).; 43 (03): 264-267.
  9. Rahmatullah M, Noman A, Hossan MS, Rashid M, Rahman T, Chowdhury MH, et al. A survey of medicinal plants in two areas of Dinajpur district, Bangladesh including plants which can be used as functional foods. American Eurasian Journal of Sustainable Agriculture, (2009); 3(4): 862-76.
  10. Rao BK, Sudarshan PR, Rajasekhar M, Nagaraju N, Rao CA. Antidiabetic activity of Terminalia pallida fruit in alloxan induced diabetic rats. Journal of Ethnopharmacology, (2003); 85(1): 169-72.
  11. Jain S, Yadav PP, Gill V, Vasudeva N, Singla N. Terminalia arjuna a sacred medicinal plant: phytochemical and pharmacological profile. Phytochemistry Reviews, (2009); 8: 491-502.
  12. Dwivedi S. Terminalia arjuna Wight & Arn.—a useful drug for cardiovascular disorders. Journal of ethnopharmacology. (2007); 114(2): 114-29.
  13. Haidry MT, Malik A. Hepatoprotective and antioxidative effects of Terminalia arjuna against cadmium provoked toxicity in albino rats (Ratus norvigicus). Biochem Pharmacol, (2014); 3(130): 2167-0501.
  14. Varghese A, Savai J, Pandita N, Gaud R. In vitro modulatory effects of Terminalia arjuna, arjunic acid, arjunetin and arjungenin on CYP3A4, CYP2D6 and CYP2C9 enzyme activity in human liver microsomes. Toxicology Reports, (2015); 2: 806-16.
  15. Soni N, Singh VK. Efficacy and advancement of Terminalia Arjuna in Indian herbal drug research: A review. Trends in Applied Sciences Research, (2019);1 4(4); 233-242.
  16. Chaudari GM, Mahajan RT. In vitro hepatoprotective activity of Terminalia arjuna stem bark and its flavonoids against CCl4 induced hepatotoxicity in goat liver slice culture. Asian Journal of Plant Science & Research. (2016);6: 10-17
  17. Gupta S, Bishnoi JP, Kumar N, Kumar H, Nidheesh T. Terminalia arjuna (Roxb.) Wight & Arn.: Competent source of bioactive components in functional food and drugs. The Pharma Innovation Journal, (2018); 7(3): 223-31.
  18. Khan S, Noor F, Sohail I, Imtiaz S, Anum F, Sarmad S, et al. Hepatoprotective role of fruit extract of Terminalia arjuna in acetaminophen intoxicated mice. Advancements in Life Sciences, (2020); 8(1): 63-7.
  19. Shakya AK, Shukla S. Evaluation of hepatoprotective efficacy of Majoon‐e‐Dabeed‐ul‐ward against acetaminophen‐induced liver damage: A Unani herbal formulation. Drug development Research, (2011); 72(4): 346-52.
  20. Fountoulakis M, Berndt P, Boelsterli UA, Crameri F, Winter M, Albertini S, et al. Two‐dimensional database of mouse liver proteins: Changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisomer 3‐acetamidophenol. ELECTROPHORESIS: An International Journal, (2000); 21(11): 2148-61.
  21. Girish C, Koner BC, Jayanthi S, Ramachandra Rao K, Rajesh B, Pradhan SC. Hepatoprotective activity of picroliv, curcumin and ellagic acid compared to silymarin on paracetamol induced liver toxicity in mice. Fundamental & clinical pharmacology, (2009); 23(6): 735-45.
  22. Subasini U, Rajamanickam G, Dubey G, Prabu P, Sahayam CS, Shabi MM, et al. Hydroalcoholic extract of Terminalia arjuna: A potential hepatoprotective herb. Journal of Biological Sciences, (2007); 7(2): 255-62.
  23. Yakubu M, Akanji M, Oladiji A. Aphrodisiac potentials of the aqueous extract of Fadogia agrestis (Schweinf. Ex Hiern) stem in male albino rats. Asian Journal of Andrology, (2005); 7(4): 399-404.
  24. Adebayo JO, Adesokan AA, Olatunji LA, Buoro DO, Soladoye AO. Effect of ethanolic extract of Bougainvillea spectabilis leaves on haematological and serum lipid variables in rats. Biokemistri, (2005); 17(1): 45-50.

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