Hepatoprotective Effect of Papaya Seed Ethanol Extract on Rifampicin Isoniazid-induced Rats

Aims: Hepatotoxicity induced by anti-tuberculosis drugs, including rifampicin and isoniazid (ATDILI, Anti Tuberculosis-Drug Induced Liver Injury), is an adverse reaction followed by significant morbidity. Several in vivo and in vitro research has confirmed that papaya seeds contain various non-essentials, minerals, and fiber. Carica papaya role in disease prevention through modulation of various processes, such as anti-inflammatory, anti-diabetes, immunomodulatory activity, and antioxidant activity, suggests a role in neutralizing free radical generation and ultimately preventing pathogenesis. This study aimed to determine the hepatoprotective effect of ethanol extract of papaya seeds on rifampicin and isoniazidinduced rats. Study Design: This study is experimental study. Methodology: The experimental animals in this study were divided into eight groups, including normal group, negative group 1, negative group 2, negative group 3, positive group, treatment group I (papaya seed ethanol extract dose of 100 mg/kgBW), treatment group II (papaya seed ethanol extract dose of 300 mg/kgBW), and treatment group III (papaya seed ethanol extract dose of 500 mg/kgBW), then the rats were dissected, and blood was taken for AST, ALT, ALP, GGT, and Bilirubin level measurements. Results: The results showed that papaya seed ethanol extract could reduce ALT, AST, ALP, GGT, and Bilirubin levels that were significantly different (P <0.05) than those in the negative control group. Original Research Article Wahfar et al.; JPRI, 33(42B): 78-83, 2021; Article no.JPRI.73305 79 Conclusion: Flavonoid contains in the extract ethanol carica papaya has vita role to prevent the liver toxicity caused by isoniazid and rifampicin.


INTRODUCTION
Tuberculosis (TB) is caused by bacillus bacteria known as Mycobacterium tuberculosis and is characterized by coughing and fever [1]. TB disease is a chronic infectious disease that causes severe problems globally and is still a significant infectious disease in Indonesia [2]. It requires a long-time comprehensive solution to treat, although various types of TB drugs have been found, as well as the Bacillus Calmette Guerin (BCG) vaccination. However, pulmonary tuberculosis is still a health problem in the world, and this disease yet cannot be eradicated. Based on data from the Ministry of Health, the prevalence of pulmonary tuberculosis cases in Indonesia in 2013 was 285 per 100,000 population, and the mortality rate was 27 per 100,000 people. Based on World Health Organization (WHO) report in 2014, the incidence of pulmonary tuberculosis in 2013 was estimated at 450,000 cases with 170,000 deaths [3].
Hepatotoxicity induced by anti-tuberculosis drugs (AT-DILI, Anti Tuberculosis-Drug induced liver injury) is a severe adverse reaction with significant morbidity. This form of toxicity has the potential to impact TB treatment outcomes in some patients. First-line anti-tuberculosis drugs, oxidative stress, and more broadly interfering with redox homeostasis along with mitochondrial dysfunction can contribute to hepatotoxicity caused by TB drugs. Some identified risk factors of poisoning, in addition to genetic factors including age, malnutrition, alcoholism, chronic hepatitis C and chronic hepatitis B infection, HIV infection, and preexisting liver disease. Importantly, these comorbid conditions are related to oxidative stress and antioxidant-related drugs, especially those for the management of mitochondrial dysfunction. Thus, joint pathogenetic mechanisms for liver injury due to the disease interactions with medications [4].
Papaya (Carica papaya Linn.), a versatile plant commonly called paw-paw, is a type of tropical evergreen tree originating from Mexico and Central America, Taiwan, and southern China, such as Hainan, Guangdong, Guangxi, Yunnan, and Fujian Province [5]. In addition to its delicious fruit flavor, papaya fruit that contains many nutrients is useful for the body and healthy digestive system. Further, the other plant parts are also used in traditional medicine systems [6]. Over the past few decades, much progress has been made concerning biological activities and the medicinal applications of papaya. It is now considered a valuable nutraceutical fruit plant [7].
This in vivo study on the hepatoprotective effect of carica papaya seed ethanol extract (EECP) on hepatoxic-induced rats with Rifampicin and INH. TB drugs that are known hepatotoxic due to oxidative stress and redox homeostasis that cause mitochondrial dysfunction, and papaya seeds that contain high antioxidant ingredients [8,9]. In this study, a comparison test was performed on Curcuma FCT tablet supplement by conducting laboratory tests on liver enzymes such as SGOT, SGPT, Alkali-phosphatase (ALP), Gama-GT (GGT), and Bilirubin.
This study used 24 Rattus norvegicus weighed 150 -200 gr. Before treatment, the rats were acclimated with a 12-hour light/dark cycle for one week at a room temperature (22-25 °C). Rats were given food ad libitum in the form of pellets and water.

Extraction of Carica Papaya
The sample used in this study was papaya seed simplicia. As much as 500 g dry powder papaya seed was dissolved in 96% ethanol with a ratio of 1:3 water and powder. The suspension was shaken occasionally for five days and was then filtered to obtain the extract. The extract was evaporated using a rotary evaporator (45-50 °C) to obtain the crude extract ethanol Carica papaya (EECP). Phytochemical screening of ethanol papaya seeds was done to determine the phenol, flavonoids, tannin and alkaloids steroids/ terpenoids, saponins contents [10].

Experimental Design
The rats were randomly divided into eight groups consisting of 6 rats each, that were normal group without inducer, positive control 1 induced with rifampicin 10.8 mg/day, positive control 2 induced with isoniazid 10.8 mg/day, positive control 3 induced with rifampicin + isoniazid 10.8 mg/day, Curcuma treatment induced with rifampicin + isoniazid 10.8 mg/day and curcuma longa 19.2 mg/day/oral, group I induced with rifampicin + isoniazid 10.8 mg/day with EECP 100 mg/kgBW, group II induced with rifampicin + isoniazid 10,8 mg/day with EECP 300 mg/kgBW, and group III induced with rifampicin + isoniazid 10.8 mg/day with EECP 500 mg/kgBW. Rifampicin, isoniazid, and EECP were given during 21 day. On day 22, rats were sacrificed by administrating ketamine 70 mg/kgBW I.P. Then, the blood was taken and centrifuged for 10 minutes at 3000-4000 rpm until obtaining two layers, serum and supernatant. Serum was taken as much as 1 ml and put into microtubes and stored in a refrigerator at -4 °C. Determination of AST, ALT, ALP, GGT, and Billirubin of blood serum was conducted at the North Sumatera regional health laboratory [11-15].

Statistical Analysis
Data were analyzed using SPSS 20. The normality test was done using Shapiro Wilk and continued with the one-way analysis of variance (ANOVA) and the Post Hoc Test using the Tukey HSD test. The p-value lower than 0.05 was considered statistically different.

RESULTS AND DISCUSSION
The result shows that the positive control 3 group with ALT 384.33 ± 13.19 U/L was significantly different (p <0.05) from the normal group with ALT 27.33 ± 2.05 U/L. The Curcuma treatment group with ALT 26.33 ± 1.24 U/L did not differ significantly (p> 0.05) from the normal group. The treatment group I with AST 250 ± 24.85 U/L was significantly different (p <0.05) from the normal group. Treatment group II with ALT 250 ± 24.85 U/L was significantly different (p <0.05) from the normal group. Treatment group III with the ALT 122 ± 9.89 U/L was significantly different (p <0.05) from the normal group. Based on Table  1, the positive control 3 group with AST 637.37 ± 44.28 U/L was significantly different (p <0.05) from the normal group with AST 105.33 ± 5.24 U/L. The Curcuma treatment group with AST 133.33 ± 6.23 U/L did not differ significantly (p> 0.05) from the normal group. Treatment group I with ALT 355 ± 8.52 U/L was significantly different (p <0.05) from the normal group. Treatment group II with ALT 250 ± 24.85 U/L was significantly different (p <0.05) from the normal group. Treatment group III with ALT 112.67 ± 7.58 U/L was significantly different (p <0.05) from the normal group.
The positive control 3 group with ALP 265 ± 8.60 U/L was significantly different (p <0.05) from the normal group with ALP 57.66 ± 1.24 U/L. The Curcuma treatment group with ALP 54 ± 6.37 U/L did not differ significantly (p> 0.05) from the normal group. The treatment group I with ALP 159.33 ± 12.76 U/L was significantly different (p <0.05) from the normal group. Treatment group II with ALP 250 ± 24.85 U/L was significantly different (p <0.05) from the normal group. Treatment group III with ALP 112.67 ± 7.58 U/L was significantly different (p <0.05) from the normal group.
The negative control group 3 with GGT 72 ± 12.02 U/L was significantly different (p <0.05) from the normal group with GGT 13.33 ± 3.68 U/L. The Curcuma treatment group with GGT 20.33 ± 4.10 U/L did not differ significantly (p> 0.05) from the normal group. The treatment group I with GGT 48.66 ± 3.09 U/L was significantly different (p <0.05) from the normal group. Treatment group II with GGT 33.33 ± 3.85 U/L was significantly different (p <0.05) from the normal group. Treatment group III with GGT 13.66 ± 2.49 U/L was significantly different (p <0.05) from the normal group.
The negative control group 3 with bilirubin 19.06 ± 0.30 U/L was significantly different (p <0.05) from the normal group with bilirubin 3.81 ± 0.50 U/L. The Curcuma treatment group with bilirubin 5.41 ± 0.37 U/L did not differ significantly (p> 0.05) from the normal group. The treatment group I with bilirubin 10.71 ± 0.78 U/L was significantly different (p <0.05) from the normal group. Treatment group II with bilirubin 7.50 ± 0.49 U/L was significantly different (p <0.05) from the normal group. Treatment group III with bilirubin 4.79 ± 0.40 U/L was significantly different (p <0.05) from the normal group.
Rifampicin is known to cause hyperbilirubinemia and liver dysfunction due to liver injury (DILI, Drug-Induced Liver Injury) and can ultimately be diagnosed as clinical exclusion. However, histologic specimens of the liver are often not obtained. Other causes of liver damage, such as acute viral hepatitis, must be sought methodically. Usually, the onset of the acute injury is within a few months after taking the drug. Agent withdrawals are allegedly offending more than double the serum alanine aminotransferase (ALT) elevation, and cessation that cause a decrease in ALT are the most reliable confirmation of diagnosis [16].
In this study, the results showed that the EECP dose of 100 mg/kgbw (group I) still showed quite high levels of ALT, AST, ALP, GGT, and Bilirubin. when compared with a dose of 300 mg/kgbw (group II) there was a decrease in ALT, AST, ALP, GGT, and Bilirubin levels but did not differ significantly from the normal group. The highest dose, namely 500 mg/kgbw, has better effectiveness when compared to the doses of 100 mg/kgbw and 300 mg/kgbw and statistically there is no significant difference when compared to normal. But the levels of AST, ALT, ALP, GGT, and Billirubin in group III could not return to normal.
In the study by Adeneye, a dose-dependent (100 -400 mg/kg/day/oral route) and a protective effect of time of 400 mg/kg/oral route from aqueous seed extracts from the Carica papaya fruit (CPE = Carica papaya fruit) was investigated in hepatotoxic carbon tetrachloride (CCl4) rats for 72 hours [20]. The results showed that the extract caused significant (p <0.05, p <0.001) dose-related attenuation in elevated liver enzyme markers in acute hepatocellular injury (ALT, AST), serum lipids (TG, TC, HDL-c, LDL-c, and VLDL-c), and serum proteins (TP and ALB). Maximum hepatoprotection is offered at an oral dose of 400 mg/kg/day extract. The biochemical results obtained are validated by improvements in the liver histological changes induced by CCl4. In addition, maximum hepatoprotection is offered at 400 mg/kg CPE up to 3 hours after CCl4 induction. Table 1

CONCLUSION
It can be concluded that extract ethanol of Papaya seed have various flavonoid and antioxidant activity that can be a hepatoprotection on rifampicin and isoniazidinduced rats by reducing liver biomarker such as ALT, AST, GGT, ALP, and billirubin. Extract ethanol of papaya seed dose 500 mg/kgbw is the most effective agent as hepatoprotective.

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
This study has received ethical clearance from the Ethics Commission of health and science commission, Universitas Prima Indonesia, Medan, Indonesia. (Ethical number 001/KEPK/ UNPRI/ II/2020).