Hepatoprotective Effect of Mountain Papaya (Vasconcellea

pubescens A.DC.) Fruit Extract against Acetaminophen-Induced

Acute Liver Damage

Heru Sasongko

*1,2

, Diah Pratiwi

, Trias Amartiwi

, Nur Rohman Efendi

and Sugiyarto

Department of Pharmacy Faculty of Mathematics and Natural Sciences Universitas Sebelas Maret. Jl. Ir. Sutami 36A

Surakarta 57126, Central Java Indonesia

Department of Biology Faculty of Mathematics and Natural Sciences Universitas Sebelas Maret. Jl. Ir. Sutami 36A

Surakarta 57126, Central Java Indonesia

Keywords: acetaminophen, hepatoprotective, mountain papaya, flavonoid, Vasconcellea pubescens

Abstract: The liver is a main organ of drugs metabolism in humans. Many studies have reported that acetaminophen

has an effect on liver injury. Targeting mitochondrial oxidant stress is a promising therapeutic option for

acetaminophen hepatotoxicity. Previous study has shown that mountain papaya (Vasconcellea pubescens

A.DC.) fruit has an effect against lipid peroxidation activity. This research aims to know the

hepatoprotective effect of mountain papaya fruit ethanolic extract (MPFE) in rats after induced by

acetaminophen. The rats were divided into six groups, with group I not administered with any treatment,

group II administered with suspension of 0.25% CMC-Na as negative control, group III was administered

with suspension of silymarin as positive control. Groups IV, V, and VI were given MPFE with variation

doses 120; 240 and 480 mg/kg body weight for 14 days. At day 14th, all groups except the normal group

were induced with acetaminophen in toxic dose (2 g/kg body weight). The liver injury was measured by

ALT, AST, bilirubin, ALP value and liver histology profile. The results showed that MPFE could

significantly decrease liver cell injury (p <0.05) by ALT, AST and liver histology profile parameters in

which each dose has the same ability.

1 INTRODUCTION

The liver is the largest gland of the carbohydrates,

lipids, proteins and xenobiotic and drug

detoxification (Kumar et al., 2013). As the center of

metabolism in the body, liver is vulnerable to

chemicals exposure which makes this organ

susceptible to injury (Muriel, 2017). Acetaminophen

overdose is the most frequent cause of liver injury

and acute liver failure in many countries (Jaeschke

and Ramachandran, 2018; Larsen and Wendon,

2014). The formation of a reactive metabolite and its

binding to cellular proteins was initially thought to

be responsible for cell death. A competing

hypothesis was introduced that questioned the

relevance of protein binding and instead suggested

that P450-derived oxidant stress and lipid

peroxidation causes acetaminophen-induced liver

injury (Jaeschke and Ramachandran, 2018).

Hepatotoxicity can occur because the metabolite n-

acetyl-p-benzoquinoneamine (NAPQI) is reactive

(Brune et al., 2015), and interact with covalent liver

macromolecule in the cysteine resulting in the onset

of oxidative stress (Vakiloddin et al., 2015).

Acetaminophen is activated by the enzyme

cytochrome P450 become metabolite N-acetyl-p-

benzoquinone imine (NAPQI) that suppress reactive

glutathione covalent bonds and liver with protein

(Walubo et al., 2004). This bond-related to the

toxicity of acetaminophen which causes liver injury

(Salhanick et al., 2006).

The liver injury can occur by structural damage

and seen from the histological profile of the liver

microscopically. Biochemical parameters in the

blood serum also can be used as an indicator when

the liver injury by an enzyme released from the

hepatic cell organelle into the blood (Amin et al.,

2010). Specific enzymes that indicate liver damage

is ALT (alanine aminotransferase), AST (aspartate

transaminase), TB (total bilirubin), ALP (alkaline

phosphatase) (Gowda et al., 2009; Limdi and Hyde,

Sasongko, H., Pratiwi, D., Amartiwi, T., Efendi, N. and Sugiyarto, .

Hepatoprotective Effect of Mountain Papaya (Vasconcellea pubescens A.DC.) Fruit Extract against Acetaminophen-Induced Acute Liver Damage.

DOI: 10.5220/0008239500660070

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 66-70

ISBN: 978-989-758-349-0

2003). Liver damage can be treated with a

hepatoprotector compound (Pradhan and Girish,

2006). Many studies associate the effects of

antioxidants with hepatoprotection consequence

(Hsiao et al., 2003; Huang et al., 2010; Tzankova et

al., 2017).

Mountain papaya (Vasconcellea pubescens

A.DC.), also called Carica pubescens, are

commonly found in the Dieng plateau, Central Java

(Sasongko et al., 2016). Mountain papayas contain

flavonoid and phenolic compounds that have

antioxidant activity (Simirgiotis et al., 2009; Uribe

et al., 2015). It has antioxidant activity with IC50

value of 8,843 to 0,983 mg/100 mL (Laily et al.,

2012). This research aims to know the

hepatoprotective effect of mountain papaya fruit

extract against acetaminophen-induced acute liver

damage.

2 MATERIALS AND METHOD

2.1 Materials

The fruit of mountain papaya (Vasconcellea

pubescens A.DC) was collected from the Dieng

Plateau, Central Java, Indonesia. Studies were

carried out using male Wistar albino rats (150-250

g). Rats were obtained and all handling procedures

havc been approved by the ethics committee of the

Faculty of Medicine, Universitas Sebelas Maret with

the number 421/V/HREC/2017. Chemical like

Alanine Aminotransferase (ALT), Aspartate

Aminotransferase (AST), alkaline phosphatase

(ALP), total bilirubin were analyzed using reagent

kits (DiaSys Diagnostic, Holzheim, Germany). All

other reagents were of analytical grade. The

instruments used were freeze dryer (VirTis

BenchTop ®) and Spektrofotometer UV-Vis (Micro

Lab 300 ®).

2.2 Sample and Extract Preparation

Mountain papaya fruit with yellowish green color

was washed and cut into small pieces. The fruit was

dried using a freeze dryer. After drying, the fruit was

ground and sieved to a uniform particle size as

sample. The sample was extracted using the

maceration method with 70% ethanol solvent for

seven days. The ratio of sample to solvent was 1:10.

The filtrate of the maceration was collected and

concentrated with a rotary evaporator at 50°C to

obtain an extract.

2.3 Phytochemical Screening

Phytochemical screening was carried out to screen

class of phytoconstituents present in the ethanolic

extract of mountain papaya stem using standard

methods reported in Harborne (2012).

2.4 Animal Experimental Design

The experimental animals were acclimatized for one

week. The rats were given aquadest drink and

standard feed. The animal were divided into six

groups with group I not administered with any

treatment, group II administered with suspension of

0.25% CMC-Na as negative control, group III was

administered with suspension of silymarin as

positive control. Groups IV, V, and VI were given

mountain papaya fruit ethanolic extract (MPFE)

with variation doses 120; 240 and 480 mg/kg body

weight for 14 days. At day 14th all groups except the

normal group were induced with acetaminophen in

toxic dose (2 g/kg body weight). After 48 hours

induction of acetaminophen, blood sample was

collected to measure ALT, AST, ALP and total

bilirubin value. The liver was taken to be analysed

for histology profile.

2.5 Histopathological Studies

Liver tissues were fixed in 10% formalin for at least

24 hours, embedded in paraffin and cut into 5 µm

thick sections in a rotary microtome. The sections

were stained with hematoxylin-eosin dye and

observed under a microscope to detect

histopathological changes in the liver (Huang et al.,

2010).

2.6 Data Analysis

The analysis was performed statistically. The

normality test used was the Shapiro-Wilk test and

variance test was done by Homogeneity of variance

test. The normally and homogeneously distributed

data were analyzed by One-way Analysis of

Variance (ANOVA). To know the differences

between each treatment groups and then continued

with Bonferroni Post Hoc test.

Hepatoprotective Effect of Mountain Papaya (Vasconcellea pubescens A.DC.) Fruit Extract against Acetaminophen-Induced Acute Liver

Damage

Table 1: The effect of mountain papaya (Vasconcellea pubescens A.DC.) fruit extract against acetaminophen-induced

on biochemical parameters.

Groups ALT

/ dl)

AST

/ dl)

ALP

/ dl)

Normal control

51.66 ± 5.25* 223.54 ± 14.67*

548,70 ± 73,72

0,502 ± 0, 01

ative control

461.36 ± 16.60 894.72 ± 22.46

774,38 ± 25,95

0,584 ± 0,02

Silymarin 100 mg/kg B.W

76.24 ± 13.54* 216.68 ± 12.33*

564,92 ± 26,84*

0,512 ± 0,02

Extract dose120 mg/kg B.W

284.23 ± 17.20* 355.68 ± 19.80*

612,60 ± 20,22

0,538 ± 0,02

Extract dose 240 mg/kg B.W

255.96 ± 14.10* 291.56 ± 19.26*

589,72 ± 18,76

0,534 ± 0,08

Extract dose 480 m

B.W

155.7 ± 15.84* 245.24 ± 18.25*

573,80 ± 12,19

0,518 ± 0,01

Symbols represent statistical significance. *p < 0.05, as compared to negative control group. n = 5 animals in each

group

3 RESULT AND DISCUSSION

3.1 Phytochemical Screening

The phytochemical compound of mountain papaya

(Vasconcellea pubescens A.DC) ethanolic extract

showed flavonoids, tannins and phenolic.

3.2 Hepatoprotective Effect

The rats’ biochemical parameter result like ALT,

AST, ALP and total bilirubin are shown in Table I.

The results demonstrated that ALT and AST were

found to be significantly increased in rats treated

with acetaminophen when compared with the

negative control group (P<0.05) but not significantly

on ALP and total bilirubin serum. The

administration of mountain papaya extract for 14

days significantly decreased the activity of serum

alanine aminotransferase and serum aspartate

transaminase in a dose-dependent manner in

acetaminophen-induced liver damage in rats

compared to that of the hepatotoxic group

(acetaminophen treatment) (P<0.05). The serum

level of ALT and AST are largely used for

determination of liver damage (Nurrochmad et al.,

2013). Serum glutamic pyruvic transaminase

(SGPT) or also called ALT (alanine

aminotransferase) is a specific enzyme that can

estimate the damage of a cell especially in the liver

(Gowda et al., 2009; Limdi and Hyde, 2003).

The mechanism of hepatotoxic from

acetaminophen is caused by the damage of hepatic

cell resulting from metabolites formed at the time of

reaction with cytochrome P450. In therapeutic dose,

the main metabolic pathway of acetaminophen is

through glucuronidation and sulfation in the liver,

and only slightly metabolized by the P450

cytochrome which produces N-acetyl quinone imine

(NAPQI). NAPQI in such amounts can be detoxified

by conjugation with glutathione (GSH). While

paracetamol is in excessive doses, it causes

saturation of the sulfate pathway, resulting in large

NAPQI formation and GSH depletion (Li et al.,

1994). Reduced amounts of glutathione will lead the

formation of Reactive Oxygen Species (ROS) and

Reactive Nitrogen Species (RNS) that cause necrosis

of hepatocytes. The presence of ROS will lead to a

loss of mitochondrial potential membrane and loss

of mitochondrial ability in synthesizing ATP. The

loss of ATP will lead the present of necrosis (Hinson

et al., 2010).

3.3 Histopathological Profile

Use the differences microscopic appearance of

hepatic cells between treatment groups with ethanol

extract of mountain papaya can be seen in Figure 1.

Figure 1. Histologic Profile of the Rat’s Liver.

Description: (A) normal Group, (B) negative control, (C)

positive control, (D) MPFE 120 mg/kg b.w, (E) MPFE 240

mg/kg b.w, (D) MPFE 480 mg/kg b.w; (a) Normal cells,

(b) Picnotic, (c) Cariorixis, (d) Kariyolysis

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

From the histological profile that showed the

condition of the hepatic cell in the treatment group

compared with the negative group, there were cells

still in normal condition, but some cells have been

damaged (necrotic). Early morphological changes

include cytoplasmic edema, dilatation of the

endoplasmic reticulum and polysomal

disaggregation. The next process occurs triglyceride

accumulation as fatty grains in the cell, progressive

mitochondrial swelling with damaging crystals and

complex biochemical swelling. The next stages may

experience hydropic degeneration, separation of cell

structures, pythonic cell nuclei, cariorixis,

karyolysis, breakage of the plasma membrane, and

eventually necrosis (Kumar et al., 2017). The dead

nucleus will shrink, have irregular and dark

boundaries. This process is called piknosis, and its

core is called piknotik. The other possibility, the

nucleus may be destroyed, leaving fragments of

chromatinic substances that is dispersed in cells

called cariorecidal. Finally, in a certain condition,

the dead nucleus loses the ability to absorb the dye

again and completely disappears, a process called

karyolysis (Wilson and Price, 2006).

4 CONCLUSIONS

The results showed that mountain papaya

(Vasconcellea pubescens A.DC) could significantly

decrease liver cell injury (p <0.05) by ALT, AST

and liver histology profile parameters in which each

dose has the same ability.

ACKNOWLEDGEMENTS

The author would like to thank Universitas Sebelas

Maret that funded this research with the Hibah

PKLP PNBP Grants scheme.

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