Effect of Lawsonia inermis Linnaeus Leaf Ethyl Acetate Extract on

Liver in Normal Rat

Tri Widyawati

, Siti Syarifah

, Dwi Rita Anggraini

Department of Pharmacology and Therapeutic, Faculty of Medicine, Universitas Sumatera Utara,

Medan, 20155, Indonesia

Department of Anatomy, Faculty of Medicine, Universitas Sumatera Utara, Medan, 20155, Indonesia

Keywords: Lawsoniainermis, leaf, Ethyl Acetate, Extract, Liver

Abstract: Previous study revealed the antihyperglycemic activity and hepatoprotective effect of Lawsonia inermis

Linn. leaf extract (EAE) dose 1 g/kgbw in streptozotocin-induced diabetic rats. The present study was

conducted to evaluate the toxic effect of EAE on hepar in normal rat. EAE was obtained by serial extraction

using n-hexane and ethyl acetate (EAE). Two groups of Wistar rats (n=5) were treated with EAE (1.25

g/kgbw) and distilled water (NC: 10 ml/kgbw), orally, daily for 14 days, respectively. After 14 days the rats

were sacrified for histopathological evaluation of liver using hematoxyilline-eosin staining.The result

showed normal apperance of liver in NC-treated rat, contrarily in EAE-treated rats showed the hydrophic

degeneration, sinusoid and central vein congestion. Multiple nodules with strict lines, consisted of fat cells

undergoing proliferation, monocytes and limphocytes infiltration were also found. These results suggest that

EAE dose 1.25 g/kgbw toxic to liver and potentially destroy its function.

1 INTRODUCTION

Our previous study showed that ethyl acetate extract

of Lawsonia inermis Linnaues (EAE) leaf which

obtained by serial extraction (nhexane-ethylacetate-

ethanol-water) was the most active as

antihyperglycemic in streptozotocin-induced

diabetic rats at dose 1 g/kgbw. It was also

demonstrated the hepatoprotective effect of this

extract. Qualitative chemical screening of EAE

traced the presence of flavonoid, tannin, saponin and

glycoside (Widyawati et al, 2018). These chemical

compounds were suggested contributed to its benefit

pharmacological activities. In order for medicinal

plants to be utilized, it must be supported not only

the efficacy, but also its safety through toxicity test.

Liver is one of organ that have important role to

detoxify the xenobiotic including herbs (Gao et al,

2008; Nasri, 2013; Teschke, 2015). The purpose of

this study is to evaluate the effect of EAE at higher

dose 1.25 g/kgbw on liver in normal rat.

2 MATERIAL AND METHODS

2.1 Plant Material Collection and

Preparation of Ethyl Acetate

Extract

L. inermisLinn. leaves were collected from

TitiKuning, Medan, Indonesia (Coordinate:

3.526093, 98.684528). The plant was identified at

“Herbarium Bogoriense”, the Research Centre for

Biology-Indonesian Institute of Science, Bogor,

Indonesia and given a herbarium identification

number - No.924/IPH.1.01/If.07/III/2017. The fresh

leaves were dried under shade and ground into

powder. About 1.5 kg of the powdered leaf was

extracted serially by maceration in n-hexane and

ethyl acetate (EAE). The freeze-dried extracts were

kept in the freezer (-20C) before used.

2.2 Animals

Healthy male Wistar rats weighing between 180-250

g were obtained from animal house of Universitas

Sumatera Utara. The animals were acclimatized at

room temperature and a 12-h dark/light cycle, and

Widyawati, T., Syarifah, S. and Anggraini, D.

Effect of Lawsonia inermis Linnaeus Leaf Ethyl Acetate Extract on Liver in Normal Rat.

DOI: 10.5220/0010100909110913

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

911-913

ISBN: 978-989-758-449-7

911

were allowed to access food and water ad libitum for

one week before being used for experimentation.The

study was performed after approved by Animal

Research Ethics Committees (AREEC), Faculty of

Mathematics and Natural Sciences (FMIPA),

Universitas Sumatera Utara.

2.3 Experimental Procedure

Rats were divided into two groups, n=5,

respectively. Group I (EAE-treated group) were

given EAE 1.25 g/kgbw, while group II were given

distilled water 10 ml/kgbw (NC-treated group). The

treatments were administered orally, at single dose

and followed for 14 days.

2.4 Preparation of Liver for

Histopathological Examination

The rats were sacrified with the carbogen gas (95%

O2 and 5% O2) and the liver was excised. The liver

was fixed in 10% buffered formaldehyde for 24

hours, followed by dehydrationusing 70% alcohol

(60 min), 96% alcohol (45 min), and absolute

alcohol (2 h). The clearing phase of the samples was

made by repeated xylene immersions, followed by

paraffin wax infiltrations.The samples were then

automatically processed with tissue processor

Thermo Scientific STP 120-3 and paraffin

embedding was prepared using modular tissue

embedding center Thermo Scientific Microm EC

350-1. The parafffin-embedded tissues were

sectioned into 5μm using the Leica RM 125RTS

microtome and mounted on a microscope slides. The

mounted slides were stained with hematoxylline (H)

and eosin (E) according to H&E staining technique.

The stained sections were then mounted in DPX

mounting medium with cover slide.

2.5 Histopathological Interpretation

Histopathological appearance of the liver was

evaluated by macroscopic and microscopic.

Degree of liver destruction were determined as

follow:

0 = normal or no destruction

+= one of these criteria was found; fatty

degeneration, or congestion of central vein and

sinusoid, focal necrosis, benign cysts with fat

++= two of these criteria were found; fatty

degeneration, or congestion of central vein and

sinusoid, focal necrosis, benign cysts with fat

+++= 3-4 criteria: fatty degeneration, or congestion

of central vein and sinusoid, focal necrosis, benign

cysts with fat

2.6 Photomicrography and Image

Analysis

Records of the histopathological results were

obtained by photomicrography using digital

photomicrographic microscope (Olympus BX 41

and  Olympus DP25 video camera) at the

Anatomic Pathology Laboratory, Department of

Anatomic Pathology, Universitas Sumatera Utara.

3 RESULTS

Table 1 showed the degree of liver destruction in

NC- and EAE-treated rats. No destruction (Degree

0) were found in normal rat. Generally, EAE-treated

rats showed level 2 of liver destruction.

Table 1: Liver destruction degree macroscopically.

Grou

ree

NC 0

EAE1 +++

EAE2 ++

EAE3 +

EAE4 ++

EAE5 ++

NC-treated group as control showed normal liver,

red to tan, well defined and soft consistency (Figure

1-NC), while grossly, the liver of EAE-treated group

showed pale tan to red, smooth and multiple cystic

(black arrow)appearance with diameter 0.2 cm. The

cysts relatively well-demarcated, white and smooth

(Figure 1-EAE).

Figure 1: Gross appearance of hepar NC- and EAE-

treated group (black arrow: cyst).

In normal hepatocyte (Figure 2-NC) showed normal

architecture with regular hepatocyte cells, round

nucleus, fine-chromatin cytoplasm eosinophilic;

normal sinusoid and central vein. Contrarily, the

liver cells of EAE-treated group showed congestion

in central vein and sinusoidal,enlargement

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

912

ofsinusoid, focal inflammation (black arrow) and

benign cysts contained proliferation of the fat cells

(red arrow) (Figure 2-EAEa-b).

Figure 2: The photomicrographs of liver section of NC-

and EAE-treated group (NC: 100x; EAE: 400x;

Hepatocyte cell (H); sinusoid (arrow); central vein (CV).

Herbal medicine derived from plant extracts are

being increasingly used to treat various of disease

(Seif, 2016). Some plant extracts and natural

compounds were found as hepatoprotective active

principles, while others adversely induced liver

toxicity (Manfo et al, 2016). The liver represents the

key "metabolic factory" is the most exposed organ

to xenobiotics including medicinal plant extracts.

This may be modulated by any compound

irrespective to the purpose of use. The

histopathological evaluation of the present study

showed that EAE dose 1.25 g/kgbw affected the

structure of liver. It was found clearly by the

changing of gross appearance of liver ie pale tan to

red and multiple cystic. This result contradictive

with our previous study that showed the

hepatoprotective effect of EAE dose 1 g/kgbw in

streptozotocin-induced diabetic rats. The higher dose

of EAE may have the role of this unwanted effect.

The action mechanisms involved in the

hepatoprotection or hepatotoxicity by the medicinal

plants are still not well elucidated.Herb induced liver

injury can be caused by the chemical compounds as

their causative agents. Elimination process for

metabolic degradation may yield hepatotoxic

metabolites that causing liver injury (Manfo et al,

2016; Frenzel and Teschke, 2016).

4 CONCLUSIONS

Ethyl acetate extract of Lawsonia inermis Linnaeus

leaf at dose 1.25 mg/kgbw toxic to the liver.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge to the

Universitas Sumatera Utara for supporting this study

(TALENTA USU GRANT 2018, No.

2590/UN5.1R/PPM/2018).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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Gao, B., Jeong, W., I., Tian, Z., 2008.Liver: an organ

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Manfo, F.,P.,T., Nantia, E.,A., Kuete, V.,

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Widyawati, T., Wahyuni, H.,S., Syarifah, S., Anggraini,

D.,R., Sari, M., I., 2018.Phytochemical Profile and

Antioxidant Assay of Ethyl Acetateof Lawsoniai

nermis (Linn) Leaf Extract. International Journal of

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