Evaluation of Blood Glucose Level and Microscopic Pancreatic Islets

of Langerhans Treated with Lawsonia Inermis Linnaeus Leaves

Ethyl Acetate Extract in Streptozotocin-induced Diabetic Rat

Dwi Rita Anggraini

, Tri Widyawati

, Siti Syarifah

, Arlinda Sari Wahyuni

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

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

Medan, 20155, Indonesia

Department of Public Health, Faculty of Medicine, Universitas Sumatera Utara, Medan, 20155, Indonesia

Keywords: blood glucose level, pancreatic islets of Langerhans, Lawsonia inermis Linnaeus, diabetic rat

Abstract: Lawsonia inermis Linnaeus leaf, is one of alternative medicine that used to treat diabetes mellitus (DM) in

Indonesia. We investigated the effect of ethyl acetate extract of Lawsonia inermis Linnaeus (EAE) on blood

glucose level (BGL) and the histopathological alterations of pancreatic islets of Langerhans (iL) in

streptozotocin (STZ)-induced diabetic rats. A number of 24 rats were divided into 6 groups, Normal control

(NC) was feed ad libitum, while STZ-induced diabetic rats (SDR) groups were treated with normal saline 10

ml/kg (P1), glibencamide 10 mg/kg (P2), EAE 250 (P3), 500 (P4) and 1000 (P5) mg/kgbw, daily orally for

14 days. Data were analyzed using one way ANOVA followed by Dunnett t test. BGL of NC-(79.75  4.95);

P2 (77.5  3.8); P3 (79.56.9); P4 (78.5  4.5); P5 (83.25  4.3) were lower than P1-treated groups (292.5

4.63) mg/dl, significantly (P<0.01). The histopathological evaluation showed that the perimeter of the islets

of Langerhans P1 were shrinked (8.59  1.8)µm, while P2(12.71  5.4); P3 (11.42  2.9); P4 (1679  11;4)

and P5 (14.373.5)µm were larger and closed to NC (19.27  3.5)µm. The present study concluded that EAE

have antihyperglycemic activy and improve the pancreatic islet of Langerhans structure.

1 INTRODUCTION

In spite of knowledge, there are great efforts that have

been made in the understanding and management of

diabetes. Today, disease related complications are

increasing day by day without any reduction in

strength (Tiwari, 2002). In spite of the presence of

known antidiabetic medicine available in the

pharmaceutical market, remedies derived from

medicinal plants are successfully used in the

treatment of this disease (Bhattaram et al., 2002;

Choubey et al., 2010)

However search for new Antidiabetic drugs

continues. The mechanism of most of the herbals used

to treat diabetes has not been defined. It has been

attributed that the antihyperglycemic effect of these

plants is due to their ability to restore the function of

pancreatic tissues by causing an increase in insulin

output or inhibit the intestinal absorption of glucose

or to the facilitation of metabolites in insulin

dependent processes. Hence treatment with herbal

drugs has an effect on protecting â-cells and

smoothing out fluctuation in glucose levels (Jia et al.,

2003; Elder, 2004)

Lawsonia inermis Linn. commonly known as

henna, is a finely ground brown or green powder

originating from dried leaves of the plant Lawsonia

inermis which is grown in dry tropical and subtropical

zones, including North Africa, India, Sri Lanka, and

the Middle East. (Borade et al., 2011).

Lawsonia inermis Linnaeus is one of the plants

commonly used in Indonesian community for the

treatment of different diseases (Widyawati et al,

2016). Previous study has been well investigated

phytochemically by various researchers such as β-

sitosterol, lawsone, esculetin, fraxetin, isoplumbagin,

scopoletin, betulin, betulinic acid, hennadiol, lupeol,

lacoumarin, laxanthone, flavone glycosides, two

pentacytic triterpenes glucoside, flavonoids,

quinoids, naphthalene derivatives, gallic acid,

coumarins, and xanthones (Chaudhary et al., 2010;

Kamal and Jawaid, 2010; Borade et al., 2011; Musa

and Gasmelseed, 2012) in Lawsonia leaves has been

reported. Earlier work establishes the use of henna as

108

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

Evaluation of Blood Glucose Level and Microscopic Pancreatic Islets of Langerhans Treated with Lawsonia Inermis Linnaeus Leaves Ethyl Acetate Extract in Streptozotocin-induced Diabetic

Rat.

DOI: 10.5220/0010039101080112

In Proceedings of the 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and Technology (ICEST 2018), pages 108-112

ISBN: 978-989-758-496-1

an alternative vegetable retanning agent (Musa and

Gasmelseed,2012).

Several study showed that effect of Lawsonia

inermis Linn ethanolic extract 500 mg/kg of body

weight was found to be better then Glibenclamide

(10mg/kgbw). These results suggest that the ethanolic

extract possess significant antidiabetic effect

(Choubey et al., 2010). Widyawati et al., 2016

showed that EAE is the most active extract as

antihyperglycemic than with n-hexane (HE),

ethylacetate (EAE), ethanol (EE), water1(WE1) and

water2 (WE2). Hence the aim of the study is to

investigate hypoglycemic effect of ethyl acetate

extract of Lawsonia inermis Linn in streptozotocin

induced diabetic rats and evaluated microscopic

pancreas of islets Langerhans.

2 MATERIAL AND METHODS

2.1 Chemical and Reagents

Streotozotocin, formalin buffer 10%, paraffin wax,

TBA reagent, heparin sodium, sodium chloride, cell

lysis buffer, aquabidest, 70% and 80% aqueous

alcohol and 96% absolute alcohol, xylol, glyserin,

Mayer”s haematoxylin, eosin, canada balsem. All

other chemical were of analytic grade.

2.2 Animals

Healthy male Wistar rats (150-200 g) were obtained

from animal house of Universitas Sumatera Utara.

The study was conducted after approved by Animal

Research Ethics Committees (AREEC), Faculty of

Mathematics and Natural Sciences (FMIPA),

Universitas Sumatera Utara (No. EC: 115/KEPH-

FMIPA/2017).

2.3 Plant Material and Preparation of

EAE

Lawsonia inermis Linn leaves were collected from

Titi Kuning, Medan, North Sumatera, Indonesia and

was authenticated by Department of Botany,

Universitas Sumatra Utrara. The fresh leaves were

dried under shade and ground into powder. The

powdered leaf then was extracted serially by

maceration in n-hexane and ethyl acetate (EAE).

2.4 Induction of Diabetes

Diabetic rats were obtain by induction STZ (55

mg/kg) intraperitoneally. Diabetic rats with fasting

blood glucose level more than 200 mg/dl were

included to the study. BGL was confirmed using

glucometer (Accu check), after 72 hours of STZ

injection.

2.5 Experimental Design

The animals were divided randomly into six groups

of four rats each and treated as follows:

 Group I (NC): Normal control rats (standard

pellets and water ad libitum) for 14 days.

 Group II (P1): Diabetic control rats were

administered with STZ, were treated with

normal saline 10 ml/kg

 Group III (P2): Diabetic rats were treated with

glibencamide 10 mg/kg

 Group IV (P3): Diabetic rats were treated with

EAE 250 mg/kgbw daily orally for 14 days.

 Group V (P4): Diabetic rats were treated with

EAE 500 mg/kgbw daily orally for 14 days.

 Group VI (P5): Diabetic rats were treated with

EAE 1000 mg/kgbw daily orally for 14 days.

2.6 Preparation Pancreatic for

Histopathological Analysis

At the end of the stipulated 14 days feeds were

withdrawn, the rats were subjected to a 12 hours fast

but had access to water. Sacrificed using chloroform

vapour. Rats were positioned on the surgical board

using pins or pin needles. The surgery started in rat

stomach by using surgical scissors. The pancreas

organ were carefully dissected out, trimmed of all fat

and connective tissue blotted dry to remove any

blood.

Within a 30 minute interval after excision, the

pancreas was immersed in buffered 10%

formaldehyde for 24 hours. The samples were fixed

in buffered 10% formaldehyde for 24 hours, followed

by dehydration

in: 1) 70% alcohol for 60 min, 2) 96% alcohol for 45

min, 3) absolute alcohol for 2 h. The clearing phase

of the samples was made by repeated xylene

immersions, followed by paraffin wax infiltrations.

The samples were automatically processed with

tissue processor Thermo Scientific STP 120-3 and

paraffin embedding was done with modular tissue

embedding center Thermo Scientific Microm EC

350-1. Next,the resulting blocks were cut at 5 μm

using the Leica RM 125RTS microtome and then

carefully placed on the microscope slides. In order to

distinguish between tissue types the sections were

stained with Haematoxylin and Eosin (H&E) staining

techniques, after which they were passed through

ascending grade of alcohol, cleared in xylene and

mount in DPX mountant, allowed to dry at room

Evaluation of Blood Glucose Level and Microscopic Pancreatic Islets of Langerhans Treated with Lawsonia Inermis Linnaeus Leaves Ethyl

Acetate Extract in Streptozotocin-induced Diabetic Rat

109

temperature and observed histopathologically under

digital light microscope

2.7 Photomicrography

Records of the Histopathological results were

obtained by photomicrography using digital

photomicrographic microscope was made with an

Olympus BX 41 microscope coupled to an Olympus

DP25 video camera at the Anatomic Pathology

Laboratory, Department of Anatomic Pathology,

Universitas Sumatera Utara.

2.8 Image Analysis

Morphometric measurements of the digitalized

images of immunostained sections were carried out

using the Image J IJ 1.46r plus image analyzer

computer system (Wayne Rasband, Maryland, USA).

The average area of the islets was determined by

measuring the area of 3 islets in each section of one

rat, and in total for 12 islets from each group (Ferreira

and Rasband, 2012)

2.9 Statistical Analysis

Data were analyzed and presented as means ± SD.

Differences between continuous data were analyzed

using one way Annova followed by Dunnett t test. p <

0.01 was considered significant.

3 RESULT AND DISCUSSIONS

3.1 Blood Gucose Level (BGL)

BGL concentration increased following STZ

injection in all groups compared with the diabetic

group during the duration of the experiment. The rats

in group treatment which received glibencamide

mg/kg (P2), EAE 250 (P3), 500 (P4) and 1000 (P5)

mg/kgbw

showed a significant decrease (P < 0.01) as

compared with the rats of the untreated diabetic group

(P1) (Table 1).

Table 1: Effect of EAE on blood glucose level in STZ-

induced diabetic rats

Grou

Blood Glucose Level (mean  SEM)

79.75  4.95***

292.5  4.63

77.5  3.8***

79.5  6.9***

78.5  4.5***

83.25  4.3***

Data was expressed as mean  SEM. ***p<0.01

The decrease of BGL in the treatment group with

EAE caused by bioactive compounds of Lawsonia

inermis Linn leaf can prevent the occurrence of

oxidation in pancreatic β cells so the damage can be

reduced. The bioactive compounds contained in

previous study has been well investigated

phytochemically by various researchers such as

polyphenols, flavonoids, alkaloids and tannins. The

role of polyphenols is thought to be capable of

protecting pancreatic β cells from the effects of free

radical toxicity produced (Chaudhary et al., 2010;

Kamal and Jawaid, 2010; Borade et al., 2011.

In line with previous studies showed the that the

feeding of 0,8mg/kg/bw of Lawsonia inermis Linn

extract ethanol decreased the glucose concentration to

normal condition after the 14th day (Syamsudin et al.,

2008). The study of Choubey et al., showed the effect

of ethanolic extract of Hena 500 mg/kgbw was found

to be better then Glibenclamide (10 mg/kgbw).

(Choubey et al., 2010)

This result so in agreement with Ojewunmi et al.,

showed that

ethanol extract of Lawsonia inermis

leaves was significantly reduced fasting blood

glucose (P<0.001) compared to the untreated diabetic

control. (Ojewunmi et al., 2014). Antika et al.,

showed the group treated ethanol extract of Lawsonia

inermis a dose of 400mg/kgbw had the lowest

decrease blood glucose levels. (Antika et al., 2017)

3.2 Evaluation of the Islets of

Langerhans (iL)

Morphometric measurements showed changes in the

mean values of iL in figure 1. The average area of iL

P1 were shrinked (8.59  1.8)µm

, while P2 (12.71 

5.4); P3 (11.42  2.9); P4 (1679  11;4) and P5

(14.373.5)µm

were larger and closed to NC (19.27

 3.5) µm

ICEST 2018 - 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and

Technology

110

Figure 1: The average area of islets of Langerhans (iL)

Figure 2: Photomicrographs of sections of the pancreas

from control group showing pancreatic acinar (PA) and

islets of Langerhans (IL) with granulated cytoplasm of islet

cells with small, dark nuclei on the periphery (alpha-cells)

(arrow), or with light and large nuclei (beta-cells) (duble

arrow); H&E staining, scale bar = 50 μm

Figure 3: Photomicrographs of sections of the pancreas

from diabetic group showing pancreatic shrinked with

degenarative change in IL espescially in center of islet

(double arrow). Irregular outlining of the islet. H&E

staining, scale bar = 50 μm

Figure 4: Photomicrographs of sections of the pancreas

from EAE group showed the nearly regular outline of islet

with apparently normal appearance of most cell. H&E

staining, scale bar = 50 μm

The examination of H&E stained sections from

the control group showed the pancreas to have a

normal histological structure. The islets of

Langerhans appeared as noncapsulated pale stained

rounded or oval areas inside the pancreatic acinar

lobules, which were formed of groups of cells

arranged in irregular, branching, and anastomosing

cords separated by blood capillaries (Figure 2). In

diabetic group (P1), STZ caused degenerative

changes in the pancreatic islets, mainly at the center

of the islets. An apparent reduction in the size and

number of islets was noticed (Figure 3). Sections

from the iL of group P4 (given 500 mg/kg bw EAE)

showed islets with nearly regular outlines and almost

normal cell morphology (Figure 4).

Streptozotocin (STZ) is an antibiotic produced by

Streptomyces achromogenes. It has been widely used

for inducing experimental diabetes mellitus in a

variety of animals, it stimulates the naturally

occurring metabolic disorder DM by causing

degeneration of pancreatic β cells. (Coskun, 2005).

The selective β cell toxicity of STZ is related to the

glucose moiety in its chemical structure, which

enables STZ to enter the cell via the low affinity

glucose transporter Glut2 in the plasma membrane

which induces an increased release of reactive oxygen

species, subsequently causing DNA damage

(Szkudelski, 2001).

This study indicated that treated of EAE L.

inermis extract caused improved destruction of iL

with increases size and number of islets caused STZ-

induced diabetic rats.

4 CONCLUSIONS

Lawsonia inermis Linn EAE have anti-

hyperglycemia effect and protective microscopic

Evaluation of Blood Glucose Level and Microscopic Pancreatic Islets of Langerhans Treated with Lawsonia Inermis Linnaeus Leaves Ethyl

Acetate Extract in Streptozotocin-induced Diabetic Rat

111

changes of islets of Langerhans in STZ-induced

diabetic rats.



Conflict of Interest Statement

The authors declare that there are no conflicts of

interest.

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