Histological Change of Pancreatic Islands Following Administration

of Saurauia vulcani Korth Leaves Extract in Alloxan-induced

Diabetic Mice

Salomo Hutahaean

, S. Ilyas

, S. Rahayu

Department of Biology, Faculty of Mathematics and Natural Sciences,

Universitas Sumatera Utara, Jl. Bioteknologi No. 1, Kampus USU, Medan, Indonesia 20155.

Keywords: Saurauia vulcani, antiglycemic, Alloxan-induced diabetes, blood glucose level

Abstract: The importance of plant-based ingredients in traditional diabetes therapy has been well known. In North

Sumatra, one of the local species used was Saurauia vulcani Korth. plant (pirdot). The study was intended to

investigate the effect of S. vulcani leaves extract on the histology of pancreatic island in alloxan-induced

diabetic mice. Thirty male mice were divided into three groups of ten mice, namely: Normal Mice (NM)

group, Non-Treated Diabetic Mice (NTDM) group, and Treated Diabetic Mice (TDM) group which is given

200 mg/kg bw leaves extract of S. vulcani daily for 21 days by oral gavage. Diabetes was induced in mice by

alloxan injection. Blood sugar levels (BGL) and body weight were measured on day 0 (baseline, time of

alloxan injection), 72 hours, and on 7, 14, and 21 days after diabetes induction. On the 21st day, the mice

were sacrificed and the pancreatic organ was isolated and 8 micron tissue sections were made and stained

with Haematoxylin and Eosin. The results showed that BGL in NTDM group increases until the end of the

experiment, while in TDM it increases first but then decreases to the level that similar to NM control group

which is relatively stable around 100 mg/dl during the study. In histological examination, irregular island

forms, loose cell population, and cells with pyknotic nucleus were found, as an indication of a damage to the

pancreatic island. These changes were not found in NM groups and TDM groups. Our result indicated that S.

vulcani extract promotes cell regeneration in pancreatic islands. The results indicated that S. vulcani leaves

extract promotes cell regeneration in pancreatic islands.

1 INTRODUCTION

Diabetes (Diabetes mellitus, DM) is a metabolic

disease with a high prevalence rate. Globally, the

number of people with diabetes in the year 2000 was

171 million (2.3%), this figure is predicted to increase

to 368 million (4.4%) in 2030 (Wild, 2004). The main

characteristic of diabetes is high blood glucose levels

(BGL). High BGL can be caused by impaired insulin

secretion in the pancreas, impaired insulin action in

pheripheral tissue, or a combination of both. The

continued effects of chronic diabetes can cause

damage, dysfunction, and failure in various organs

(Saikh, 2016). The treatment of diabetes includes

injecting insulin, using drugs that can increase

pancreatic secretion activity, and drugs that can

increase tissue response to insulin. Diabetes

medications such as sulfonylurea analogues, alpha-

glucosidase inhibitors, and biguanides have side

effects, such as lowering BGL to a very low levels

(hypoglycemia effect), hepatotoxicity, lactic acidosis,

and diarrhea. (Fowler, 2007). Therefore, there is a

need to look for new agents that meet the

requirements as an ideal antidiabetic compound. The

candidates compound which is ideal as an anti-

diabetic drug is an agent that can reduce blood sugar

levels and simultaneously increasing the pancreatic

beta cell population. One strategy that can be done is

to test the antihyperglycemic activity and the effect

on pancreatic organ of plants extract that have

traditionally been used by people as a drug for

diabetes.

In one province in Indonesia (North Sumatra

province), the leaves of pirdot plants (Saurauia

vulcani Korth.) have long been used as a drug for

diabetes (Situmorang., 2015). This article reports the

effect of S. vulcani leaf extract on the histology of

pancreatic island in alloxan-induced diabetic mice.

Hutahaean, S., Ilyas, S. and Rahayu, S.

Histological Change of Pancreatic Islands Following Administration of Saurauia vulcani Korth Leaves Extract in Alloxan-induced Diabetic Mice.

DOI: 10.5220/0010104010951098

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

1095-1098

ISBN: 978-989-758-449-7

1095

2 MATERIALS AND METHODS

2.1 Preparation of Plant Extract

The leaves of S. vulcani were obtained from the

forest of Sintongmarnipi, Toba Samosir, North

Sumatra Province, Indonesia. The leaves are picked,

cleaned, and separated from the petiolus, then dried

in open air for about one week. The dried leaves are

powdered into fine flour using an electric blender.

The extraction process was carried out by maceration

method using ethanol as a solvent. The maceration

results obtained are then filtered and the solvent was

evaporated using a rotary evaporator at 40 ° C

(Sitorus, 2015).

2.2 Experimental Animals

The experimental animals used were mice (Mus

musculus L.), male, healthy, aged ± 3 months,

weighing 25-30 grams. Animal experiments were

obtained from and maintained in animal cages

Department of Biology FMIPA USU Medan. Mice

are kept in a special cage for experimentation, given

pellet feed and drinking water (tap water) on an ad

libitum basis. The animals were adjust to a new

environment by kept them in the cage for two weeks

before experimentation.

Induction of diabetes in mice was carried out by

giving a single injection of alloxan (100 mg/kg body

weight) intraperitoneally. Mice were fasted 12 hours

before the injection. Selection of mice was done 72

hours after the alloxan injection. Only mice that have

a blood glucose level (BGD) >200 mg/dl are included

in the experiment.

The experiment was designed in completely

randomly designed (CRD).Thirty diabetic mice (BGL

>200) were randomly assigned to three treatment

groups of 10 mice each. The treatment groups were:

normal mice as control (NM groups), Non-Treated

Diabetic Mice (NTDM groups), and Treated Diabetic

Mice (TDM groups) that received S. vulcani leaves

extract 200 mg/kg body weight. The treatment was

given daily by oral gavage for 21 days.

2.3 Blood Glucose Level and Body

Weight

Blood glucose level was measured on blood removed

from the tail of the mouse. BGL determination is

performed with a glucometer (EasyTouch). BGL

measurements were carried out on day 0 (baseline) at

the time of alloxan injection, then 72 hours after the

injection, and then on the 7th, 14th, and 21st days.

About 5 mm the tip of the mouse's tail was cut with

scissors, then the blood was left to drip to a special

test strip for glucose and after about 10 seconds the

BGL number will appear on the glucometer screen.

The body weight was weighed on the same day

as the BGL determination day by using an electronic

scale.

2.4 Tissue Preparation

Animals were sacrificed at the end of the experiment.

Pancreatic organs were isolated and fixed in

formaldehyde. Tissue section was prepared by the

paraffin method. Eight μm thick tissue sections were

stained with HE and used for pancreatic island

observation.

2.5 Statistic Analysis

Parametric data were analyzed with ANOVA, the

Duncan 'test post hoc was apllied for all ANOVA

significant result. The differences between means

were considered significant at p<0.05.

3 RESULT AND DISCUSSION

Results from ANOVA showed that at the time of

alloxan injection (baseline, day 0) blood glucose level

(BGL) between treatment groups was not

significantly different (p>0.05). In the group of

normal mice (NM) the BGL baseline was around 100

mg/dl. This level was stable in NM group until the

end of the experiment.

Significant differences in BGL between

treatments began to be seen in observations 72 hours

after alloxan injection, whereas mice injected with

alloxan increased their blood glucose levels up to>

200. In figure 1, BGL in the NTDM group continued

to increase to> 250 mg/dl. The increasing level of

BGL in NTDM group was believed due to the

development of diabetic condition. In contrary, the

BGL in the TDM group was dropped after the animal

received S. vulcani leaves extract. The BGL

decreases was detected on day-7, but the significant

different to NTDM group (p<0.05) started on day-14.

The level continues to decrease until it reaches a level

that is not significantly different from the control

(NM group) at the end of the study (p>0.05).

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

1096

Figure 1. The change in mean of blood glucose level in

normal mice group (NM) group, Non-Treated Diabetes

Mice (NTDM), and Treated Diabetes Mice (TDM) at

different time of observation. The TDM mice received 200

mg/kg body weight leaves extract (Saurauia vulcani Korth).

The effect of treatments on body weight is shown

in figure 2. There is no significant difference in body

weight between treatments both at baseline and at 72

hours after alloxan injection, but on day 7 the body

weight in NTDM and TDM groups is lower than body

weight in control (NM) (p<0.05). These conditions,

especially in the NTDM group, continued until the

end of the study, while in the TDM group the body

weight improved to levels that were not different

significantly from controls (NM).

These results indicated that alloxan induces

diabetes in mice characterized by an increase in BGL

and then followed by weight loss. The results

obtained was agree with the previous studies

(Ewenighi,., 2015; Hutahaean., 2018). Uncontrolled

diabetes causes an increase in glycogenolysis,

lipolysis, and gluconeogenesis. Biochemical

activities through these three pathways are basically

the use of a new energy source for the body to

compensate for the low glucose entering the cell. The

use of energy sources from body fat and protein in

these processes is believed to be the pathway for the

mechanism of weight loss in diabetes.

The mechanism of diabetes induction by alloxan

is by damaging the pancreatic beta cells through the

resulting free radical effects. Alloxan is a glucose

analog which is specifically accumulates in the

pancreatic beta cells. By intracellular thiol activity,

especially glutathione, alloxan produces reactive

oxygen species (ROS) in a cyclic redox reaction with

its product, dialuric acid. Dialuric acid autoxidation

produces superoxide radicals, hydrogen peroxide

and, hydroxyl radicals. Hydroxyl radicals are

responsible for the death of beta cells that have very

low antioxidant defense abilities. As a thiol reagent,

alloxan also selectively inhibits glucose-induced

insulin secretion through its ability to inhibit glucose

sensor in beta cells (Szkudelski, 2001; Lenzen, 2008).

Histological observation showed the damage of

pancreatic cells in the NTDM group (figure 3).

Figure 3. The change of pancreatic island feature after

Saurauia vulcani leaves extract treatment in alloxan-

induced diabetic mice. A1 and A2: normal mice (NM); B1

and B2: Non-Treated Diabetes Mice (NTDM); C1 and C2:

Treated Diabetes Mice (TDM). The TDM mice received

200 mg/kg body weight leaves extract (Saurauia vulcani

Korth). Staining HE; 400 X.

Figure 2. The change in mean body weight in normal mice

group (NM) group, Non-Treated Diabetes Mice (NTDM),

and Treated Diabetes Mice (TDM) at different time o

observation. The TDM mice received 200 mg/kg bod

weight leaves extract (Saurauia vulcani Korth).

Histological Change of Pancreatic Islands Following Administration of Saurauia vulcani Korth Leaves Extract in Alloxan-induced Diabetic

Mice

1097

The damages were cells with a pyknotic nucleus,

cell populations reduction which was characteristics

of a more loose group of cells in the island (figure 3,

B1 and B2). In NTDM group, island cell population

reduced, the boundaries of the islands appear

irregular, and cells damage of pyknotic type found

(black arrow). Those were not found in control (NM)

and in mice treated with Saurauia vulcani leaves

extract (TDM group).

In addition, the forms of islands also appear

irregular compared to the control group (NM). In the

TDM group, there were indications of improvement

in the morphological structure of the pancreas which

was characterized by the denser features of island

cells, no more pyknotic cells, and more regular island

boundaries. The morphological changes obtained was

supporting the findings of BGL repair due to the

treatment of S. vulcani leaves extract.

ACKNOWLEDGEMENTS

This research was funded by the Directorate of

Research and Community Service of the Directorate

General of Research Strengthening and Development

of the Ministry of Research, Technology and Higher

Education, in accordance with the Research Funding

Agreement and Community Service in Fiscal Year

2018.

REFERENCES

Ewenighi C, U Dimkpa, J Onyeanusi, L Onoh, G

Onoh, U Ezeugwu. 2015. Estimation of glucose

level and body weight in Alloxan Induced

Diabetic Rat treated with Aqueous extract of

Garcinia Kola Seed. Ulutas Med J. 1(2): 26-30.

doi: 10.5455/umj.20150507042420.

Fowler, M. J. 2007. Diabetes treatment, Part 2: Oral

agents for glycemic management. Clin. Diabetes

25, 131–134.

Hutahaean, S., Tanjung, M., Sari, D. P. & Ningsih, V.

E. 2018. Antihyperglycemic and

antihyperlipidemic effects of pirdot (Saurauia

vulcani Korth.) leaves extract in mice. IOP

Conference Series: Earth and Environmental

Science, Vol. 130, Conference 1.

Lenzen S. 2008. The mechanisms of alloxan- and

streptozotocin-induced diabetes. Diabetologia.

Feb;51(2):216-26.

Shaikh, H., Shrivastava, V. K. & Amir, M. 2016.

Diabetes Mellitus and Impairment of Male

Reproductive Function: Role of Hypothalamus

Pituitary Testicular Axis and Reactive Oxygen

Species. 8, 41–50.

Sitorus, P. 2015. Characterization Simplisia and

Ethanolic Extract of Pirdot ( Saurauia Vulcani ,

Korth ) Leaves and Study of Antidiabetic Effect

in Alloxan Induced Diabetic Mice. International

Journal of ChemTech Research 8, 789–794.

Situmorang, R., Harianja, A., & Silalahi, J. 2015.

Karo’s Local Wisdom: The Use of Woody Plants

for Traditional Diabetic Medicines. Indonesian

Journal of Forestry Research, 2(2), 121-130.

doi:http://dx.doi.org/10.20886/ijfr.2015.2.2.121-

130.

Szkudelski, T. 2001. The mechanism of alloxan and

streptozotocin action in B cells of the rat pancreas.

Physiol. Res. 50, 537–546.

Wild, S. 2004. Global Prevalence of Diabetes

Estimates for the year 2000 and projections for

2030. World Health 27, 1047–1053.

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

1098