Antihyperglycemic and Pancreatic Protective Effect of Squalene

in Streptozotocin-induced Diabetic Rat

Tri Widyawati

, Siti Syarifah

, Muhammad Ichwan

, Dwi Rita Anggraini

, Arlinda Sari Wahyuni

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

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

Keywords: antihyperglycemic, pancreatic islets of Langerhans, squalene, diabetic rat

Abstract: Squalene (Sq), is one compound that reported found in antidiabetic plant (Syzygium polyanthum Wight Walp

leaf). The aim of this study was to evaluate its antihyperglycemic activity and potential pancreactic protective

effect in streptozotocin-induced diabetic rats (SDR). Twenty Wistar rats (male, 180-230 g) were divided into

four groups (n=5) and treated once daily for 14 days p.o. Group I (Sq 160 mg/kg), Group II (metformin (M)

500 mg/kg), Group IV (diabetic control (DC) distilled water 10 ml/kg) and Group III (normal control (NC)

distilled water 10 ml/kg). Blood glucose level (BGL) and a histological study of the pancreas were performed.

As compared to DC, SQ showed significant reduction (p<0.05-0.01) at day6-day14. Interestingly,

histopathological assessment showed the restoration of the STZ-induced pancreatic islet cells damage. The

present study concluded that Sq have antihyperglycemic activity and pancreatic protective effect.

1 INTRODUCTION

Diabetes is a major public health concern (Ramadan

et al., 2017) that being the number one killer among

all chronic diseases in the world (Widyawati et al.,

2015a).

In spite of continuous new drug development to

treat diabetes, medicinal plants remain a potential

alternative therapy as antidiabetic agent (Ramadhan

et al., 2017). The potential of antidiabetic plant is not

only for its antihyperglycemic activity but also for

identification of the responsible active compounds.

Previous study (Widyawati et al, 2015b) identified

squalene (Sq) as one of compounds in antidiabetic

plant ie. Syzigium polyanthum leaf. Squalene, a

triterpene, that has been implicated in several studies

as a compound that contributes to the

antihyperglycemic activities of plants (Baskar et al.,

2011; Jananie et al., 2011; Widyawati, 2015a)

Considering that diabetes is associated with impaired

pancreatic function, in this study we investigated the

effect of Squalene (Sq) on blood glucose level and

pancreatic structure in streptozotocin-induced

diabetic rats.

2 MATERIAL AND METHODS

2.1 Chemical

Squalene, streptozotocin and tween 80 were

purchased from Sigma Aldrich (St. Lous, MAU,

USA).

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

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 (No. EC: 115/KEPH-

FMIPA/2017).

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

Antihyperglycemic and Pancreatic Protective Effect of Squalene in Streptozotocin-induced Diabetic Rat.

DOI: 10.5220/0010045204830486

In Proceedings of the 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and Technology (ICEST 2018), pages 483-486

ISBN: 978-989-758-496-1

483

2.3 Diabetes Induction in Experimental

Diabetic Rats

STZ (55 mg/kg, prepared in a 0.9% NaCl solution)

was injected intra-peritoneally to 16-hrs-fasted rats. ,

administered at single dose of 120 mg/kg body weight

intraperitoneally. Diabetes was confirmed by

determining the blood glucose concentration using

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

injection. The rats that had BGL above 200 mg/dl

were included for the study (Yusoff et al, 2017).

2.4 Experimental Set up

Diabetic rats were divided randomly into three groups

(n=5). The first group (Sq) was administered Sq (160

mg/kg). The second group (M) was given metformin

(500 mg/kg) to serve as the positive control. The third

group (DC) was treated with normal saline (10 ml/kg)

and served as the negative control. Normal control

(NC) rats were included to the study that received

normal saline (10 ml/kg). All treatments were

dissolved in NaCL 0.9% and tween 80 5% before

administration.

2.5 Preparation Pancreatic for

Histopathological Analysis

The 14-days treated diabetic rats were sacrified with

the carbogen gas (95% O2 and 5% O2) and the

pancreas was excised for histological studies. The

pancreas was fixed in 10% buffered formaldehyde for

24 hours, followed by dehydration using 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 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 5 μm 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.

2.6 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.

2.7 Statistical Analysis

Data was expressed as mean ± standard error of the

mean (S.E.M). The results were analysed using

Kruskall Wallis

followed by Mann-Whitney. p <0.5 was

considered significant.

3 RESULT AND DISCUSSIONS

3.1 Blood Gucose Level (BGL)

The effect of once daily oral administration of Sq on

the BGL is presented in Table 1. Administration of Sq

(160 mg/kg) showed significant blood glucose

reduction at day6 to day14 (p<0.05-0.01) compared

to DC-treated group. Metformin as the positive

control decreased the BGL significantly at day6 to

day14 as well (p<0.01). However, only NC-treated

groups showed significant different from day3-day14

(p<0.01).

Table 1. Effect of 14 days daily oral administration of

Squalene on the blood glucose level of SDR

BGL

(mg/dL)

Group

Sq M NC DC

67.2  1.4* 75  2* 80  4.8 80.8  2.2

Day0

327  9** 354.8  24** 79  5.1** 249.6  14.6

Day3

280.4  10.8 271.8  15.9 81.2  3.7** 284.4  8.5

Day6

241.4  16.8* 181.8  24.7** 81.4  3.9** 293.6  17.6

Day9

184  11.7* 144.2  14.2** 79.4  3.4** 300.2  10.9

Day12

136.2  9.2* 108.4  8.2** 81  4.1** 307  20

Day14

85.2  1.6** 80.4  5.7** 78.4  4** 318.4  26

Data is expressed as mean  S.E.M, n=5.*p<0.05, **p<0.01, ***p<0.001versus DC.

Sq has been reported in several studies as one of

compounds found in the plants that have

antihyperglycemic activity (Baskar et al, 2011;

Jananie et al, 2011; Widyawati, 2015b; Ragasa et al,

2014). The present study support the previous study

that showed dose-dependently antihyperglycemic

activity of Sq (Widyawati, 2015b). However, the

present study evaluate the effect after 14 days

treatment that longer than the previous study.

3.2 Evaluation of the islets of

Langerhans (iL)

Figure 1 shows a digital visualization of H & E

staining of normal pancreas rat. The islet of

Langerhans (IL) was clearly distinguished from the

surrounding exocrine tissue by a continuous

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

Technology

484

conncetive tissue capsule. Exocrine area is the area

outside of the IL. The islet cells were appeared

rounded with prominent blue-black nuclei and

various light pink cytoplasma. The figure 1 also

shows the IL with granulated cytoplasm of islet cell

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

and with light and large nuclei (beta-cells), and

pancreatic acinus (PA).

Figure 1. Histological appearance with H&E staining of the

islet of Langerhans of normal rats (40x10 magnification).

*alpha-cells (arrow), beta-cells (double arrow), pancreatic

acinus (PA)

Figure 2 shows a digital visualization of H & E

staining of DC-treated pancreas rat. The figure shows

that the size of IL of a diabetic rat decreased of size

and the outline was irregular and shrinked.

Figure 2. Histological appearance with H & E staining of

the islet of Langerhans of diabetic control rats (40x10

magnification).

*alpha-cells (arrow), beta-cells (double arrow), pancreatic

acinus (PA)

Figure 3 shows a digital visualization of H & E

staining of Sq-treated pancreas rat. The figure shows

the nearly regular outline of an IL with apparently

normal appearance of most cells.

Figure 3. Histological appearance with H&E staining of the

islet of Langerhans of SQ-treated rats (40x10

magnification).

*alpha-cells (arrow), beta-cells (double arrow), pancreatic

acinus (PA)

H & E staining at the present study revealed

significant changes in the general histological

organisation of the pancreatic tissue between NC and

DC (Figure 1 & 2). The STZ-demonstrated its ability

to destroy the structure of pancreatic IL following

STZ administration. The IL clearly seen in both

normal and diabetic rats. It was due to the

surrounding exocrine cells that were not affected by

the induction (Razak et al., 2010). In NC, the IL

featured circular shape with regular cell lining and no

degenerated cells were observed. This appearance

support the previous reported studies by Juarez et al.,

(2012) and Andrade-Cetto et al., (2008). On the other

hand, DC showed a different histological changes of

IL. The IL showed a shrunk with degeneration of islet

cells. Treatment with Sq at the dose of 160 mg/kg

restored the histological appearance of the IL as the

outline and most of cells appeared nearly regular and

normal.

4 CONCLUSIONS

Squalene have anti-hyperglycemic activity and

pancreatic protective effect in STZ-induced diabetic

rats.

Conflict of Interest Statement

The authors declare that there are no conflicts of

interest.

Antihyperglycemic and Pancreatic Protective Effect of Squalene in Streptozotocin-induced Diabetic Rat

485

REFERENCES

Andrade-Cetto, A., Becerra-Jiménez, J., Cárdenas-

Vázquez, R., 2008. Alfa-glucosidase-inhibiting activity

of some Mexican plants used in the treatment of type 2

diabetes. Journal of ethnopharmacology, 116(1): 27-32.

Bhaskar, A., Nithya, V., Vidhya., V.G., 2011.

Phytochemical evaluation by GC-MS and

antihyperglycemic activity of Mucuna pruriens on

streptozotocin-induced diabetes in rats. Journal of

Chemical and Pharmaceutical Research, 3: 689-696.

Janani, R.K., Priya, V., Vijayalakshmi, K., 2011.

Phytoconstituents evalauation by GC-MS and anti-

hyperglycemic activity of Cynodon dactylon on

streptozotocin-induced diabetes in rats. Journal of

Chemical and Pharaceutical Research, 3 (4): 460-466.

Juárez-Rojop, I. E., Díaz-Zagoya, J. C., Ble-Castillo, J. L.,

Miranda-Osorio, P. H., Castell-Rodríguez, A. E.,

Tovilla-Zárate, C. A., Bermúdez-Ocaña, D. Y., 2012.

Hypoglycemic effect of Carica papaya leaves in

streptozotocin-induced diabetic rats. BMC

Complementary and Alternative Medicine, 12(1): 236.

Razak, A., Mariam, A., Amirin, S., Mohd Zaini, A., 2010.

Assessment on functionality and viability of β cells

following repetitive dosage administration of ethanolic

extracts of Andrographis paniculata on streptozotocin-

induced diabetic rats. International Medical Journal,

9(1): 21-26.

Ragasa, C. Y., Torres, O. B., Shen, C. C., Lachica, M. K. E.

G., Sulit, A. B., Chua, D. B. D. L., Raga, D. D., 2014.

Triterpenes from the Leaves of Syzygium

polycephalum, S. cumini, and S. samarangense.

Chemistry of Natural Compounds, 50(5): 942-944.

Ramadan, B. K., Schaalan, M. F., Tolba, A. M., 2017.

Hypoglycemic and pancreatic protective effects of

Portulaca oleracea extract in alloxan induced diabetic

rats. BMC complementary and alternative medicine,

17(1): 37.

Widyawati, T., Yusoff, N. A., Asmawi, M. Z., Ahmad, M.,

2015. Antihyperglycemic effect of methanol extract of

Syzygium polyanthum (Wight.) leaf in streptozotocin-

induced diabetic rats. Nutrients, 7(9): 7764-7780.

Widyawati, T., 2015. Anti-diabetic activity-guided studies

of Syzygium polyanthum (Wight) leaf extracts and

elucidation of their mechanisms of cction (Doctoral

dissertation, Universiti Sains Malaysia).

Yusoff, N. A., Lim, V., Al-Hindi, B., Abdul Razak, K. N.,

Widyawati, T., Anggraini, D. R., Asmawi, M. Z., 2017.

Nypa fruticans Wurmb. Vinegar’s aqueous extract

stimulates insulin secretion and exerts hepatoprotective

effect on STZ-induced diabetic rats. Nutrients, 9(9):

925.

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

Technology

486