Puguntano Extract Supplementation Enhances Insulin Secretion and

Decreases Insulin Resistance in High Fat

Diet/Streptozotocin-induced Type 2 Diabetic Rats

Santi Syafril

, Dharma Lindarto

Department of Internal Medicine, Faculty of Medicine, Universitas Sumatera Utara-H. Adam Malik General Hospital,

Medan, Indonesia

Santi Syafril. Department of Internal Medicine, Faculty of Medicine, Universitas Sumatera Utara-H. Adam Malik Hospital,

Taman Setiabudi Indah Blok L no.18 Medan, 20122, Indonesia.

Keywords: Puguntano, HOMA-, HOMA-IR, T2DM

Abstract: BACKGROUND: Secondary metabolites of puguntano (Curangafel-terrae Merr.) which contains

flavonoids, saponins, tannins, and steroids/ terpenoids, increased insulin secretion and decreased insulin

resistance in type 2 diabetes mellitus (T2DM). AIM: This study investigated the effect of puguntano leaf

extract to insulin secretion and insulin resistance in high-fat diet/streptozotocin-induced type 2 diabetic rats.

METHODS: A combination of high-fat diet-feeding (HFD) and multiple low dose intraperitoneal injections

of streptozotocin(STZ) was used to induced T2DM in 48 male Wistar rats, which were then randomly

divided into control and treatment groups (n = 24 per group). Puguntano leaf extract was administered to the

treatment group once daily (200 mg/kg.bw) for 10 days. Insulin secretion (HOMA-) and insulin resistance

(HOMA-IR) were measured from fasting glucose and fasting serum insulin concentration by homeostasis

model assessment (HOMA) in control group after becoming T2DM and in the treatment group after 10 days

of puguntano treatment. Data were analyzed using the Wilcoxon test. RESULTS: HOMA- was

significantly higher in the treatment group than in the control group, while HOMA-IR was significantly

lower in the treatment group than in the control group (p<0.05). CONCLUSION: This study demonstrated

that puguntano leaf extract treatment may significantly enhance insulin secretion and decreased insulin

resistance in T2DM rats.

1 INTRODUCTION

Impaired insulin secretion from β-cells of the

pancreas, resistance to tissue actions of insulin, or a

combination of both is the pathophysiology of type 2

diabetes mellitus (T2DM). The spectrum of the

disease originally is arising from tissue insulin

resistance and gradually progressing to a state

characterized by complete loss of secretory activity

of the beta cells of the pancreas (Chaudhury et al.,

2017). In T2DM, both its action and secretion are

impaired (Irsad et al., 2011). Homeostatic model

assessment (HOMA) is a method for assessing  cell

function (HOMA-) and insulin resistance (HOMA-

IR) from basal (fasting) glucose and insulin

concentrations (Wallace et al., 2004)

The incidence of diabetes is alarmingly

increasing throughout the world. One of the worst

affected areas appears to be Asia where diabetes

could rise two- to three-fold in the near future. It is

crucial to identify effective and low-cost

medications for treating diabetes considering the

economic constraints. Alternative strategies to the

current pharmacotherapy of diabetes such as herbal

medicine are urgently needed because of the

enormous cost and limited access to modern

therapies for many rural populations in developing

countries(Hafizur et al., 2012)

Puguntano (Curangafel-terrae Merr.) is a plant

belonging to the Scrophulariaceae family found in

TigaLingga village, Dairi regency, Sumatera Utara

province, Indonesia (Sitorus et al., 2014). Puguntano

traditionally has been used as a drug of colic,

malaria, diuretic, fever, and skin disease. Modern

pharmacological investigations indicated that the

extract of puguntano exerts diuretic, antipyretic,

hepatoprotective, cardioprotective, antidiabetic,

Syafril, S. and Lindarto, D.

Puguntano Extract Supplementation Enhances Insulin Secretion and Decreases Insulin Resistance in High Fat Diet/Streptozotocin- Induced Type 2 Diabetic Rats.

DOI: 10.5220/0009862001370141

In Proceedings of the 2nd International Conference on Tropical Medicine and Infectious Disease (ICTROMI 2019), pages 137-141

ISBN: 978-989-758-469-5

137

antioxidant, anti-inflammatory, anthelmintic,

antiasthma, and analgesic activities (Sitorus et al.,

2017).

Phytochemical analysis of puguntano showed

that the major phytochemicals are flavonoids,

saponins, tannins, and steroids/terpenoids, which

have anti-diabetic activity (Harahap et al., 2013)..

Tannins decrease insulin resistance through

enhancing the glucose uptake via mediators of the

insulin-signaling pathways (Kumari et al., 2012).

and enhance insulin secretion through inducing β-

cell regeneration (Kim et al., 2017). Flavonoids

enhance insulin secretion by preventing β-cell

apoptosis and promoting β-cell proliferation and

enhance insulin activity (Vinagayan et al., 2015).

Terpenoidsdecrease insulin resistance through

increasing glucose transporter-4 (GLUT-4)

expression and translocation viaproliferator-

activated receptor gamma (PPAR- Υ) activation

(Goto et al., 2010;Song et al., 2004;Monsalve et al.,

2013). Saponindecrease insulin resistance through

increasing insulin signaling via increasing GLUT-4

expression and increase insulin secretion from the

beta cell islets (Elbarky et al., 2017).

Lindarto et al., reported that insulin resistance is

decreased in newly diagnosed T2DM patients after

treatment with puguntano leaf extract for 12 weeks,

showed by the significant a significant decrease in

fasting blood glucose (FBG) levels and HOMA-IR,

a significant increase in adiponectin levels, while

there is the increase in HOMA- but is not

statistically significant (Lindarto et al., 2016).

In this study, we investigate the effect of

puguntano leaf extract to insulin secretion and

insulin resistance in high-fat diet/streptozotocin-

induced type 2 diabetic rats.

2 MATERIAL AND METHODS

Male Wistar rats (180–200 g) were used throughout

the study. The rats were kept and maintained in

laboratory experimental animal units under standard

conditions of temperature and humidity with a 12h

light:12 h darkness cycle. The rats were maintained

in clean cages with ad libitum access to water and

food. All animals were treated according to the

guideline for care and use of laboratory animals with

the approval of the Ethics Committee of Universitas

Sumatera Utara, Medan, Indonesia (Reference

42/TGL/KPEK FK USU-RSUP HAM/2018). A

high-fat diet (HFD) feeding for 5 weeks, followed

by two intraperitoneal injections of streptozotocin

(STZ) (30 mg/kg; Sigma-Aldrich, Munich,

Germany) was given to induced T2DM. After this,

FBG levels were determined in blood obtained from

a lateral tail vein using a glucometer (Roche

Diagnostics). The rats with FBG levels of more than

200 MD/dL were used for this study (Zhang et al.,

2008)

Forty-eight diabetic rats were randomly divided

into two groups (a control group and a treatment

group) of twenty-four rats each. The treatment group

was treated with 200 mg/kg/day ethanolic extract of

puguntano leaves using an orogastric cannula for 10

days. Control rats were sacrificed on the day their

diabetes was confirmed, while the puguntano-treated

rats were sacrificed after 10 days treatment period

was complete.

After anesthesia with ketamine, the rats were

decapitated, and blood was obtained from the left

ventricle for the measurement of FBG by

spectrophotometry and fasting insulin using a

sandwich ELISA. Insulin secretion was assessed

using HOMA- equation and insulin resistance was

assessed using HOMA-IR equation, which is

calculated using fasting insulin and glucose

concentrations. The HOMA-IR and HOMA-β were

calculated using the formula given below [3]:

HOMA-IR= [(fasting plasma insulin in μU/L) x

FBG in mg/dL)/405]

HOMA-β = [(360 x fasting plasma insulin in

μU/L) / (FBG in mg/dL - 63)].

The study was conducted in the Molecular Genetics

Laboratory, Medical Faculty of Universitas

Padjajaran. The ethanolic extract of puguntano

leaves was obtained by maceration methods in the

Department of Biological Pharmacy, Faculty of

Pharmacy, Universitas Sumatera Utara, Medan,

Indonesia (Kemenkes RI,2013)

2.1 Statistical Analysis

Statistical analyses were performed by using the

SPSS 22.0. All values were expressed as the median

(minimum-maximum). Statistical difference

between the groups was assessed by the Wilcoxon

test. P values <0.05 were considered significant.

3 RESULTS

As shown in Table 1, FBG, fasting insulin was

significantly lower in the treatment group than in the

control group.

ICTROMI 2019 - The 2nd International Conference on Tropical Medicine and Infectious Disease

138

Table 1. FBG and fasting insulin in the control and

treatment group

Group

Control

(n=24)

Med (min-

max

Treatment

(n=24)

Med (min-max)

FBG

(mg/dl)

384

(207-490)

122

(95-213)

0.001*

Fasting

insulin

(lU/ml

)

56.56

(49.63 –

73.67)

51.31

( 47.77-59.00)

0.001*

Wilcoxontest.

Significantif p < 0.05

Figure 1.HOMA-β in the control and treatment group

Figure

As shown in Figure 1, HOMA- β was significantly

higher in the treatment group than in the control

group, while HOMA-IR was significantly lower in

the treatment group than in the control group.

4 DISCUSSION

An HFD combined with multiple low doses of

streptozotocin with the dose 30 mg/kg at weekly

intervals for 2 weeks, proved to be a better way for

developing an animal model of T2DM which closely

simulates the natural pathogenesis of T2DM and is

widely used in studies of the efficacy of anti-diabetic

drugs. HFD is a better way to initiate the insulin

resistance which is one of the important features of

T2DM and the low-dose STZ has been known to

induce a mild impairment of insulin secretion which

is similar to the feature of the later stage of T2DM.

Feeding the animal with HFD following low-dose

STZ would closely mimic the natural history of the

disease events (from insulin resistance to β cell

dysfunction) as well as metabolic characteristics of

human T2DM (Okita et al., 2013)

The secondary metabolites of puguntano leaf

have the anti-diabetic activity, which tannins

decrease insulin resistance and enhance insulin

secretion, flavonoids enhance insulin secretion and

enhance insulin activity, terpenoids decrease insulin

resistance and saponin decrease insulin resistance

and increase insulin secretion from the beta cell

islets. Because of these in vitro results, we were

tempted to investigate the scientific basis of the use

of puguntano leaf for the management of T2DM by

the TigaLingga people. This was performed by

looking at the antidiabetic activity of puguntano leaf

extract on HFD/STZ-induced T2DM rats.

The two main causes of hyperglycemia in T2DM

are impaired insulin secretion and increased insulin

resistance. Evaluation of insulin resistance (or

sensitivity) and β-cell function is important for

understanding the disease status and selection of

pharmacologic treatment (Akbarzaedah et al., 2018)

The insulin resistance was calculated using the

HOMA-IR and the steady state of pancreatic β-cell

function was measured by calculating the HOMA-

β(Sumantri et al., 2017).

This present study has demonstrated significantly

lower FBG, fasting insulin levels, and HOMA-IR,

and significantly higher HOMA-β in the treatment

group with puguntano leaf extract than in the control

group. This may be explained by an effect of one or

more secondary metabolites the tannins, flavonoids,

triterpenoids, and saponins present in puguntano leaf

extract to insulin secretion and insulin sensitivity.

A previous study showed that suspension of

puguntano extract of 100 mg/kg bb decreased blood

glucose level similar to the 10 mg/kg

bwglibenclamide suspension (Thakare et al., 2017).

Glibenclamide is a second generation of

sulfonylureas with the mechanism of action to

increase insulin secretion primarily augment the 2nd

phase of insulin secretion with little effect on the 1st

phase . Another study reported that after 12 weeks

0,5

1,5

2,5

Control Treatment

HOMA‐IR

Control

Treatment

Puguntano Extract Supplementation Enhances Insulin Secretion and Decreases Insulin Resistance in High Fat Diet/Streptozotocin- Induced

Type 2 Diabetic Rats

139

treatment with puguntano leaf extract in newly

diagnosed T2DM patients, there was the decrease of

insulin resistance as shown as the significant

reduction in FBG levels and HOMA-IR (p=0.012,

p=0.033; respectively) but there was no significant

increase of insulin secretion (HOMA-β)(p=0.262)

(Lindart et al., 2016).

This study is the first study to evaluate the effect

of puguntano leaf extract (Turanga feel- terrae

Merr.) on insulin secretion and insulin resistance in

such a rat model of T2DM.

In conclusion, this study revealed, for the first

time, the antidiabetic activity of puguntano leaf

extract to insulin secretion and insulin resistance in

an animal model of T2DM. Our data strongly

suggest that the antidiabetic activity is due to

enhanced insulin secretion and improvement in

insulin resistance.

ACKNOWLEDGMENT

The authors acknowledge the assistance of the

Molecular Genetics Laboratory, Faculty of

Medicine, UniversitasPadjajaran, Bandung

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Type 2 Diabetic Rats

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