Antidiabetic Activity Of Diterpene Lactone Fraction Of “Sambiloto”

(Andrographis Paniculata Nees.) On Mice (Mus Musculus) Induced By

Alloxan

Rani Maylana, Wiwied Ekasari, Lusiana Arifianti, Hadi Poerwono, Sukardiman*

Department of Pharmacognosy and Phytochemistry , Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia

Keywords A. paniculata Nees., Diterpene Lactone Fraction, Antidiabetic Activity, in vivo, Alloxan

Abstract Objective: The present study was carried out to evaluate the antidiabetic activity of diterpene lactone

fraction of “Sambiloto” (A. paniculata Nees.) Methods: Antidiabetic was induced experimentally by a

single intraperitoneal administration of 3.8 mg/20 g body weight of Alloxan-monohydrat in each male mice.

Three days after alloxan administration, blood glucose level was measured using a glucometer. Mice with

blood glucose levels above 200 mg/dL were considered diabetic and were used in this study. Then the mice

were randomly divided into five experimental groups of six mice each. First group as positive control

(Glibenclamide 0.06 mg/20 g body weight). Second group as negative control (CMC-Na 0.5%). Samples

were given by oral route at three dosage levels (7.4 mg, 14.9 mg, and 22.3 mg/20 g body weight). After

treatment finished, blood sampling was done by sterilizing the tail with alcohol and then nipping the tail at

the start of the experiment and this was repeated after 2

, 4

, 6

, and 24

h. Results: Results were

expressed as Mean±SEM of the blood glucose levels per number of animals used in every study point. One-

way ANOVA and post-ANOVA (LSD) test were used to compare the means in each group. Conclusion:

Diterpene lactone fraction of “Sambiloto” had antidiabetic activity and the most effective dose was at 14.87

mg/20 g body weight of mice.

1 INTRODUCTION

Diabetes mellitus is one of the common metabolic

disorders with micro-and macrovascular

complications that results in significant morbidity

and mortality. It is considered as one of the five

leading causes of death in the world [1]. In modern

medicine no satisfactory effective therapy is still

available to cure diabetes mellitus. There is

increasing demand by patients to use natural

products with antidiabetic activity due to side effects

associated with the use of insulin and oral

hypoglycemic agents [2].

Andrographis paniculata Nees (Acanthaceae) is

a traditional medicinal herb, grown as shrub in the

moist soil, shady areas of India, China, Indonesia

and throughout Southeast Asia. It has been used as

antihyperglycemic [3,4].

The past research shown that an extract of

“Sambiloto” (A. paniculata Nees.) with the main

content of diterpene lactone was andrographolide

efficacious as antidiabetic drugs [5]. Therefore

further research, antidiabetic activity using diterpene

lactone fraction of “Sambiloto” in mice induced by

alloxan.

2 MATERIALS AND METHODS

2.1. Plant Material

The diterpene lactone fraction of “Sambiloto” got

from Department of Pharmacognosy and

Phytochemistry, Faculty of Pharmacy Airlangga

University Surabaya Indonesia.

2.2. Preparation Diterpene Lactone

Fraction of “Sambiloto”

“Sambiloto” herbs was cutted into small pieces and

dried under shade at room temperature, then blended

up into powder. Then the powder was extracted by

maceration method. It used 2 L of 96% ethanol for

24 h. After extraction, the extract was filtered using

a Buchner funnel to obtain filtrate in the form of

Maylana, R., Ekasari, W., Ariﬁanti, L., Poerwono, H. and Sukardiman, .

Antidiabetic Activity of Diterpene Lactone Fraction of "Sambiloto" (Andrographis paniculata Nees.) on Mice (Mus musculus) Induced by Alloxan.

DOI: 10.5220/0009845800002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

liquid extract. Maceration performed four times. The

filtrate which four times maceration were collected

and evaporated using a rotary evaporator with

temperature not exceeding 50 °C and low pressure ±

2% initial volume. The ethanol extract was

measured as much as 100 mL dissolved in water :

ethyl acetate (1:1) during three times. Then a

solution obtained were separated by a separating

funnel to obtain ethyl acetate fraction at the top and

ethanol-water at the bottom. Ethanol-water fraction

was shaken again with ethyl acetate four times.

Ethyl acetate fraction which collected were dried

with Avicel and Cab-O-Sil (4:1) [3].

2.3. Experimental Design

2.3.1. Antidiabetic of Diterpene Lactone

Fraction from “Sambiloto” by

Alloxan-Induced Diabetic Model

Experimental animals: The study used male

BALB/c mice (8-10 weeks old) that weighed 20-40

g with a mean weight of 30 g. The mice were housed

at a temperature of 25°C with 12 h light/12 h

darkness photoperiod.

Induction of diabetes: Hyperglycemia was

induced experimentally by a single intraperitoneal

administration of 186.9 mg/kg body weight of a

freshly prepared alloxan-monohydrate (2, 4, 5, 6

tetraoxypyrimidine; 5-6-dioxyuracil). Forty-eight h

after alloxan administration, blood glucose level was

measured using a glucometer. Mice with blood

glucose levels above 200 mg/dL were considered

diabetic and used in this study. Prior to initiation of

this experiment, the animals were fasted for 8-12 h

but allowed free access to water until the end of the

experiment [6].

2.3.2. Procedure

Experimental design: The mice were randomly

divided into six experimental groups of five mice

each. These groups included positive control (0.06

mg/20 g body weight glibenclamide), negative

control (0.5% CMC-Na) and three tested dose levels

(7.4 mg, 14.9 mg, and 22.3 mg/20 g body weight)

diterpene lactone fraction of “Sambiloto”. All of

groups were given by oral route.

Blood sampling: Blood sampling was done by

sterilizing tail with alcohol and nipping tail at the

start of the experiment. It was repeated after 2

, 4

and 24

h. Every time the blood glucose levels

were determined with a glucometer [6].

Data management and statistical analysis:

Data was entered in the Microsoft

Excel spread

sheet, cleaned and exported to Statistical Package

for Social Sciences (SPSS Version 17.0) software.

The results were expressed as Mean ± Standard

Error of Mean (SEM) of the blood glucose levels per

number of animals used in every study point. One-

way ANOVA and post-ANOVA (LSD) test were

used to compare the means in each group. p≤0.05

was considered statistically significant.

3. RESULTS AND DISCUSSIONS

Table 1. The effect of Diterpene Lactone Fraction of

“Sambiloto” on Glucose Level in Mice with Alloxan

Induced Diabetes

Mice

Group

Treatment

Blood Glucose Levels (mg/dL)

0 h

2 h

4 h

6 h

Diabeti

control

0,5 %

CMC Na

563 ±

15.5

559

14.0

584

9.99

591

9.33

582

6.8

Diabeti

control

Glibencla

mide (0.06

mg/20 g

body

weight)

597 ±

3.33

466

30.6

326

19.3

247

13.1

334

15.

Diabeti

treated

7.4 mg /20

g body

weight

462 ±

64.7

477

72.3

439

79.3

423

81.7

402

66.

14.9 mg/20

g body

weight

409 ±

52.5

302

57.8

318

42.9

301

55.3

265

48.

22.3 mg/20

g body

weight

431 ±

60.1

356

65.5

388

54.9

368

73.3

321

55.

The comparison between groups was done using one way

ANOVA, Values were in mean ± SEM; Number of

animals in each group = 6

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

Fig. 1 Effect of Diterpene Lactone Fraction of

“Sambiloto” (A. paniculata Nees.) on Glucose Level

The result showed that positive control treated

with glibenclamide suspension was obtained an

average reduction in blood glucose levels of 263 ±

14.3 (44.1%). Negative control, giving a suspension

0.5% CMC Na, didn’t give effect in decreasing

blood glucose levels of -18.7 ± 13.7 (-3.64%) and it

become a baseline of this experiment. Based on

SPSS statistical analysis, it showed that positive

control had a significant difference to negative

control with p calculated value of 0.000 (p <0.05). It

indicated that positive control was able to lower

blood glucose levels significantly over an 24-hour

and antidiabetic test method used in this study was

correct.

The first treatment group that containing 7.4

mg/20 g BW diterpene lactone fraction resulted in

an average reduction in blood glucose levels of 59.7

± 18.2 (14.8%). It had p calculated value at 0.013 (p

<0.05) when compared to negative control using

statistical analysis Anova One Way and Post Hoc

LSD. It showed that group 1 was able to lower blood

glucose levels significantly.

The second treatment group that containing 4.9

mg/20 g BW diterpene lactone fraction resulted in

an average reduction in blood glucose levels of 144

± 34.6 (35.9%). Based on statistical analysis Anova

One Way and Post Hoc LSD result, it had p

calculated value at 0.000 (p <0.05) when compared

to negative control. It showed that group 2 was able

to lower blood glucose levels significantly.

The third treatment group that containing 22.3

mg/20 g BW diterpene lactone fraction resulted in

an average reduction in blood glucose levels of 111

± 15.1 (27.6%). Based on statistical analysis Anova

One Way and Post Hoc LSD, it had p calculated

value at 0.000 (p <0.05) when compared to negative

control. It showed that group 3 was able to lower

blood glucose levels significantly.

Comparising data between groups 1 and 2 was

obtained significantly different with a significance

value of 0.008 (p <0.05), whereas between group 1

and 3 and group 2 and 3 were no significant

differences with a significance value of 0.095 (p>

0.05) and 0.260 (p <0.05), respectively. It can be

concluded that group 2 was the most effective dose

for lowering blood glucose levels.

Comparising data between positive control and

three treatment groups to negative control was

obtained no significant difference (p count <0.05),

which means positive control and three treatment

groups can lower blood glucose levels in mice

significantly for 24 hours. Then three treatment

groups compared to positive control showed

significant difference (p count <0.05) and it had

greater average value of a decreasing blood glucose

levels than positive control. It can be concluded that

the fraction of diterpene lactone (group 1, 2 and 3)

had antidiabetic activity but their decreasing blood

glucose levels were not as big as a positive control.

Thus, the hypothesis in this study accepted that

diterpene lactone fraction can decrease blood

glucose levels. Compound suspected which has

activity lowers blood glucose levels is

andrographolide with triggering mechanism

secretion of insulin so glucose metabolism increases,

it can cause blood glucose levels to decline [7].

The results of this study can be used as scientific

information and supporting data for further studies

in order to develop diterpene lactone fraction as

dosage forms of antidiabetic phytopharmaceutical

drugs or raw materials. It had been found the

effective dose of diterpene lactone fraction that can

be used as traditional or complementary drug

candidate for antidiabetic drugs is safe. Furthermore,

it can be done praformulation study (physical

characteristics, physicochemical characteristics,

biofarmasetika characteristics, compatibility, and

stability) and formulations diterpene lactone fraction

from “sambiloto”.

4 CONCLUSION

Diterpene lactone fraction of “Sambiloto” at 7.4

mg/20 g body weight, 14.9 mg / 20 g body weight,

and 22.3 mg / 20 g body weight had antidiabetic

activity in mice induced by alloxan. The most

effective dose was 14.9 mg/20 g body weight.

Antidiabetic Activity of Diterpene Lactone Fraction of "Sambiloto" (Andrographis paniculata Nees.) on Mice (Mus musculus) Induced by

Alloxan

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