Indonesian J. Pharm. Vol. 27 No.

3 : 139 144
ISSN-p : 2338-9427
DOI: 10.14499/indonesianjpharm27iss3pp139
Research Article

ANTIDIABETIC ACTIVITY OF ETHANOLIC EXTRACT OF

Kalanchoe pinnata LEAVES IN ALLOXAN INDUCED
HYPERGLYCAEMIC RATS
Tri Yuliani, Indah D. Dewijanti, Sofna D.S. Banjarnahor

Research Center for ABSTRACT

Chemistry, Indonesian Diabetes remains a major burden in health care both
Institute of Sciences (LIPI), in developed and developing countries. Kalanchoe pinnata
Kawasan PUSPITEK, Muncul, has been used as a traditional medicine to treat diabetes. The
Serpong, Kota Tangerang aim of this study to find scientific evidence of antidiabetic
Selatan, Banten 15314 activity of Kalanchoe pinnata extract (KPE) through hypoglycemic
effect using animal model of diabetes mellitus. Hyperglycaemia
Submitted: 13-5-2016
was developed in rats using alloxan 150mg/kgBW. Three
Revised: 25-07-2016
Accepted: 11-09-2016 days after alloxan injection, rats having fasting blood glucose
(FBG) >200mg/dL were divided into six groups, namely
*Corresponding author HG (hyperglycaemia), HG+KPE high-dose (hyperglycaemia+
Tri Yuliani KPE 33.2mg/kg), HG+KPE medium-dose (hyperglycaemia+
KPE 11.6mg/kg), HG+KPE low-dose (hyperglycaemia+
Email: KPE 5.8mg/kg), standard drug 1 (hyperglycaemia+glibenclamide
[email protected] 1.35mg/kg), standard drug 2 (hyperglycaemia+acarbose
13.5mg/kg). Then, FBG was measured every 5 days recorded as
t1, t2, and t3 to determine fluctuations in blood glucose. At the
end of the study, rats were sacrified, pancreas was collected and
number of pancreatic beta cell langerhans was determined. KPE
11.6mg/kg showed best hypoglycemic effect and improvement of
the number of pancreatic beta cell langerhans. KPE has
hypoglycemic effect through improvement of the number of
pancreatic beta cell langerhans but not in dose dependent
manner.

Key words: Antidiabetic, Kalanchoe pinnata, hyperglycaemic, alloxan,

pancreatic

INTRODUCTION insulin production. This condition leads to

WHO (2015) reported 9% people aged insulin dependent DM. In the other hand, type
18+ worldwide suffered from diabetes, while in 2 DM was caused by inadequate action of
Indonesia the prevalence was only 1.5% insulin in action site (Hossain et al., 2016).
(Kemenkes RI, 2013). It is worth noticing that Pharmacotheraphy in DM patient does
this disease is a burden of health care in both not eliminate the cause of the disease but its
developed and developing countries (Abdulazeez targets on the symptom using insulin and
et al., 2013). Diabetes develops from prolong hypoglicemic agent such as sulfonylurea and
glucose imbalance between intracellular and biguanids (Abdulazeez et al., 2013; Verma et al,
extracellular resulted in hyperglycaemia state in 2015). However, sulfonylurea exert several
which blood glucose can not be utilized by adverse effect such as hypoglicemia and weight
living cells. These conditions lead to vascular gain, in addition to toxic effect on liver and
complication through increasing oxidative stress renal, while metformin cause lactic acidosis
and inflammation. Vascular complication is the (Verma et al., 2015).
leading cause of death in both diabetes type Alloxan is widely used in experimental
1and type 2 patient (Domingueti et al., 2015). pharmacology to induce diabetes in rat. The
Diabetes mellitus (DM) are classified as pathological mechanism is by destruction of
two major disease: type 1 DM and type 2 DM. insulin producing pancreatic -cells. Alloxan
Type 1 DM is an autoimmune disease that undergo redox reaction resulted in increase
immune system of the body attack insulin increasing of radical species. In this case,
producing pancreatic cells resulting in low pancreatic -cells is the most sensitive organ

Volume 27 Issue 3 (2016) 139

Tri Yuliani

towards these radical attacks. Hyperglycaemia clearance certificate number: 663/UN2.F1/

state does not occur immediately. At first, ETIK/2016).
alloxan induces insulin release independent of
glucose level. However, this effect is followed by Induction of diabetes
suppression of the islet respons to even high level Induction of diabetes was performed
of glucose leading to hyperglycaemia state according to Misra and Aiman (2012), with
(Szkudelski et al., 2001). modification. Base line FBG was measured
Nowadays, the need for safe and before alloxan injection (t(-1)). Alloxan
effective drugs has brought natural product as monohydrate (Sigma Aldrich; stored at 4C)
the promising source of companion drug for was dissolved in normal saline at room
diabetes (Sharma and Gupta, 2015). Kalanchoe temperature (freshly prepared) and injected to
pinnata has been traditionally used worldwide as 30 overnight fasted male Sprague-Dawley rats
medicinal plants, including to treat diabetes at a dose of 150mg/kg intraperitonially. The
(Patil et al., 2013). Our previous study reveals its animals were then kept for the next 7 days on
antidiabetic activity through inhibition of 10% glucose after alloxan administration. After
-glucosidase with IC50~16.12 ppm. 72h of alloxan injection (t0), FBG was
Meanwhile, radical scavenger activity using determined using GlucoDr glucometer strips.
DPPH method revealed IC50 ~29.61ppm of Animals with FBG >200mg/dL were
ethanol fraction, 23.15 ppm of buthanol considered to have developed experimental
fraction, and 17.27 ppm of ethyl acetic fraction diabetes as shown by hyperglycaemia state.
(Indah et al., 2013). Then, FBG was measured every 5 days
Therefore, current study aims to recorded as t1, t2, and t3 to determine
determine antidiabetic activity of ethanolic fluctuations in FBG. Blood was collected from
extract of Kalanchoe pinnata in alloxan-induced tail vein. As standard reference, Glibenclamide
diabetic rats. was given at a dose of 1.35mg/kg orally per
day, while Acarbose was given at a dose of
MATERIALS AND METHODS 13.5mg/kg orally per day.
Kalanchoe pinnata leaves were collected
from Kawasan Puspiptek Serpong and were Animal experimentation
authenticated by Research Center for Biology, In the present study the animals
Indonesian Instititute of Sciences (LIPI). were distributed into 7 groups (n=5) as
follows:normal control, HG (hyperglycaemia),
Preparation of plant extracts HG+KPE high-dose (hyperglycaemia+
The leaves were dried using blower oven KPE 33.2mg/kg), HG+KPE medium-dose
<50C for 24h, crushed, and extracted using (hyperglycaemia+KPE 11.6mg/kg), HG+KPE
70% ethanol (3x24h maseration). Ethanol low-dose (hyperglycaemia+KPE 5.8mg/kg),
extracts obtained were then concentrated in standard drug 1 (hyperglycaemia+ glibenclamide
rotary evaporator under vacuum. The yield of 1.35mg/kg), standard drug 2 (hypergly-
ethanolic extract of Kalanchoe pinnata (KP) dry caemia+acarbose 13.5mg/kg). The study was
leaves was 15.7%. conducted for 15 days to evaluate the potential
of the extracts to lower FBG level. Body
Animals weights of the rats were monitored weekly
The Male Sprague Dawley rats (150- during the study period.
350g) were procured from Fakultas Kedokteran
Hewan IPB, Bogor and housed under standard Pancreas histological observation
conditions of temperature and relative humidity Pancreas histological observation was
with 12h light/dark cycle. Animals were fed on conducted on day 15, at the end of the
standard commercial pellet diet and water ad experimental period. Rats were euthanized by
libitum. The ethical clearance of the experiment ether and the pancreas were collected and fixed
has been approved by Health Research Ethics with bouins solution for 24h. The pancreas
Commitee, Universitas Indonesia and Cipto was then stained with Hematoxylin eosin (HE) for
Mangunkusumo Hospital, Indonesia (Ethical observation of number of beta cell Langerhans.

140 Volume 27 Issue 3 (2016)

 Antidiabetic Activity

Tabel I. Effect of KPE on FBG during study

FBG SD (mg/L)
Group
t (-1) To t1 t2 t3
Normal 103.6715.33 117.928.07 107.837.15 97.4213.75 93.58.24
HG 1152.45 429106.88 267106.10 207104.24 13016.85
HG+KPE (high dose) 112.212.76 384137.48 174.6111.84 111.224.22 11613.93
HG+KPE (medium dose) 11210.05 259.448.53 134.430.00 115.817.88 116.210.40
HG+KPE (low dose) 112.26.57 353.8163.69 142.435.67 113.616.36 124.610.26
HG+ Acarbose 109.516.99 26236.79 121.8322.25 105.6714.51 978.65

Statistical analysis treatment (glibenclamide or acarbose) reduced

Numeric data were expressed as mean FBG similar to normal while KPE treatment
SD. Data were analyzed using one-way analysis did not reduced FBG significantly.
of variance (ANOVA) (p<0.05) followed by Histological observation using HE
Multiple LSD. staining showed that all treatment, but KPE
high-dose, improved the morphology of cells
RESULTS DISCUSSION in comparison to the HG group based on the
Base line FBG (t(-1)) was determined number of cell (Figure 2). However, only KPE
before alloxan injection. All rats showed medium and low dose increased the number of
normal FBG with no significant difference cells significantly.
among the groups (Table I, Figure 1). K. pinnata has traditionally been used
Three days after alloxan injection (t0), as medicinal plant to treat various ailments
hyperglycaemia state was confirmed by FBG around the world, including diabetes (Patil et al.,
>200mg/dL in all the rats. However, FBG 2013). Therefore, several study have been
increase was not significant in HG+KPE conducted to evaluate its bioactivity, such as
medium dose group and acarbose group antidiabetics, anticonvulsant, antinociceptive,
compared to normal group. Meanwhile, no antiedematogenic, antiinflammatory, anticancer
significantly difference was observed among and antiHPV, antileishmanial (Patil et al.,
groups that received alloxan injection. 2013; Mora-Perez et al., 2016; Ferreira et al.,
FBG reduction were detected after 5 2014; Mahata et al., 2012; Muzitano et al.,
days (t1) alloxan injection in all groups. 2006).
While 5 days treatment brought FBG to a K. pinnata leaves contain anthocyanins
level <200mg/dL in all groups, HG groups that considered responsible for its antidiabetic,
remained suffered from hyperglycaemia as anticancer, cardiovascular, and neurological
shown by FBG >200mg/dL. As a result, activity (Cruz et al., 2012). Anthocyanins are
only HG group differed significantly from water soluble flavonoid compounds that gives
normal group. In the other hand, all treatment color in plant (Cruz et al., 2012).
reduced FBG significantly, but not high-dose Phytochemistry study of the KPE used in this
KPE. study revealed flavonoids and steroids
Ten days after treatment (t2), all rats compound and isolated quercetin glycosides
FBG kept decreasing. However, HG group (Fajriyah, 2011).
remained in hyperglycaemia state as shown by We suggest that the bioactivity of
FBG >200mg/dL. As a result, only HG group K. pinnata in reducing FBG and improving
differed significantly from normal group and all morphology of the islets of Langerhans
treatments reduced FBG significantly. and cells were caused by its various
Fifteen days after treatment, all rats natural compounds constituent, for example
FBG went back to normal as shown by quercetin through antioxidant mechanism
FBG <200mg/dL. However, only standard (Figure 3).

Volume 27 Issue 3 (2016) 141

Tri Yuliani

Figure 1. Effect of KPE treatment on FBG level in alloxan induced rat. HG=Alloxan,
KPE= Kalanchoe pinnata extract, high dose = 33.2mg/kg BW, medium dose= 11.6mg/kg BW,
low dose= 5.8mg/kg BW, Glibenclamide = 1.35mg/kg bw, Acarbose = 13.5mg/kg BW. t(-1)
=base line, t 0= after alloxan injection, t1 or t2 or t3 = 5 or 10 or 15 days after test sample
administration. Data are expressed as mean SD. * & x, p < 0.05 (* indicates comparison between
normal and HG; x indicates comparison between HG and treatments).

Figure 2. Effect of KPE treatment on number of pancreatic beta cell langerhans in alloxan induced
rat. HG=Alloxan, KPE= Kalanchoe pinnata extract, high dose = 33.2 mg/kgbw, medium dose= 11.6
mg/kg bw, low dose= 5.8 mg/kg bw, Glibenclamide = 1.35 mg/kg bw, Acarbose = 13.5 mg/kg
bw. x, p < 0.05. (x indicates comparison between HG and treatments).

Figure 3. Pancreatic cell Langerhans stained with Hematoxylin eosin (HE).

142 Volume 27 Issue 3 (2016)

 Antidiabetic Activity

It is worth noting that oxidative stress plays a anthocyanins in Kalanchoe brasiliensis

role in the pathogenesis of diabetes mellitus. Camb. and Kalanchoe pinnata (Lamk.)
Hyperglycaemia was characterized with increase Pers, Anais da Academia Brasileira de
oxidative stress leading to defect in insulin Cincias. 84(1): 211-217
action and insulin secretion. Quercetin as Domingueti CP., Dusse LM., Carvalho MD., de
antioxidants reduced oxidative stress leading to Sousa LP., Gomes KB., Fernandes AP.
protection of cells of the pancreas resulted in 2015. Diabetes mellitus: The linkage
the increase of insulin production and between oxidative stress, inflammation,
decreased of FBG. Several studies reported hypercoagulability and vascular
quercetin mechanism of action in diabetes such complications. J Diabetes Complications.
as decreases lipid peroxidation, increases Dec 18.
antioxidant enzymes activity, inhibits insulin- Ferreira RT., Coutinho MAS., CarmoMalvar
dependent activation of phosphoinositol-3- D., Costa EA., Florentino IF., Costa SS.,
kinase (PI-3K), and reduces intestinal glucose Vanderlinde FA. 2014. Mechanisms
absorption by inhibiting GLUT (Sunarwidhi et Underlying the Antinociceptive,
al., 2014). Antiedematogenic, and Anti-
Based on pharmacological and Inflammatory Activity of the Main
histopathological studies, we suggested that the Flavonoid from Kalanchoe
hypoglycemic effect of KPE was optimum at pinnata.Evidence-Based Complementary
medium dose (11.6mg/kg bw). and Alternative Medicine: 429256.
However, it is difficult to conclude that Global status report on noncommunicable
hypoglycemic activity of KPE in this study is diseases. 2014. Geneva, World Health
only caused by quercetin. Various active Organization.
compounds in KPE may synergistically increase Hossain MK., Dayem AA., Han J., Saha SK.,
hypoglycemic effect. Yang G., Choi HY., Cho S. 2016. Recent
Therefore, the mechanism of action of Advances in Disease Modeling and Drug
each active compounds in the extract needs Discovery for Diabetes Mellitus Using
further investigation, including immuno Induced Pluripotent Stem Cells. Int. J.
histochemical observation on pancreatic insulin Mol. Sci. 17: 256.
expression. Indah DD., Euis F, Megawati, Tri Y. 2012. The
Antidiabetic Activity of Cocor Bebek
CONCLUSION Leaves (Kalanchoe pinnata Lam.Pers.)
This study showed that KPE possesed Ethanolic Extract from Various Areas.
bioactivity in lowering blood glucose and J.Trop. Life Sciences. 2(2) : 37 39.
improving the morphology of cells of Kementrian Kesehatan RI. Badan Penelitian
Langerhans islet. KPE is potential to be dan Pengembangan. Riskesdas. 2013.
developed as a blood glucose-lowering agent Jakarta.
for diabetic patients. Mahata S., Maru S., Shukla S., Pandey A.,
Mugesh G., Das BC., Bharti AC. 2012.
ACKNOWLEDGMENTS Anticancer property of Bryophyllum
We thank the Program Insentif Riset pinnata (Lam.) Oken. leaf on human
Peneliti dan Perekayasa Tahun 2011 for cervical cancer cells. BMC Complementary
financial support in the study. and Alternative Medicine. 12:15
Misra M. and Aiman U. 2012. Alloxan: An
REFERENCES unpredictable drug for diabetes
Abdulazeez SS. 2013. Diabetes treatment: A induction? Indian J Pharmacol.44(4): 538
rapid review of the current and future 539.
scope of stem cell research. Saudi Pharm. Muzitano MF, Tinoco LW, Guette C, Kaiser
J. 23: 333340. CR, RossiBergmann B, Costa SS. The
Cruz BP., Chedier LM., Peixoto PHP.,Fabri antileishmanial activity assessment of
RL., Pimenta DS. 2012. Effects of light unusual flavonoids from Kalanchoe
intensity on the distribution of

Volume 27 Issue 3 (2016) 143

Tri Yuliani

pinnata. Phytochemistry, 2006; 67: 2071- Ethanolic Extracts Standardized by

2077 Rutin and Quercetin in Alloxan-induced
Patil SB., Dongare VR., Kulkarni CR., Joglekar Hyperglycaemia Rats. Adv Pharm Bull.
MM., Arvindekar AU. 2013 Antidiabetic 4(Suppl 2): 613-618.
activity of Kalanchoe pinnata in Szkudelski T. 2001.The Mechanism of Alloxan
streptozotocin-induced diabetic rats by and Streptozotocin Action in B Cells of
glucose independent insulin secretagogue the Rat Pancreas. Physiol. Res. 50: 536-
action. Pharm Biol. 51(11):1411-8. 546.
Sharma SB., Gupta R. 2015. Drug development Verma RK., Mishra G., Singh P., Jha KK.,
from natural resource: a systematic Khosa RL. 2015. Anti-diabetic activity of
approach. Mini Rev Med Chem. 15(1):52-7. methanolic extract of Alpinia
Sunarwidhi AL., Sudarsono S., Nugroho AE. galanga Linn. aerial parts in
2014. Hypoglycemic Effect of streptozotocin induced diabetic
Combination of Azadirachta indica A. rats. Ayu. 36(1):91-95.
Juss. and Gynura procumbens (Lour.) Merr.

144 Volume 27 Issue 3 (2016)