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 in
Institute of Sciences (LIPI), developed and developing countries. Kalanchoe pinnata has
Serpong,Tangerang Selatan, been used as a traditional medicine to treat diabetes. We try to
Indonesia find scientific evidence of antidiabetic activity of Kalanchoe
pinnata extract (KPE) through hypoglycemic effect using animal
Submitted: 13-5-2016
model of diabetes mellitus. Hyperglycaemia was developed in
Revised: 25-07-2016
rats using alloxan 150mg/kgBW. Three days after alloxan
Accepted: 11-09-2016
injection, rats having fasting blood glucose (FBG) >200mg/dL
*Corresponding author were divided into six groups, namely HG (hyperglycaemia),
Tri Yuliani HG+KPE high-dose (hyperglycaemia+KPE 33.2mg/kg), HG+KPE
medium-dose (hyperglycaemia+KPE 11.6mg/kg), HG+KPE
Email: low-dose (hyperglycaemia+KPE 5.8mg/kg), standard drug 1
anik_baik @yahoo.com (hyperglycaemia+glibenclamidee 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 dependent DM. In the other hand, type

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

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release independent of glucose level. However, Induction of diabetes

this effect is followed by suppression of the islet Induction of diabetes was performed
respons to even high level of glucose leading to according to Misra and Aiman (2012), with
hyperglycaemia state (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 4°C)
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 α- 10% glucose after alloxan administration. After
glucosidase with IC50 16.12ppm. Meanwhile, 72h of alloxan injection (t0), FBG was
radical scavenger activity using DPPH method determined using GlucoDr glucometer strips.
revealed IC50 29.61ppm of ethanol fraction, Animals with FBG >200mg/dL were
23.15ppm of buthanol fraction, and 17.27 ppm considered to have developed experimental
of ethyl acetic fraction (Indah et al., 2013). diabetes as shown by hyperglycaemia state.
Therefore, current study aims to Then, FBG was measured every 5 days
determine antidiabetic activity of ethanolic recorded as t1, t2, and t3 to determine
extract of Kalanchoe pinnata in alloxan-induced fluctuations in FBG. Blood was collected from
diabetic rats. tail vein. As standard reference, Glibenclamide
was given at a dose of 1.35mg/kg orally per
MATERIALS AND METHODS day, while Acarbose was given at a dose of 13.5
Kalanchoe pinnata leaves were collected mg/kg orally per day.
from Kawasan Puspiptek Serpong and were
authenticated by Research Center for Biology, Animal experimentation
Indonesian Instititute of Sciences (LIPI). In the present study the animals
were distributed into 7 groups (n=5) as
Preparation of plant extracts follows: normal control, HG (hyperglycaemia),
The leaves were dried using blower oven HG+KPE high-dose (hyperglycaemia+
<50°C for 24h, crushed, and extracted using KPE 33.2mg/kg), HG+KPE medium-dose
70% ethanol (3x24h maseration). Then, ethanol (hyper-glycaemia+KPE11.6mg/kg),HG+KPE
extracts obtained were concentrated in rotary low-dose (hyperglycaemia+KPE 5.8mg/kg),
evaporator under vacuum. The yield of standard drug 1 (hyperglycaemia+ gliben-
ethanolic extract of Kalanchoe pinnata (KP) dry clamidee 1.35mg/kg), standard drug 2
leaves was 15.7%. (hyperglycaemia+acarbose 13.5mg/kg). The
study was conducted for 15 days to evaluate the
Animals potential of the extracts to lower FBG level.
The Male Sprague Dawley rats (150- Body weights of the rats were monitored
350g) were procured from Fakultas Kedokteran weekly during the study period.
Hewan IPB, Bogor and housed under standard
conditions of temperature and relative humidity Pancreas histological observation
with 12h light/dark cycle. Animals were fed on Pancreas histological observation was
standard commercial pellet diet and water ad conducted on day 15, at the end of the
libitum. The ethical clearance of the expreriment experimental period. Rats were euthanized by
have been approved by Health Research Ethics ether and the pancreas were collected and fixed
Commitee, University of Indonesia and Cipto with bouin’s solution for 24h. The pancreas
Mangunkusumo Hospital, Indonesia (Ethical was then stained with Hematoxylin eosin (HE)
clearance certificate number: 663/UN2.F1/ for observation of number of beta cell
ETIK/2016). Langerhans.

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Tabel I. Effect of KPE on FBG during study

FBG± SD (mg/L)
Group
t (-1) to t1 t2 t3
Normal 103.67±15.33 117.92±8.07 107.83±7.15 97.42±13.75 93.5±8.24
HG 115±2.45 429±106.88 267±106.10 207±104.24 130±16.85
HG+KPE (high dose) 112.2±12.76 384±137.48 174.6±111.84 111.2±24.22 116±13.93
HG+KPE (medium dose) 112±10.05 259.4±48.53 134.4±30.00 115.8±17.88 116.2±10.40
HG+KPE (low dose) 112.2±6.57 353.8±163.69 142.4±35.67 113.6±16.36 124.6±10.26
HG+ Acarbose 109.5±16.99 262±36.79 121.83±22.25 105.67±14.51 97±8.65

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, except KPE
high-dose, improved the morphology of β 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 β 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).
increase was not significant in HG+KPE Therefore, several study have been
medium dose group and acarbose group conducted to evaluate its bioactivity, such as
compare to normal group. Meanwhile, no antidiabetics, anticonvulsant, antinociceptive,
significant difference was observed among antiedematogenic, antiinflammatory, anticancer
groups that received alloxan injection. and antiHPV, antileishmanial (Patil et al.,
FBG reduction were detected after 5 2013; Mora-Perez et al., 2016; Ferreira et al.,
days (t1) alloxan injection in all groups. 2014; Mahata et al., 2012; Muzitano et al.,
While 5 days treatment brought FBG to a 2006).
level < 200mg/dL in all groups, HG groups K. pinnata leaves contain anthocyanins
remain suffered from hyperglycaemia as that considered responsible for its antidiabetic,
shown by FBG >200mg/dL. As a result, anticancer, cardiovascular, and neurological
only HG group differs significantly from activity (Cruz et al., 2012). Anthocyanins are
normal group. In the other hand, all treatment water soluble flavonoid compounds that gives
reduced FBG significantly, but not high-dose color in plant (Cruz et al., 2012).
KPE. Phytochemistry study of the KPE used in this
Ten days after treatment (t2), all rats’ study revealed flavonoids and steroids
FBG kept decreasing. However, HG group compound and isolated quercetin glycosides
remain in hyperglycaemia state as shown by (Fajriyah, 2011).
FBG >200mg/dL. As a result, only HG group We suggest that the bioactivity of
differs significantly from normal group and all K. pinnata in reducing FBG and improving
treatments reduced FBG significantly. morphology of the islets of Langerhans and β
Fifteen days after treatment, all rats’ cells were caused by its various natural
FBG went back to normal as shown by compounds constituent, for example quercetin
FBG <200mg/dL. However, only standard through antioxidant mechanism (Figure 3).

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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 Beta cell Langerhans stained with Hematoxylin eosin (HE).

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It is worth noting that oxidative stress plays a Camb. and Kalanchoe pinnata (Lamk.)
role in the pathogenesis of diabetes mellitus. Pers, Anais da Academia Brasileira de
Hyperglycaemia was characterized with increase Ciências. 84(1): 211-217
oxidative stress leading to defect in insulin Domingueti CP., Dusse LM., Carvalho MD., de
action and insulin secretion. Quercetin as Sousa LP., Gomes KB., Fernandes AP.
antioxidants reduced oxidative stress leading to 2015. Diabetes mellitus: The linkage
protection of β cells of the pancreas resulted in between oxidative stress, inflammation,
the increase of insulin production and hypercoagulability and vascular
decreased of FBG. Several studies reported complications. J Diabetes Complications.
quercetin mechanism of action in diabetes such Dec 18.
as decreases lipid peroxidation, increases Ferreira RT., Coutinho MAS., CarmoMalvar
antioxidant enzymes activity, inhibition of D., Costa EA., Florentino IF., Costa SS.,
insulin-dependent activation of Vanderlinde FA. 2014. Mechanisms
phosphoinositol-3-kinase (PI-3K), reduces Underlying the Antinociceptive,
intestinal glucose absorption by inhibiting Antiedematogenic, and Anti-
GLUT (Sunarwidhi et al., 2014). Inflammatory Activity of the Main
Based on pharmacological and Flavonoid from Kalanchoe
histopathological studies, we suggested that the pinnata.Evidence-Based Complementary
hypoglycemic effect of KPE was optimum at and Alternative Medicine: 429256.
medium dose (11.6mg/kg bw). Global status report on noncommunicable
However, it is difficult to conclude that diseases. 2014. Geneva, World Health
hypoglycemic activity of KPE in this study is Organization.
only caused by quercetin. Various active Hossain MK., Dayem AA., Han J., Saha SK.,
compounds in KPE may synergistically increase Yang G., Choi HY., Cho S. 2016. Recent
hypoglycemic effect. Advances in Disease Modeling and Drug
Therefore, the mechanism action of each Discovery for Diabetes Mellitus Using
active compounds in the extract needs further Induced Pluripotent Stem Cells. Int. J.
investigation, including immunohistochemical Mol. Sci. 17: 256.
observation on pancreatic insulin 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 Langerhans islet Kementrian Kesehatan RI. Badan Penelitian
and β cells. KPE is potential to be developed as dan Pengembangan. Riskesdas. 2013.
a blood glucose-lowering agent for diabetic Jakarta.
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
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