Activity Test of Chloroform Extract of Kenitu Leaves

(Chrysophyllum cainito L.) On Lowering Blood Sugar Levels in White Male Wistar Rat
(Rattus norvegicus L.) Induced with Alloxan
1
* Burhan Ma’arif Z A, 2 Novi Yusro Maulidiyah, and 3Meilina Ratna D.
1
Department of Pharmacy, Faculty of Medical dan Science Healty, Maulana Malik Ibrahim Islamic State
Universtity Malang,
Jl. Gajayana No. 50 Malang 65144

* Corresponding author: noviyusromaulidiyah@gmail.com

Co-author 1, email: burhan.maarif@farmasi.uin-malang.ac.id.com
Co-author 2, email: xxxxxxx@xxxxxxxxxxx

ABSTRACT

Chrysophyllum cainito L. generally known by the people of East Java with the term
kenitu. Kenitu leaf have contain some compounds, there are alkaloid, sterol and triterpenoids.
There are known usefull for lowering blood sugar levels. This purpose research to determine
the activity of extrac choloform leaf kenitu and optimal dose in mice againts the decrease in
blood sugar levels that heve increased blood sugar levels due to alloxan.
This treatment was performed on mice experiments with 5 groups. The treatments
were negative control (induction of alloxan without therapy), positive control (therapy with
metaformine), therapy of kenitu leaf extract with 3 dose variations, that 25, 50, and 75
mg/KgBB. Blood sugar level measurements were performed using the Easy Touch tool.
Statistical analyzes were performed using Normality, Homogenity, Kruskal-willis tests
and Mann-Whitenay tests. The results of the analysis showed that there was a significant
difference in each treatment group, significant differencess were significant between the
negative and positive control groups, 25, 50 and 75 mg/KgBB, that indicated with significant
value p = 0,04. The results showed that chloroform kenitu leaf extract give effect to the
decrease of blood sugar level in rats induced by alloxan. The optimal dose of kenitu leaf
extract to decreassed blood sugar levels was indicated by at a dose of 50 mg/KgBB
experimental animals.
Keywords: Kenitu Leaf (Chrysophyllum cainito L), Blood Sugar Level, Alloxan.

INTRODUCTION
Diabetes Mellitus is a collection of symptoms that arise in a person caused by an increase
in blood glucose levels due to a decrease in progressive insulin secretion from the background of
insulin resistance (1). Disorders of the hormone insulin is the basis of symptoms in diabetes
mellitus. Insulin is produced by pancreatic organs located near the liver and plays a role in
releasing and storing body fuel (2). Diabetes mellitus type 2, in contrast to type 1 diabetes
mellitus, is not problematic with insulin, but with insulin receptors ( 3).
Chrysophyllum cainito L. is commonly known by the people of East Java with the term
kenitu, while in its home region (Central America) is called star apple. This plant is included in the
family Sapotaceae and grow in the area with high rainfall and humid that is at an altitude of 5-1000
meters above sea level. It is a type of tree plant whose height ranges from 10-30 meters, chronically
(perenial). Includes hermaphrodite plants (4).
The plant is nutritious as a medicine such as bark, sap, fruit and seeds. Fresh fruit is
consumed to reduce inflammation in the throat and lungs While infusion of the skin of the
fruit is rich in tannin substances that can be used for tonics, stimulants, diarrhea drugs,
dysentery, stop bleeding, inflammation and gonorhoe drugs. Seeds that taste bitter are used as
a febrifuge, tonic and diuretic by pounding. The sap of a tree in brazil is used to treat an
abscess, while elsewhere it is used as a diuretic, a febrifuge and a drug for dysentery (5). Fruit
has high antioxidant activity, among which are: kenitu produces antioxidant anthocyanins,
and cyanidin-3-O-ß-glucopyranoside (6). Leaves are used as traditional antidiabetic herbs by
the Aboude-Mandeke tribe. It contains alkaloids, sterols or triterpene which play a role in
lowering glucose levels by antioxidant mechanisms (7).
So far, there has been no publication of the leaf antidiabetic activity of kenitu using
chloroform leaf extract of kenitu which can decrease blood sugar level of male white rod
induced wistar strain with alloxan. Based on the above uraiaan conducted a study of white
blood glucose levels of male rats indirectly wistar strains induced with alloxan.

MATERIAL AND METHOD

Materials
The materials used in this research are leaf-simplisia powders obtained from Materia
Medika Stone, alloxan, 0.9% NaCl, chloroform, 70% alcohol, CMC-na, and white rats wistar
strains obtained from the Laboratory of the Faculty of Biology Islamic University Negeri
Malang.

Instrumentation
The instruments used in this study are digital scales (Shimadzu Uni Bloc), Sonica®
Ultrasonic Cleaner, moisture analyzer, rotary evaporator (IKA RV 10 Basic), hotplate (IKA
RW 20 Digital), oven (Memmert UN 55), funnel, erlenmeyer , injection tools, cotton,
intubation tools, surgical scissors, amputation tools, easy touch tools, mouse cages and places
to eat and drink.

Preparation of Diabetes Mellitus Mice (DM) and Control

Before alloxan-induced rats, the test animals were empowered first but still fed. This
was performed in accordance with the experimental protocols that stated that the tested
animals for an 8-12 hour period were more likely to have diabetes than non-empowered test
animals (8). During the removal of the husks are removed from the cage so as not to be eaten
by the animal try. First, the measurement of fasting levels to determine blood sugar levels of
animal testing before alloxan. Second, alloxan monohydrate solution induced in group K (-),
K (+), P1, P2, and P3 mice at 32 mg / 200 gBB doses intraperitoneally by positioning the
supine mice to the visible abdomen. In the abdomennya mice smeared 70% alcohol in order to
avoid infection, then pinched to feel the muscle (9). After the injection of rats were fed and
drunk as usual.
Blood glucose measurements in mice were repeated on day 7 after alloxan induction to
ensure that mice had permanent diabetes.
Procedure
The data collection procedure in the research that will be done consists of material
preparation, material extraction, and antidiabetic activity test with chloroform leaf extract
kenitu.

Preparation of Leaf Material

The fresh leaves are washed thoroughly, then dried with oven until the leaves are
completely dried at 400C so that the form changes (wet kenitu leaves become crisp but leaves
leaf still look green), after dry simplicia is milled with a grinding machine so it becomes
powder.

Making Left Chloroform Extracts

The materials used in this research are leaves of kenitu plant obtained from Materia
Medika, Batu, Malang.Weigh the dried powder of soybean leaves as much as 75 g then
diultrasonic with 1500 ml of chloroform for 2 minutes, then filtered, then the diultrasonic
residue again. The work is repeated, so overall the extracting is done for 3 times every 25
grams using 500 ml solvent.The filtrate or liquid extract produced is evaporated /
concentrated using a rotary evaporator until it is able to extract viscous (concentrated).The
concentrated extract obtained by the antidiabetic test.

Antidiabetic Activity Test

Rats are fasted for 8-12 hours, but they are still drinking. Measured fasting blood sugar
(H0). Alloxan-induced mice with a dose of 32 mg / 200 gBB intraperitoneally administered
by means of rats positioned towards the frontal to the visible portion of the abdomen.
Measured blood sugar levels of fasting rats, mice are said to have fasting diabetes when their
blood sugar levels> 126 mg / dl. The negative control group was given alloxan (K-), the
positive control group was given alloxan and oral antidiabetic drug ie metformine (K +), the
test group 1 was alloxan and chloroform leaf extract with dose 25 mg / kgBW (P1), the test
group 2 was given allocyanate of chloroform leaf extract with a dose of 50 mg / kgBW (P2)
and the test group 3 were alloxan and chloroform leaf extract with a dose of 75 mg / kgBW
(P3). Treatment was administered once daily for 14 days, then blood sugar measurements
were performed on days 3, 7, and 14.

Measurement of Blood Sugar Levels

Rats tail smeared 70% alcohol and ditoreh with needles or cut the tip of the tail to
form a small wound. Then the blood drops on the glucometer strip. The results obtained from
glucometer is the blood glucose level of the rat.

RESULTS AND DISCUSSION

Preparation Simplisia Leaf Kenitu
The samples used leaf kenitu obtained from Balai Materia Medika Kota Batu, East
Java, the leaves are dark green top, while the bottom is fluffy and golden. Based on the results
of leaf extraction kenitu using chloroform solvent this research is a thick brown thick viscous
form with weight 14.20 gram and randemen chloroform leaf extract kenitu equal to 18,94%.
The water content of the sample after repeating 3 times is 6.75%. From this value it is
known that the powder of simplicia has good moisture content because less than 10%.
According to the POM (2002) the smaller the value of water content, the withdrawal of active
compounds by the solvent is more effective when the extraction process. The percentage of
10-12% is safe water content for dry matter, while less than 10% is good water content (10).
Identification Test of Alkaloid Compounds, Sterols and Triterpenoids
Result of identification test of compound in leaf extract kenitu showed that leaf extract
kenitu contain alkaloid compound, sterol and triterpenoid, shown in table 1.
To isolate an alkaloid compound in a plant requires a good solvent to extract the
alkaloids, ie organic solvents such as ether, alcohol, benzene and the like (11). While on the
extract if it contains free alkaloids when viewed under 365 nm UV rays berkluoresensi green /
orange by using thin layer chromatography method or commonly called with TLC (12).
Meanwhile, to know the existence of terprnoid content is done by compound class test
using thin layer chromatography method, this compound shows different color with
compound 1 that is red color as terpenoid group (13). Furthermore, this compound is
determined by the LierbermanBurchard method, showing the reddish-green color as a group
of steroids (13).

Test Activity of Leaf Extracts to Reduced Blood Sugar Levels

Research on chloroform activity of chloroform leaf extract of kenitu against blood
sugar level is done with the aim to know the existence of antidiabetes activity on chloroform
leaf extract kenitu and know optimal dose of chloroform leaf extract kenitu in rat to decrease
glucose level.
In this study blood used for antidiabet test was measured blood sugar level of fasting
rats. Blood glucose measurements were performed on days 0, 3, 7, and 14. Graph 1 was
obtained.
Based on the results obtained in the study showed a change in blood sugar levels after
administration of alloxan, elevated blood sugar levels is caused by β-cell necrosis of the
pancreas gland by alloxan. The selective mechanism of destruction by alloxan according to
Szkudelski (2001) is that alloxan binds to Glut-2 which facilitates the entry of alloxan into the
pancreatic β-cell cytoplasm, increasing depolarization of mitochondria as a result of Ca2 + ion
intake followed by excess energy use resulting in a lack of energy in cell.
The mechanism of alloxan toxicity begins with the entry of alloxan into pancreatic
beta cells and the retrieval rate will determine the alloyan diabetogenic properties. Damage to
ß cells occurs through several processes simultaneously, ie through the oxidation of sulfidryl
groups and the formation of free radicals. The alloxan alloying mechanism produces damage
to pancreatic ß cells primarily invading cellular compounds containing sufidril groups, amino
acids cysteine and proteins binding to SH groups (14).
Decrease in blood sugar levels is due to the provision of metformin dose 9 mg
metformin. Due to the presence of specific mechanisms of metformin in lowering blood sugar
levels. Metformin mechanisms in lowering blood sugar levels include direct stimulation of
glycolysis in peripheral tissues by increasing glucose expenditure from the blood, reducing
glycogenogenesis of the liver, slowing the absorption of glucose from blood, reducing
glucagon levels in plasma and increasing insulin binding to insulin receptors. The mechanism
of action of metformin in lowering blood sugar levels does not depend on the presence of
functioning pancreas cells (15).
Alkaloids potentially lower blood glucose levels by reducing insulin resistance in the
presence of C-dependent up-regulation protein kinase on insulin receptors, increasing
glycolysis, stimulating GLP-1 secretion and inhibiting DPP-4 (16).
While on the work of triterpenoids and sterols as decreased antidiabet by increasing
the activity of insulin secretion in pancreas islet (7). Triterpenoids can work as anti-diabetic
with an insulin-dependent stimulating effect and protect pancreatic β cells from oxidative
stress and also act as anti-insulin resistant (17). With the improvement of the pancreas tissue,
there is an increase in the amount of insulin in the body so that blood glucose will enter into
the cell resulting in decreased blood glucose in the body. From the results of observation, it
can be concluded that giving chloroform leaf extract kenitu able to improve the morphology
of Langerhans island better.

Data analysis
Blood glucose measurement data in rats tested for normality with Shapiso Wilk test
showed blood glucose levels in normal distributed mice with significant values (p> 0.05).
Normality test results are considered normal if the value of p-value> 0.05. P-value test result
of normality of measurement of blood glucose level found in table 2.
Based on Table 5.4, the p-value of all four formulas> 0,05, then the value of blood
glucose level in normal mice. After being declared normal, it is followed by homogeneity test
using Levene's test. Given the results of blood glucose measurements in mice there is data that
is not homogeneous with the value (p - value <0.05) that is found on the 7th day
measurements of p = 0.03. Homogeneity test results can be seen in table 3.
Due to the non-homogeneous data, it is followed by Kruskal-Wallis nonparametric
test, to know the significant difference in each treatment group.
The result of measurement test of Kruskal-Wallis nonparametric test data can be seen
in table 4. Result of data analysis of blood glucose measurement with Kruskal-Wallis
nonparametric test obtained sigification value (p <0,05) that there is significant difference in
negative control group with treatment group has a value <0.000 on day 7. There was a
significant difference in the negative control group with the treatment group having a value
<0.02 on day 3, and no significant difference in the negative control group with the treatment
group had a value <0.384 on day 0. It was followed by Mann-Whitenay nonparametric test to
observe significant differences in each treatment group with negative control group.
The result of measurement test of Mann-Whitenay nonparametric test data can be seen
in table 5. Result of data analysis of blood glucose measurement with Mann-Whitenay
nonparametric test obtained sigifikasi value (p <0,05) that there is significant difference in
control group with control group positive, dose group 25 mg / KgBB, group of dose 50 mg /
KgBB, and group dose 75 mg / KgBB with significant value equal to p = 0,04 at day 7. Table
6.
The purpose of Mann-Whitenay nonparametric test is to know the existence of
significant differences in each treatment group, so that from the analysis result can be
concluded that there is a significant difference between the control group and the treatment
group shown by significant value p = 0.04 on the 7th day.

CONCLUSION
Based on the results and the discussion obtained by the researcher, it can be concluded
that the leaf chloroform extract has activity on the decrease of blood sugar level shown in
dose group 50 mg / KgBB> 75 mg / KgBB> 25 mg / KgBB and> positive control group on
day 7th. And the optimal dose of leaf kenitu on chloroform leaf extract therapy that gives
effect to decrease blood sugar levels in rats induced with alloxan is at dose 50 mg / KgBB.
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Grafik 1. pengukuran kadar gula darah

Tabel 1. Uji Identifikasi Senyawa

Uji Hasil
Fase Hasil sinar
Identifikasi Fase Gerak sinar Keterangan
Diam UV 366
Senyawa UV 254
Noda
Kloroform : 9 Adanya
Alkaloid berwarna Positif
Metanol : 1 bercak
jingga
Noda
n-Heksan : 7 berwarna Adanya
Sterol Silika Positif
Etl-asetat : 3 Merah bercak
Gel
kehijauan
n-Heksan : 2
Noda
Eter : 3 Adanya
Terpenoid berwarna Positif
Etil-asetat: 3 bercak
Merah
Etanol : 2

Tabel 2. P-value normalitas Shapiro-Wilk

P-value normalitas Keterangan
Pengukuran
Shapiro-Wilk
Hari ke-0 0,81
Hari ke-3 0,97 Normal
Hari ke-7 0,43

Tabel 3. P-value homogenitas Levene’s test

P-value homogenitas Keterangan
Pengukuran
Levene’s test
Hari ke-0 0,72 Homogen
Hari ke-3 0,85
Hari ke-7 0,03 Tidak homogeny

Tabel 4. P-value nonparametrik Kruskal-Wallis

P-value nonparametrik Keterangan
Pengukuran
Kruskal-Wallis
Kontrol positif
25 mg/KgBB
0,000 Berbeda signifikan
50 mg/KgBB
75 mg/KgBB
Tabel 5. P-value nonparametrik Mann-Whitenay
 P-value nonparametrik Keterangan
Pengukuran
Mann-Whitenay
Kontrol positif
25 mg/KgBB
0,04 Berbeda signifikan
50 mg/KgBB
75 mg/KgBB

Tabel 6. Pengukuran spss

P-value Hari P-value Hari P-value Hari
Pengukuran
ke- 0 ke- 3 ke- 7
Negatif 0,127 0,827 0,046*
Dosis 25 mg/KgBB 0,827 0,127 0,827
Positif
Dosis 50 mg/KgBB 0,827 0,127 0,827
Dosis 75 mg/KgBB 0,827 0,275 0,275
Negatif 0,827 0,127 0,046*
Dosis 25 Positif 0,127 0,127 0,827
mg/KgBB Dosis 50 mg/KgBB 0,827 0,827 0,513
Dosis 75 mg/KgBB 0,827 0,827 0,827
Negatif 0,827 0,050 0,046*
Dosis 50 Positif 0,127 0,127 0,827
mg/KgBB Dosis 25 mg/KgBB 0,827 0,827 0,513
Dosis 75 mg/KgBB 0,827 0,275 0,827
Negatif 0,827 0,050 0,046*
Dosis 75 Positif 0,127 0,275 0,275
mg/KgBB Dosis 25 mg/KgBB 0,827 0,827 0,827
Dosis 50 mg/KgBB 0,827 0,275 0,827
*) adanya pebedaan yang signifikan dibawah p-value 0,05