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
Novi Yusro Maulidiyah, 2Burhan Ma’arif Z A, 3Meilina Ratna D.
1
Departement of Pharmacy, Maulana Malik Ibrahim State University Malang
Jl. Gajayana No. 50 Malang 65144

Corresponding author: [email protected]

Co-author 1, email: [email protected]

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.

INTODUCTION
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 ( Soegondo dkk., 2009). 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
(Herqutanto. 2009). Diabetes mellitus type 2, in contrast to type 1 diabetes mellitus, is
not problematic with insulin, but with insulin receptors (BP PERKENI, 2011).
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 (USDA, 2003).
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 (Morton, 1987). Fruit has high antioxidant activity,
among which are: kenitu produces antioxidant anthocyanins, and cyanidin-3-O-ß-
glucopyranoside (Einbond et al., 2004). 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 (Koffi et al., 2009).
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.

METHODOLOGY
Materials and Methods
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.

Materials
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.

RESEARCH METHODS
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 (Fitriani, 2011). 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 (Shofia, 2013). 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.

Prosedur Penelitian
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 (Puspita, 2009).

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 (Ayuni,
2013). 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 (Handayani, 2006).
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 (Nurhidayah, et al 2015).
Furthermore, this compound is determined by the LierbermanBurchard method,
showing the reddish-green color as a group of steroids (Nurhidayah, et al 2015).
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 (Szkuldelski, T. 2001).
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 (Katzung, 2007).
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
(Azhari, et al., 2016).
While on the work of triterpenoids and sterols as decreased antidiabet by
increasing the activity of insulin secretion in pancreas islet (Koffie et al., 2009).
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 (Guttierez, 2013). 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

Grafik pengukuran kadar gula darah

700
600
Kadar gula darah

500 Negatif
400 Positif
25 mg/kgBB
300
50 mg/kgBB
200
75 mg/kgBB
100
0
KGD0 mg/dl KGD3 mg/dl KGD7 mg/dl KGD14 mg/dl

Waktu 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