Tests of Cholesterol Decreasing in White Mice (Mus musculus) With In-Vivo Using Methanol Extract

Umbi Talas (Colocasia esculenta L) as Cardiovascular Disease Prevention Effort

Samsul Rizal Umami1) Sarifa Siti Hapizah1) Rosita Fitri2) Aliefman Hakim1)
1) Chemistry Education FKIP University of Mataram
2) Biology Education FKIP University Mataram

Abstrack: This study aimed to determine the effect of taro root extract (Colocasia esculenta L)
to decrease cholesterol levels of white mice (Mus musculus). Induction hiperkolesterolemik done by
providing quail egg yolk, broiler chicken feed and administration of propylthiouracil (PTU) in white
mice (Mus musculus). In this study using extraction methods and test cholesterol-lowering with taro
root extract. The mice were divided into 5 experimental groups consisting of 3 treatment groups (P1,
P2, and P3) and two control groups (K + and K-). In the group P1 doses used were 100 mg / kg body
weight, P2 dose of 200 mg / kg, and P3 is 300 mg / kg. While the group K + and K- simvastatin dose
given only distilled water. The results showed that at a dose of 100 mg / kg showed elevated levels of
cholesterol by 5.2 mg / dL, the K- increased by 3.8 mg / dL. While at P2 decreased quite high by 27.9
mg / dL, P3 decreased by 2.4 mg / dL and K + also decreased by 2.2 mg / dL. Based on these results,
a dose of 200 mg / kg showed a dose reduction is better than another dose.
Keywords: Hiperkolesterolemik, PTU, Bulbs Talas, Extraction, Cholesterol

1. INTRODUCTION
Cholesterol is currently not only a health problem faced by developed countries but also developing
countries. Cholesterol is one of the leading causes of cardiovascular disease that is a deadly disease
and has become a serious problem in both developed and developing countries. The World Health
Organization (WHO) and the World Heart Federation (World Heart Federation) predict that heart
disease will be the leading cause of death in Asian countries by 2010 [12]. In Indonesia alone,
cardiovascular disease is the number one killer along with dietary changes that tend to be high in fat
and low in fiber.

High cholesterol levels will lead to the formation of plaques starting with fat-protein / LDL
infiltration (Low Density Lipoprotein) into the arterial wall causing atherosclerosis. Recent research
has shown that inflammation is involved in all stages of atherosclerosis [7]. Currently a lot
on the market, cholesterol-lowering drugs, both natural and synthetic drugs. However, since the use of
modern or synthetic drugs has side effects such as urinary retention with hyponatremia,
gastrointestinal disorders [4], the current trend of people prefer natural medicine because natural
medicines are believed to be safer, cheaper and easier to find in the community, synthetic [8]. This
phenomenon is of particular concern among natural materials researchers.

In the nature of Indonesia there are approximately 30,000 species of plants from about 40,000 species
of plants in the world, just found 940 species of which are medicinal plants believed to be cure by the
public can cure the disease [9]. Knowledge of medicinal plants based on experience and skills that
have been passed down from one generation to the next generation [11].

Taro tuber is one of the nutritious medicinal plants commonly used as food intake of people with
diabetes mellitus. Based on research conducted by Prajapati (2011) reported that taro ethanol extract
(Colocasia esculenta L) has pharmacological effects such as hypoglycemic, antifungal, anticancer,
hypolipidemic, antiinflammatory, and neuroprotective properties [10]. Based on the above
information, the researcher is interested to do research about cholesterol drop test on white mice (Mus
musculus) in-vivo using metasol extract of taro tuber (colocasia esculenta L) as prevention of
cardiovascular disease.

2. RESEARCH METHODOLOGY

2.1 Tools and Materials

This research uses tools such as knives, taro tuber, blender, filter, spoon, beaker, oven, stirrer,
measuring cup, spray bottle, seker, mortar and pestle, rotary evaporator, injection syringe, falkon,
scissors , sonde, animal scales (analytical scales), animal cages, glass funches, erlenmeyer flasks, test
tubes, centrifugation tools, micropipes, volumetric pipettes, and cholesterol tests (GCUs). While the
ingredients to be used are taro tuber (Colocasia esculanta L.), white mice (Mus musculus), technical
methanol, aquades, aluminum foil, quail egg yolk, feed birds chirping, broiler chicken feed,
simvastatin, propiltiourasil, gloves , mask, and tissue.

2.2 Research Procedures

2.2.1 Taro Extraction (Colocasia esculanta L)
Extraction of taro tubers was done by maseration method (immersion). A total of 5 kg of taro tubers
that have been purchased on the market thinly sliced and dried using an oven. then taro tubers blend to
be used as powder. Finished powder is used for extraction. Extraction was done by immersing 470
grams with methanol solvent of 1410 mL. This immersion uses a 3: 1 ratio between solvent With
powder. The taro tubers were fed into two erlenmeyers of 1000 mL each of 235 grams and 705 mL of
methanol solvent was added. The maserate is stirred using a sieve at 150 rpm for 1 day. The resulting
filtrate was evaporated using a rotary evaporator for 3 days with a temperature of 69-70 º C to obtain a
thick extract of taro tuber extract.

2.2.2 Test Animal Preparation

Test animals used in this study were 25 white mice (Mus musculus), which was approximately 3
months old with weight 20-30 grams. The test animals were divided into 5 groups, ie 3 treatment
groups (P1, P2, P3), and two control groups (K + and K-). Each group consists of 5 mice [13]. Prior to
treatment, all test animals were acclimatized for 3 days to adapt the mice to environmental conditions.
After that, the mice were fed a standard UN Feed (Bird Feed) for 7 days.

2.2.3 Increased White Mice Cholesterol Levels

Feeding of hypercholesterol was done for 25 days, hypercholesterol feed consisted of 30 grams of
quail yolk, PTU (Propyltiouracil) 0.1% and water to 1000 mL and 5 kg of broiler chicken feed.
Preparation of hypercholesterol feed is by way of PTU 100 mg dissolved into 1000 mL of aquades,
quail eggs dissolved with PTU solution that has been made. For 15 days this feed is given to the mice
with each volume of 0.5 per mouse in a way to be condensed. Next 10 days ahead given broiler
chicken feed to raise the cholesterol level of test animals. After 25 days, checking all mice (25 mice)
for known cholesterol of mice after feeding of hypercholesterolemia. Checking cholesterol is done by
using GCU (cholesterol test). Before checking, the mice are fasted for 10-12 hours, the measurement
of cholesterol mouse is total cholesterol of mice after hypercholesterol feed.

2.2.4 Dosing Planning

The dose of methanol extract of taro tuber used was in group P1 used 100 mg / gBB, P2 with dose of
200 mg / gBB, P3 with dose 300 mg / gBB, positive control (K +) with dose of simvastatin 0,026 mg /
gBB (conversion factor 0, 0026) and for negative control (K-) given aquades.

2.2.5 Test of Cholesterol Decrease in White Mice (Mus musculus)

Test animals were given treatment to each group of doses that had been made with 0.5 mL per mice
for 12 days. Cholesterol levels are checked after 12 days of treatment. Before that, the mice are fasted
for 10-12 days.

2.2.6 Measurement of Cholesterol Levels

Measurements are made using the GCU Easy Touch tool The tool is calibrated first with a code
number adapted to the test strip used. Test strips inserted in a special place on the tool, then on the
screen will appear a picture of "blood drops" that
indicates the tool is ready for use. After the tail of the mouse was disinfected with 70% ethanol, the
tail tip was cut, the first drop of blood was discarded, the next drop was dripped onto the test strip
tucked into the tool. A certain amount of blood will be absorbed according to the absorbent capacity
of the test strip until a beep is heard, after which the mice bleeding is stopped. Results will be seen on
screen after 150 seconds for cholesterol test.

3. RESULT AND DISCUSSION

3.1 Research Results
Cholesterol levels of the rats group fed a high-cholesterol diet diet for 25 days on average have shown
hypercholesterolaemia. Furthermore, each group was treated with taro tuber extract for 12 days. Data
of total cholesterol level of each group of mice after fed with hiperkolesterol and taro tuber extract can
be seen in Table 1.

Cholesterol levels after feeding of

hypercholesterolemia (mg / dL) Cholesterol levels after dosing (mg / dL)

No. E1 E2 E3 C+ C- E1 E2 E3 C+ C-

1 155 193 202 192 183 129 155 216 172 230

2 165 215 209 192 187 230 188 228 184 200

3 203 168 165 179 227 152 163 156 124 169

4 207 193 214 184 193 188 138 207 216 190

5 135 178 206 176 183 129 165 177 216 203

Rata- 173 189,4 199, 184,6 194,6 165,6 161,6 196,8 182,4 198,4
Rata±SD ±31,17 ±17,81 ±19,61 ±7,33 ±18,56 ±43,33 ±18,10 ±29,59 ±38,01 ±22,12

Table 1. Cholesterol levels after feeding of hypercholesterolemia and dosing

3.2 Discussion
Based on the results of research mentioned above can be said that, the average lowest cholesterol
levels after given feed hypercholesterol is at P1 that is 173 mg / dL while the highest at P3 is 199.2
mg / dL. This value is not much different from the average K-cholesterol level of 194.6 mg / dL.
While the P2 group had an average cholesterol level of 189.4 mg / dL. while for K + is not much
different from P2 that is equal to 184,6 mg / dL.
Results of the analysis with One Way Anova showed that cholesterol levels in the negative control
group differed significantly with positive control group and 3 treatment groups. This suggests that the
negative control group given hypercholesterolic feed and only given aquades showed elevated
cholesterol.

Administration of doses in each of the experimental groups showed that there was a decrease in
cholesterol levels in the P2, P3, and K + groups. While in the group P1 and K- there was an increase
in cholesterol levels. At P2 given a dose of 200 mg / kgBB showed a decrease from 189.4 mg / dL to
161.5 mg / dL with an average decrease of 27.9 mg / dL. While at P3 given dose of 300 mg / kgBB
decreased from 199.2 mg / dL to 196.8 mg / dL with an average decrease of 2.4 mg / dL. And for K +
given doses of simvastatin which is a commercial cholesterol-lowering drug can lower mice
cholesterol levels from 184.6 mg / dL to 182.4 mg / dL with an average decrease of 2.2 mg / dL.
While in the P1 group there was an increase in cholesterol levels with an average increase of 5.2 mg /
dL. While in the K-group there was also an increase of 3.8 mg / dL. In this study induction of
hypercholesterolemia was done by giving quail egg yolk, broiler chicken feed and PTU
administration. The purpose of this gift is to make the mouse hypercholesterol. Propylthiouracil
(PTU) is a thyroid hormone antagonist. Under normal circumstances, thyroid hormones can increase
fat metabolism by increasing the formation of LDL receptors in liver cells, resulting in rapid removal
of LDL from plasma and cholesterol lipoprotein secretion by liver cells [12].

Based on research data, showed that there was a decrease in cholesterol level of mice after giving taro
tuber extract. According to [6] one of the content of taro tuber is a flavonoid compound. Flavonoids
are known to have antioxidant activity. Flavonoids can act with free radicals through direct capture of
oxygen free radicals and inhibit the enzymes that cause free radicals such as cyclooxygenase and
lipoxygenase. In lowering cholesterol, the antioxidant compound is thought to work by inhibiting the
HMG-CoA reductase enzyme that acts as a catalyst in the formation of cholesterol and increases the
activity of Lechitin Cholesterol Acyl Taransferase (LCAT) [5].

This decrease in cholesterol levels occurs at doses of 200 mg / kgBW, 300 mg / kgBW, and in
positive controls given simvastatin dose. The simvastatin used as a benchmark also has an
anticholesterol mechanism by competitively inhibiting the HMG-CoA reductase enzyme which has a
function as a catalyst in the formation of cholesterol. But the K + group gave a lower decrease
compared to the P2 and P3 groups, and the P3 group gave a lower decrease compared to the P2 group.
While for the K-group showed elevated cholesterol levels. This happens because in group K- only
given aquades without given dose. For P1 showed elevated cholesterol levels also given a dose of 100
mg / kgBW.

The highest test dose of 300 mg / kgBW in this study only slightly reduced mean cholesterol levels in
mice compared to 200mg / kgBW. This condition is a fairly common phenomenon encountered in
testing a new drug candidate, where dose optimization meaning a pharmacological response has a
maximum effect on a given dose [3]. This suggests that a dose of 300 mg / kgBW is not optimal to
lower total blood cholesterol levels.

4. CONCLUSION
From the results of this study and cover all that is in the scope of this study we can draw the
following conclusions:
1. Giving taro tuber extract in this study can reduce cholesterol levels of white mice.
2. Dose 200 mg / kgBB taro tuber extracts provide better cholesterol levels than doses of 100 mg /
kgBW, 300 mg / kgBW, and simvastatin dose.

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