Submitted : 06-03-2021
Revised : 29-08-2021
Accepted : 18-10-2021
Trad. Med. J., September-December 2021
Vol. 26(3), p 155-160
ISSN-p : 1410-5918 ISSN-e : 2406-9086
In Vivo Antihypercholesterolemic Potential of Uncaria cordata (Lour.)
Merr as Ethanolic Extract
Meiriza Djohari*, Mira Febrina,Tri Wahyuni
Sekolah Tinggi Ilmu Farmasi Riau
ABSTRACT
The present investigation aims to evaluate the antihypercholesterolemic potential of Uncaria cordata
(Lour.) Merr. as an ethanolic extract in diet-induced hypercholesterolemic male white mice model. In this
study, white mice were segregated into 6 groups; all the groups except the normal control group were given
a high-fat diet to induce hypercholesterolemia. After induction of cholesterolemia, normal and negative
control groups were treated with NaCMC, the positive control group was treated with atorvastatin, and the
remaining three groups received ethanolic extract Uncaria cordata (Lour.) Merr. in three doses (100, 200,
and 400 mg/Kg BW) for a treatment period of 29 days. Measurement of cholesterol levels was performed
on days 0, 15, 22, and 29 using EasyTouch® GCU digital devices. The results were analyzed by one way
ANOVA test and ANOVA Repeated test. The results showed that the ethanol extract of the Uncaria cordata
(Lour.) Merr. root plant dose 100, 200, and 400 mg/Kg BW effect in lowering total cholesterol in male white
mice significantly (P <0,05). Ethanol extract from Uncaria cordata (Lour.) Merr. a dose of 200 mg/Kg BW
showed a better decrease in cholesterol levels on day 29 compared to day 22.
Keywords: Cholesterol; Flavonoid; Uncaria cordata (Lour.) Merr.
INTRODUCTION
Cholesterol is one of the plasma lipid
compounds found in tissues in the form of plasma
lipoproteins. Cholesterol can be in a state of free
cholesterol or combined with long-chain fatty
acids, forming cholesterol esters. These
compounds have an important role in the plasma
membrane and also as precursors of adrenal cortex
hormones and vitamin D and bile acids (Murray
et al, 2009).
Hypercholesterolemia is a condition that
signed with Increased cholesterol levels in the
blood and this is the main factor of atherosclerosis
associated with coronary heart disease which can
lead to many cases of death (Muhtadi et al, 2013).
Normally, the body produces the right
amount of cholesterol, but the tendency to
consume animal foods with high fat can trigger
excess cholesterol in the blood (Bertram, 2010).
Coronary heart disease (CHD) is a
dysfunction of the heart due to a heart muscle lack
of blood due to narrowing of the coronary arteries.
Based on Basic Health Research (RisKesDas) in
2018, coronary heart disease was ranked as the
seventh non-communicable disease in Indonesia
and it is estimated that in 2020 CHD became the
first common killer of 36% of all world deaths
(DepKes, 2006; Kemenkes, 2019) and because of
that, we need antihypercholesterolemic.
*Corresponding author : Meiriza Djohari
Email : meirizadj@gmail.com
The use of modern or synthetic drugs in the
statin class is now often used for cholesterol
treatment. However, the disadvantage of the statin
group is that they have side effects in the form of
gastrointestinal disorders, myopathy, allergic
reactions,
and rhabdomyolysis (Liman and
Hartadi, 2002). Thus, people today prefer natural
medicine because natural medicine is believed to
be safer, cheaper, and easily found in the
community around synthetic drugs (Muhtadi et al,
2013). Based on this, research on herbal medicines
used as antihypercholesterolemic still needs to be
developed (Azhari et al., 2017).
The genus Uncaria has the potential to
decrease blood cholesterol levels. Based on
research conducted by Frinanda (2014), the best
dose of Uncaria gambir in inhibiting the increase in
total cholesterol and stabilizing the blood value of
mice is 50 mg/kg BW. In Asia, the popular genus
Uncaria is used for antidiabetic, immune system
stimulants, hypo cholesterol agents and to reduce
the risk of strokes, heart attacks, and hypertension
(Heizment et al, 2005).
Kaik-kaik Root Plant (Uncaria cordata
(Lour.) Merr.) is the genus Uncaria found in
Indonesia, especially in the Indigenous Prohibition
Forest of Kampar Regency, Riau Province. The
leaves of Kaik-kaik Root (Uncaria cordata (Lour)
Merr.) Contain secondary metabolites in the form
of flavonoids, saponins, terpenoids, steroids, and
phenolics (Sefralisa, 2015). Flavonoid compounds
play a role in reducing blood cholesterol levels.
Traditional Medicine Journal, 26(3), 2021 | DOI: 10.22146/mot.64531
155
Meiriza Djohari
The flavonoids are thought to work by inhibiting
the work of the enzyme HMG-CoA reductase which
functions as a catalyst in the formation of
cholesterol (Sekhon, 2012).
Based on the above description, the
researchers were interested in conducting a study
on the effect of ethanol extract of leaves of Kaikkaik Root plants at doses of 100, 200, and 400
mg/kg BW in reducing levels of the total
cholesterol of male white mice (Mus musculus L.).
The method used in this study is POCT (Point Of
Care Testing), using an Easy Touch® tool equipped
with a blood cholesterol test strip. This method is
easy for daily use and still gives accurate data.
To increase cholesterol levels of mice, quail 10
mL/kg BW was used (Kusuma et al ., 2016). The
selection of ethanol solvents for extraction in this
study is based on the fact that besides being nontoxic, ethanol is a universal solvent so that it can
attract polar and nonpolar compounds, the
compounds expected to be attracted by these
solvents are flavonoids (Marjoni, 2016).
METHODOLOGY
Materials
The tools used in this study are a set of
distillation equipment, vacuum rotary evaporator,
analytical scales (Shimadzu Auw 220), dark
bottles, animal scales, animal cages, filter paper,
aluminum foil, funnels, oral sonde, hot plates,
surgical scissors, glasses measuring, alarm, watch
glass, drip pipette, mortar and buffer, vial,
parchment, test tube (Pyrex Iwaki®), drip plate,
measuring device for total cholesterol levels and
strip test (Easy Touch®).
The material used in this study was 96%
ethanol, mice food (standard feed No. 552), highfat feed (quail egg yolk), swab alcohol, Sodium
Carboxymethyl Cellulose (Na CMC), atorvastatin
10mg, aqua dest, chloroform, ammonia, 2 N
sulfuric acid, Mayer reagent, concentrated
hydrochloric acid, 1% iron (III) chloride,
magnesium metal, norit, anhydrous acetic acid,
concentrated sulfuric acid.
Methods
Fresh leaves of kaik-kaik root plant 1 kg
cleaned and dried air. The dried samples obtained
450 g were sorted to remove dirt that was still left
behind during the drying process. The extraction
process is done by maceration or immersion with
96% ethanol solvent which has been distilled. A
sample of 250 g which had been dried and chopped
before was put into a dark bottle and then
immersed in ethanol solvent which had been
distilled until the sample was completely
submerged. Sample containers are stored at room
156
temperature and protected from direct sunlight.
The immersion process lasts for five days while
stirring occasionally. The ethanol extract was
filtered using filter paper and the pulp was
macerated in the same way for 5 days to three
repetitions. The results of maceration were
collected, then concentrated with a vacuum rotary
evaporator.
Animal Test Preparation
Before the experiment, the animals were
acclimatized for about 7 days to adapt to the
environment. The test animals used were male
white mice (Mus musculus L.) with an average
weight above 20 grams of body weight. Animals are
grouped into four groups, namely the control
group (Na CMC 1%), doses of 100, 200, and 400
mg/Kg BW. Grouping was done randomly in such a
way that the distribution of body weight was
evenly distributed for all groups with variations in
body weight not more than 20% of the average
body weight. Animals are weighed every day to
determine the volume of test preparation to be
given
Na CMC powder was weighed 0.05 mg. Then
sprinkled on hot water 20 times (1 mL) in the hot
mortar and left for 15 minutes. Then crushed until
homogeneous, then added little by little the
ethanol extract of leaves of kaik-kaik root plant
that have been weighed according to the planned
dose, then crushed to homogeneous and added
aqua dest to a volume of 5 mL. The route for giving
the test preparation is given orally. This treatment
is given every day once a day for 28 days and
measurements are taken on days 0, 15, 22, and 29.
Measurement of Total Cholesterol Levels
Measurements were made with a
cholesterol measuring device, namely easy touch®
GCU. The tool is calibrated first with the code
number corresponding to the test strip. The test
strip is inserted in a special place on the device,
then on the screen, a picture of blood droplets will
appear indicating the device is ready for use.
Before being measured, mice have fasted for 12-14
hours. According to Murray et al (2009), this
fasting process aims to reduce the activity of HMGCoA reductase. After the mice's tail was disinfected
with 70% ethanol the tail end was cut until the
blood came out and enough to use, the first blood
droplet was removed to avoid the wrong results,
the next droplet was applied to the test strip until
it sounded and 150 seconds will appear on the
screen cholesterol with mg / dL. The cholesterol of
the experimental animals was measured on the day
after the 8th day of acclimatization (T0), day 22
(T15) after 14 days of induction, day 29 (T22),
Traditional Medicine Journal, 26(3), 2021
In Vivo Antihypercholesterolemic Potential Of Uncaria cordata (Lour.)
Figure 1. leaves of Kaik-kaik Root plants
and day 36 (T29) after treatment. Contain a brief
but sufficiently complete description of procedures
and materials to allow the experiment to be
repeated. Only new procedures should be
described. Previously published procedures
should be referenced. Significance materials
should be described in detail.
RESULT AND DISCUSSION
One of the genus Uncaria which is thought to
have the potential to reduce cholesterol levels is
Kaik-kaik root plant. In this study, Kaik-kaik root
plants were taken from the Rumbio Traditional
Ban Forest of Kampar Regency, Riau Province.
Taking plants in the forest to match the sampling
sites in the study of the previous kaik-kaik root
plants. The part of the plant used is the part of the
leaf (figure 1).
Experimental
mice
were
made
hypercholesterolemia by feeding a high-fat diet in
the form of quail egg yolk as much as 10 ml/Kg BW
for 28 days. In this study, we only investigated
feeding.
Taking blood of mice is carried out through
the tail vein (Lateralis). The blood is taken by
cutting a bit of the tip of the mice which has been
given 70% alcohol, then removing the first drop of
blood, and then the blood is dripped onto a
cholesterol test strip that is installed on the device.
This cholesterol level measurement uses the Point
of Care Testing (POCT) method. POCT is a simple
laboratory examination using a small amount of
blood sample and the results can be known quickly
Traditional Medicine Journal, 26(2), 2021
because without using sample preparation. The
principle of inspection with Point of Care Testing
(POCT) uses biosensor technology that produces
electric charges from chemical interactions
between certain substances in the blood and strip
electrodes. Changes in electrical potential that
occur as a result of the reaction of these two
substances will be measured and converted to a
number that corresponds to the amount of electric
charge produced. The numbers produced in the
examination are considered to be equivalent to the
levels of substances measured in the blood
(Kemenkes, 2010).
The observational data was processed using
one-way Analysis of Variance (ANOVA) statistical
analysis consisting of one dependent variable
(cholesterol level) and one independent variable
(treatment), while to find out the differences in the
measurement time of cholesterol levels in each
group used statistical analysis repeated ANOVA.
Statistical methods ANOVA is a comparative
analysis technique to find significant differences
from the mean (average) data that is more than
two variables or groups (Dahlan, 2019).
Based on the results of measurements made,
the average total cholesterol level of male white
mice (Mus musculus L.) in the normal control
group, negative controls, positive controls, doses of
100, 200, and 400 mg/kg BW respectively at T0 is
116, 8; 166.6; 156.6; 142.4; 149 and 151.6 mg / dL.
These levels are appropriate when compared with
the normal total cholesterol level of male mice
(Mus musculus L.), which is 90-170 mg / dL.
The average total cholesterol level in T15 is 121;
206.4; 219.4; 174,2; 183 and 196.6 mg / dL. The
average total cholesterol level in T22 is 116; 223.6;
143.4; 151,6; 138; 149.8 mg / dL and at T29 is
121.2; 228.6; 137.2; 145; 131.8 and 143.6 mg / dL
(see table I) and we can see in Figure 2, that there
was an increase in cholesterol levels in all
treatment groups on day 15 due to the induction of
high-fat feed, and began to experience a decrease
in cholesterol levels on days 22 and 29 in the
positive control group and the treatment group
except for the negative control group.
Based on the subset of homogeneity of the
results of the Tukey Post Hoc test, the difference in
total cholesterol levels on the 22nd day to day 15
and 29 to day 15, in the negative control group
showed significantly different results (p <0.05)
with all groups. It can be said that the cholesterol
level of the negative control group increased
because it was induced with high-fat feed.
The administration of ethanol extract of
leaves of kaik-kaik root at a dose of 100 mg / Kg
BW showed significantly different results (p <0.05)
with the negative control group, positive control,
157
Meiriza Djohari
Figure 2. Average of cholesterol levels
normal control, 200 and 400 mg kg BW doses.
It can be said that on the 22nd and 29th-day
ethanol extract of leaves of kaik-kaik root plant
dose of 100 mg / Kg BW was able to reduce total
cholesterol levels because it was significantly
different (p <0.05) with a negative control group
but had not been able to reduce comparable
cholesterol levels with a positive control group.
The administration of ethanol extract of
leaves of kaik-kaik root plant 200 mg/kg BW
showed results that did not differ significantly
(p> 0.05) with a group dose of 400 mg/kg BW and
showed significantly different results (p <0.05)
with a negative control group, positive control,
normal control and dose of 100 mg/kg BW.
This can be interpreted that on the 22nd day and
29th day the ethanol extract of leaves of kaik-kaik
root plant at a dose of 200 mg/Kg BW had activity
in reducing total cholesterol levels which were
comparable to the group dose of 400 mg/Kg BW
but not comparable with the positive control group
given atorvastatin.
The administration of ethanol extract of
leaves of kaik-kaik root at a dose of 400 mg/kg BW
showed significantly different results (p <0.05)
with the negative control group and positive
controls. This can be interpreted that the ethanol
extract of leaves of Kaik-kaik root plant at a dose of
400 mg/kg BW can reduce total cholesterol levels
but is not comparable with the positive control
group given atorvastatin.
In this present study, we found based on the
results of the repeated ANOVA test, that the total
cholesterol levels of all groups at T0, T22, and T29
158
were significantly different (p <0.05) with
cholesterol levels in T15. This shows that in T15
there was an increase in mice cholesterol after an
induced high-fat feed, but in the normal control
group, there was no significant difference (p> 0.05)
because in the normal control group only Na CMC
was given without induction of high-fat feed,
so that cholesterol levels same as T15. Total
cholesterol levels of mice given ethanol extract of
kaik-kaik root leaves, doses of 100 and 400 mg/Kg
BW showed results that were not significantly
different (p> 0.05) at T22 with T29. This can be
interpreted that the administration of ethanol
extract of leaves of kaik-kaik root of doses of 100
and 400 mg/Kg BW was not able to reduce total
cholesterol levels in T29, but in the 200 mg/Kg BW
dose group there were significant differences
(p <0.05) on T22 with T29. This shows that the
ethanol extract of leaves of kaik-kaik root plant
dose of 200 mg/Kg BW is still able to reduce total
cholesterol levels in T29.
This decrease in cholesterol levels is
thought to be due to the presence of secondary
metabolites as flavonoids, phenolics, and saponins
contained in the extract. According to Sekhon
(2012), flavonoids can reduce levels of cholesterol
by inhibiting the activity of the enzyme HMG-CoA
reductase, flavonoids are also thought to inhibit
ACAT
activity
(Acyl-CoA
Cholesterol
Acyltransferase) in the liver. HMG-CoA reductase
enzyme has a role in the synthesis of cholesterol
so that when the enzyme's work is inhibited,
cholesterol synthesis will decrease. The
mechanism of inhibition by flavonoid compounds
Traditional Medicine Journal, 26(3), 2021
In Vivo Antihypercholesterolemic Potential Of Uncaria cordata (Lour.)
Table I. Effect of Ethanol Extract of Kaik-kaik Roots (Uncaria cordata (Lour.) Merr.) On Total Cholesterol
Levels of Male White Mice (Mus musculus L.).
Group
Number of Animals
Normal
1
2
3
4
5
average±SD
1
2
3
4
5
average±SD
1
2
3
4
5
average±SD
1
2
3
4
5
average±SD
1
2
3
4
5
average±SD
1
2
3
4
5
average±SD
Negative
Positive
Dose 100
mg/kg BW
Dose 200
mg/kg BW
Dose 400
mg/kg BW
t0
119
119
118
116
112
116,8±2,949
168
164
166
169
166
166,6±1,949
155
157
155
157
159
156,6±1,673
142
144
139
145
142
142,4±2,302
150
148
151
150
146
149±2
152
152
150
150
154
151,6±1,673
occurs when an analog of the HMG-CoA reductase
enzyme with its substrate, HMG-CoA, is converted
into mevalonic acid. This shows that flavonoids act
as competitive inhibitors with HMG-CoA so that the
HMG-CoA reductase enzyme tends to bind to
flavonoids as a result of a decrease in the formation
of mevalonic acid which plays a role in cholesterol
biosynthesis.
Flavonoids and phenolic compounds can be
antioxidants because of their ability to donate
hydrogen atoms, free radical scavengers, and metal
ion chelating. LDL is a lipoprotein that contains a
lot of cholesterol, this cholesterol will be partially
Traditional Medicine Journal, 26(2), 2021
Cholesterol Level (mg/dL)
t15
t22
120
117
125
120
115
112
120
112
125
119
121±4,183
116±3,807
207
224
204
228
208
225
206
221
207
220
206,4±1,516 223,6±6,949
211
139
218
142
222
146
224
145
222
145
219,4±5,176 143,4±2,880
175
151
174
154
175
145
177
157
174
151
175±1,224
151,6±4,449
183
139
184
137
186
138
182
139
180
137
183±2,236
138±1
193
155
196
150
197
145
198
145
199
154
196,6±2,302 149,8±4,764
t29
123
129
116
123
129
124±5,385
224
237
230
230
222
228,6±5,899
137
136
140
137
136
137,2±1,643
142
139
146
150
148
145±4,472
132
132
136
131
128
131,8±2,863
140
145
145
140
148
143,6±3,507
taken to the liver, other steroidogenic tissues that
have receptors for LDL cholesterol and some will
experience oxidation and are captured by the
scavenger-A receptor (SR-A) in macrophages and
will become foam cells (foam cell), so the more LDL
cholesterol levels inside the plasma, the more that
will experience oxidation and captured by
macrophagic cells which play a role in the
formation of atherosclerosis (Sudoyo et al, 2009).
It can be said that flavonoids and phenolics as
antioxidants can inhibit the oxidation of
cholesterol so that foam cells are not formed.
Based on research conducted by Rocio et al (2013)
159
Meiriza Djohari
Saponin compounds reduce plasma cholesterol
levels by reducing absorption from cholesterol in
the intestine.
CONCLUSION
Based on the research that has been done, it
can be concluded that the ethanol extract of leaves
of kaik-kaik root plant (Uncaria cordata (Lour.)
Merr.) For 14 days in male white mice (Mus
musculus L.) at doses of 100, 200. and 400 mg/Kg
BW has an affects on the decrease in cholesterol
levels. The ethanol extract of leaves of kaik-kaik
root at a dose of 200 mg/Kg BW on the 29th day
still reduced cholesterol levels compared to the
22nd day. The group dose of 200 mg/Kg BW on
days 22 and 29 can lower cholesterol levels greater
than the group dose of 100 and 400 mg/Kg BW.
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Traditional Medicine Journal, 26(3), 2021