Anti-Hyperlipidemic Effect of 70% Ethanol Extract from Mesona

palustris Blume Leaves on Male Hamsters

Dwitiyanti, Ni Putu Ermi Hikmawanti*, Kriana Efendi, Riana Puspa Dewi, Fernita Afriyani

Faculty of Pharmacy and Science, Universitas Muhammadiyah Prof Dr. Hamka, Jakarta, Indonesia

Keywords: extract, black cincau, black grass jelly, Mesona palustris Blume, anti-hyperlipidemic

Abstract: In Indonesia, Mesona palustris Blume is known as the black cincau (black grass jelly). This plant has

been used for various health condition. This study aims to determine the effectiveness of 70% ethanol

extract from black cincau leaves as anti-hyperlipidemic on hyperlipidemic hamsters. The dried leaves

were extracted using 70% ethanol by maceration method. Syrian hamsters were divided into 7 groups of 4

hamsters), i.e. normal control group, positive control group (fenofibrate 1.235 mg/Kg body weight), positive

control group (atorvastatin 2.4 mg/Kg body weight), negative control group, test groups with 3 dose

variations (780, 1560, and 3120 mg/Kg body weight each day). All groups except normal control group

were induced with high-fat diet for 28 days. Parameters measured were decreased total blood cholesterol,

LDL, triglycerides level, and increased blood HDL level. Treatment was performed for 14 days. The 70%

ethanol extract of black cincau with a dose of 3120 mg/Kg body weight was able to decrease blood total

cholesterol level by 51.49%, blood LDL level by 49.92%, and blood triglyceride level by 45.70%. Extract

with the dose is also able to increase blood HDL level by 29.30%. Fenofibrate was able to decrease blood

triglyceride level by 50.62% and increase blood HDL level by 34.82%, whereas atorvastatin was able to

decrease blood total cholesterol level by 54.34% and blood LDL level by 51.13%. It could be concluded

that the 70% ethanol extract from black cincau leaves is effective as an anti-hyperlipidemic agent in

hyperlipidemic hamster equivalent to positive control group.

1 INTRODUCTION

The highest prevalence of cardiovascular disease in

Indonesia is coronary heart disease, which is 1.5%,

with the highest rate is in East Nusa Tenggara

province (4.4%) and the lowest in Riau Province

(0.3%) (Ministry of Health of Republic of Indonesia,

2013). One of the causes of coronary heart disease is

atherosclerosis - an accumulation of lipids and

fibrous tissue in the coronary arteries that narrows

the lumen of the blood vessels. When the lumen

narrows the resistance to blood flow increases (Price

and Wilson, 2006).

Hyperlipidemia is a condition when one or more

of the fat components such as cholesterol,

phospholipids or triglycerides, increase. The

hyperlipidemic condition is characterized by an

increase concentration of lipoprotein - a substance

for fat transport in plasma (Priyanto, 2009). An

increase in the concentration of lipoproteins are

characterized by increased total cholesterol,

triglycerides, Low Density Lipoprotein (LDL)

levels, and decreased of High-Density Lipoprotein

(HDL) level (Price and Wilson 2006). Cholesterol is

a component of fat or lipids, which is continuously

formed or synthesized in the liver. Approximately

70% of cholesterol in the blood is synthetized in the

liver, while the rest comes from food intake (Anies,

2015).

An increase in the lipid levels in the blood can

occur due to poor lifestyle, such as eating foods high

in fat (Hardman and Limbird, 2012). This condition

can be overcome by improving lifestyle and

consuming anti-hyperlipidemic drugs, which are

used to treat the hyperlipidemic condition by

reducing the total cholesterol, triglycerides, LDL, or

increasing HDL levels. Unfortunately, drugs such as

fibrates and statin groups generally cause harmful

side effects such as muscle aches, itchy skin, rashes,

and other effects such as visual impairment, and

peripheral neuritis. Fibrate groups can also aggravate

the condition of liver function disorders so that they

cannot be given to patients with liver failure. Statins

such as simvastatin can cause undesirable side

Anti-Hyperlipidemic Effect of 70 .

DOI: 10.5220/0008239400610065

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 61-65

ISBN: 978-989-758-349-0

effects such as muscle pain, muscle weakness

(myopathy) and chest pain (Junaedi, 2012). The use

of natural ingredients becomes an alternative to

avoid the side effects arising from synthetic drugs.

Plants that have been shown to have a lipid-lowering

and antioxidant activity are black cincau or black

grass jelly (Mesona palustris Blume) (Amelia and

Tri, 2014).

Black cincau is a traditional food that contains

antioxidants agents and has been used empirically

for various health conditions. Black cincau contains

phenolic components such as protocatechuic acid, p-

hydroxybenzoic, vanillic acid, and syringic acid,

flavonoids, polyphenols, saponin glycosides,

terpenoids and steroids and gel-forming components

that are natural polysaccharides (Maslukhah et al.,

2016). The antioxidants in the phenolic compounds

enable black cincau to reduce blood fat (Fauzziyah

et al., 2016). Previous research has shown that black

jelly leaves extract with a dose of 130 mg/200 g

body weight can decrease cholesterol level by

22.44%, triglyceride by 26.95%, LDL by 42.39%

and the increase of HDL by 27.62% in white rats

(Amelia and Tri, 2014). Seventy percent ethanol

extract of black cincau leaves has higher in vitro

antioxidant activity compared to water extract

(Widyaningsih, 2013).

This study aims to determine the activity of 70%

ethanol extract of black cincau leaves with three

different dose variations in male hamsters with

hyperlipidemic condition. Fenofibrate and

atorvastatin are used as positive controls.

Observations were performed on total cholesterol,

triglycerides, LDL, and HDL levels.

2 MATERIALS AND METHODS

2.1 Extraction of Black cincau Leaves

The dried black cincau leaves obtained from Balai

Penelitian Tanaman Obat dan Aromatik

(BALITTRO), Bogor in March 2017. It was

extracted by maceration method using 70% ethanol

solvent. The filtrate was evaporated using vacuum

rotary evaporator (EYELA) until extract could be

obtained. The quality of the extract was determined

by identification of organoleptic, water content, and

phytochemical screening

2.2 Animal Preparation

Twenty-four male Syrian hamsters (Mesocricetus

auratus) aged 3-4 months around 50-100 g were

obtained from Research Animal Breeder, Bekasi.

This study used six groups with each group

consisting of four animals. Before treatment, the

animals were acclimatized for seven days. At this

stage, the animals were given standard drink and

feed. Except normal control groups, all the other

groups were made into hyperlipidemic conditions

with high-fat feeding (40% quail yolk, 10%

vegetable oil, and standard feed for hamsters’ ad

100%) for 28 days. The protocol no. 17-05-0488

was approved by the Health Research Ethics

Committee of the Faculty of Medicine, Universitas

Indonesia no. 459/UN2.F1/ETIK/2017.

2.3 Animal Test Treatment

All groups were treated for 14 days. Group I (normal

control group) was given standard feed each day,

group II (negative control group) was given Na-

CMC 0.5% each day, group III (positive control

group) was given fenofibrate at 1235 mg/Kg body

weight each day, group IV (positive control group)

was given atorvastatin at 2.4 mg/Kg body weight

each day, group V (dosage 1 test group) was given

the black cincau leaves extract at 780 mg/Kg body

weight each day, group VI (dosage 2 test group) was

given the black cincau leaves extract at 1560 mg/Kg

body weight each day, group VII (dosage 3 test

group) was given the black cincau leaves extract at

3120 mg/Kg body weight each day. On day 29 and

day 44, the animals were fasted and then

anaesthetized with an injection of ketamine with a

dose of 74 mg/Kg body weight. Blood sample was

taken through the orbital sinus. Blood sample was

collected in a microtube, then centrifuged for 15

minutes at 4000 rpm to obtain the blood serum for

measurement of total cholesterol, triglycerides, LDL

and HDL levels by enzymatic methods.

2.4 Measurement of Total Cholesterol

Level

Ten μl of serum was added with 1000 μl of enzyme

reagent, then homogenized using vortex and

incubated for 5 minutes at 37°C and analyzed with a

Microlab-300 clinical spectrophotometer.

2.5 Measurement of LDL Level

One hundred μl serum was added with 1000 μl of

precipitation reagent (a mixture of heparin and

sodium citrate solution). The mixture was

homogenized using a vortex and incubated at 37ºC

for 5 minutes, then was centrifuged at 4000 rpm and

was allowed to stand for 1 hour until LDL

precipitation was formed. One hundred μl of the

supernatant was mixed with 1000 μl of cholesterol

enzyme reagent. The mixture was homogenized

using a vortex and then incubated for 5 minutes at

37°C and analyzed with a Microlab-300 clinical

spectrophotometer.

2.6 Measurement of Triglyceride Level

Ten μl of blood serum was mixed with 1000 μl

triglyceride reagent kit, then was homogenized with

vortex and was incubated for 5 min at 37ºC.

Furthermore, it was analyzed using a Microlab-300

clinical spectrophotometer.

2.7 Measurement of HDL Level

Two hundred μl of serum was added with 500 μl of

precipitation reagent. The mixture was homogenized

using a vortex and was incubated at room

temperature for 10 minutes. It was centrifuged at

4000 rpm for 10 minutes. 100 µl of supernatant was

added with 1000 µl cholesterol reagent then was

incubated at 37ºC for 5 minutes. It was analyzed

with a Microlab-300 clinical spectrophotometer.

2.8 Data Analysis

The data were analyzed for normality and

homogeneity then were continued with one-way

ANOVA with 95% significance level (p<0,05). The

Tukey test was conducted to see a significant

difference between each group (Priyatno 2010).

3 RESULTS AND DISCUSSION

A total of 600 g of dried black cincau leaves was

extracted with 70% ethanol solvent produced 187.7

g extract. It was blackish-brown, viscous, and had

distinctive smell extract. The water content of the

extract was 8.7%. The extraction of black cincau

leaves with 70% ethanol revealed compounds of

flavonoids, saponins, phenolics, and

triterpenoids/steroids. The results of the chemical

content identification can be seen in Table 1.

Based on the test, the 70% ethanol extract of

black cincau with a dose of 3120 mg/kg body weight

could decrease total cholesterol level by 51.49%;

LDL level by 49.92%; and triglyceride level by

45.70%. It could also increase HDL level by 29.30%

in male hamsters. Fenofibrate was able to decrease

triglyceride level by 50.62% and increase HDL level

by 34.82%. Atorvastatin was able to decrease total

cholesterol level by 54.34% and LDL level by

51.13%. These results can be seen in Figure 1 to

Figure 4.

The 70% ethanol extract of black cincau leaves

contain compounds that could alter the lipid profile

Figure 1: The reduction percentage of cholesterol total

level from black cincau leaves 70% ethanol extract with

dosage variations and control groups. Error bars show the

standard deviation from the average of four data.

Figure 2: The reduction percentage of triglyceride level

from black cincau leaves 70% ethanol extract with dosage

variations and control groups. Error bars show the

standard deviation from the average of four data.

Table 1: Chemical content of 70% ethanol extract from

black cincau leaves

Chemical content Result

Alkaloids -

Flavonoids +

Saponin +

Phenolic +

Triterpenoids/Steroids +

Note: (+) = positive; (-) = negative

Anti-Hyperlipidemic Effect of 70

by reducing blood total cholesterol, triglycerides,

LDL and improving HDL levels. High consumption

of flavonoids has previously been reported to

contribute to a decreased risk of coronary heart

disease by lowering the serum cholesterol and

triglycerides in rats. The plasma cholesterol

concentration was lowered by rutin (one of

flavonoids groups). It has potential biological effect

on lowering the plasma cholesterol and hepatic

HMG-CoA reductase activity (Park et al., 2002).

Ruel et al., (2006) reported that flavonoid

consumption may be cardioprotective, and give a

favourable impact on circulating HDL-cholesterol

concentrations. Plant components that are known to

have the ability to decrease intestinal lipid

absorption are polyphenols, saponins and plant

sterols. Saponins from dietary Momordica dioica

powder prevent the development of fatty liver by the

inhibition of intestinal lipid absorption as a lipase

inhibitor in rats (Sato et al., 2011). Triterpenoidal

saponins provide inhibition of pancreatic lipase

enzymes (Lunagariya et al., 2014). Bioactive

terpenoids can modulate the activities of ligand-

dependent transcription factors, namely, peroxisome

proliferator-activated receptors (PPARs). Because

PPARs are dietary lipid sensors that control energy

homeostasis, daily eating of these terpenoids may be

useful for the management of obesity-induced

metabolic disorders such as type 2 diabetes,

hyperlipidemic, insulin resistance and cardiovascular

diseases (Goto et al., 2010).

4 CONCLUSIONS

Based on the research, 70% ethanol extract of black

cincau leaves at dose 3120 mg/Kg body weight is

able to decrease total cholesterol, triglyceride, LDL

and increase HDL level in male hamsters with

hyperlipidemic condition comparable to fenofibrate

and atorvastatin.

ACKNOWLEDGEMENTS

The authors would like to thank Lembaga Penelitian

dan Pengembangan Universitas Muhammadiyah

Prof. Dr. Hamka for internal research fund.

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Figure 4: The improvement percentage of HDL level from

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Anti-Hyperlipidemic Effect of 70