The Potency of Binahong Leaves (Anredera cordifolia (Ten.) Steenis)

Subfraction with Ethanol 70% as an Antihyperuricemic Agent

Vera Ladeska, Ani Pahriyani and Monika Silviani Gunawijaya

Faculty of Pharmacy and Science, Universitas Muhammadiyah Prof. DR. HAMKA, Jakarta, Indonesia

Keywords: Uric acid, Anredera cordifolia (Ten.) Steenis, antihyperuricemic, Binahong leaves.

Abstract: Hyperuricemia is an abnormally high level of uric acid in a blood. Binahong leaves (Anredera cordifolia

(Ten.) Steenis) is one of the plants traditionally used as an antihyperuricemic remedy. This study aims to

determine the ethanol 70% subfraction activity of binahong leaves on the uric acid level of male white mice.

Antihyperuricemia assay was conducted for 36 days by dividing 24 mice into six groups. There are normal

control who was given standard feed and Na CMC 0.5%, positive control was given purine and allopurinol

0.8 mg/20 g BW and the assay group was given a purine feed and binahong leaves subfraction SF 3 with a

dose of 1.83 mg/20gBW, 3.60 mg/20gBW, and 5.40 mg/20gBW. Blood sampling was conducted by orbital

sinus after 2 hours from induction of potassium oxonate. Blood sampling was measured with an enzymatic

method using a clinical spectrophotometer. The result showed that the third dose had no significant

difference to the positive control with a percentage of decrease of 56.6%. The conclusion is that binahong

leaves subfraction has the same activity as an antihyperuricemic agent with allopurinol at dose 5.40

mg/20gBW.

1 INTRODUCTION

Indonesia is currently facing health problems of

Non-Communicable Diseases (NCDs), which tend

to increase every year. The Ministry of Health RI

(2016) reported that the number of death from NCDs

rose from 37% in 1990 to 57% in 2015. The

increased deaths can be caused by changes in diet

with imbalance nutrition (Kemenkes RI, 2011). One

sign of NCDs is due to dietary changes that lead to

increased levels of purine in the body causing

hyperuricemia (Purwaningsih, 2009). According to

an epidemiological survey conducted in Bandungan,

Central Java in a WHO-COPCORD collaboration

(2015) 4,683 samples aged between 15-45 years the

prevalence of hyperuricemia was 24.3% in males

and 11.7% in women. This disease can be grouped

into primary gout that commonly occurs (90% of

cases) and which cause is unknown clearly, and

secondary gout (10% of cases) that is experienced

by women after menopause due to hormone

imbalance (Daniati, 2015).

The number of side effects that arise from the

use of synthetic drugs and the long duration of

therapy has become a problem in the health field.

The development of herbal medicine can be a

solution to the problems considering the widespride

existance of medicinal plants in Indonesia. Binahong

leaves (Anredera cordifolia (Ten.) Steenis) are

traditionally used to treat gout, heart, diabetes,

stroke, asthma, acne, influenza, stiff, burn and so on

(Susetya, 2012). Binahong leaves contains active

compounds such as saponins, polyphenols,

flavonoids and polysaccharides (Rachmawati, 2008).

Flavonoid compounds are suspected to inhibit the

enzyme xanthine oxidase, which can inhibit the

formation of uric acid (Lin, 2002).

Binahong leaves extracted with ethanol 70% has

proven to decrease the level of uric acid at dose 200

mg/kgBW 91,83% from 3,56 mg/dl to 178 mg/dl

(Lidinilla, 2014). Fraction binahong leaves with

ethanol 70% at dose of 3,66 mg/20gBW has been

proven to decrease the level of uric acid from 4.048

mg/dl to 1.403 mg/dl. Based on the results of

previous research (Mutiarini, 2015), it is necessary

to further research into the subfraction stage in order

to produce a purer and cleaner compound of impure

compounds by column chromatography method.

The Potency of Binahong Leaves (Anredera cordifolia (Ten.) Steenis) Subfraction with Ethanol 70 .

DOI: 10.5220/0008240801430146

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 143-146

ISBN: 978-989-758-349-0

143

2 MATERIAL AND METHODS

2.1 Material

Mice, Vacuum Rotary Evaporator, Microcentrifuge,

column chromatography, Vortex Mixer, micropipet,

Clinical Spectrophotometer (Microlab 300), TLC

plate (silica gel GF 254), binahong leaves (Anredera

cordifolia (Ten.) Steenis), potassium oxonate (Sigma

Aldrich Chemical), allopurinol, ketamine.

2.2 Methods

2.2.1 Sample Preparation

Binahong leaves taken from BALITTRO was dried

by sun and covered with black cloth. Binahong

leaves were then powdered and sieved with mesh

number 40.

2.2.2 Mice Preparation

Twenty-four mice (Indonesian Institute of Sciences,

region Indonesia) were acclimatized and were fed

with standard feed. They were then divided into six

test groups of four mice for antihyperurecemic test.

2.2.3 Extraction

The simplicia powder was macerated with ethanol

70% for three times, then filtered. The resulting

mixture was collected and was evaporated with a

vacuum rotary evaporator until a viscous extract was

obtained.

2.2.4 Fractionation

A total of 170 g of binahong extract was fractionated

with n-hexane and ethanol - water in a separating

funnel – and was shaken for 15 minutes. After that it

was allowed to stand to form 2 layers (n-hexane at

the top and ethanol-water at the bottom). The

ethanol coating: water was fractionated back with an

ethyl acetate solvent, and then was rehydrated for 15

minutes. After that, it was allowed to stand to form

two layers (ethyl acetate at the top and ethanol:

water at the bottom). Each treatment was repeated

until the top layer was clear then all the fractions of

n-hexane, ethyl acetate and ethanol were evaporated

with a vacuum rotary evaporator.

2.2.5 Subfraction Proccess

An ethanol fraction was used as much as 20 g by

making wet column chromatography using a mixture

of n-hexane gradient solvent: ethyl acetate and ethyl

acetate - methanol in a ratio of 10: to 0:10.

2.2.6 Phytochemical Screening

Phytochemical screening was performed to test the

presence of groups of alkaloids, saponins, tannins,

flavonoids, and terpenoids with TLC method. The

stationary phase employed was a GF254 silica gel

plate with a mobile phase system and a detection

reagent adjusted to each of the detected compounds.

Silica Gel GF245 as a stationary phase and ethyl

acetate – methanol - ammonia (4-1-1) as a mobile

phase. The principle of separation on TLC based on

absorption and partition. TLC method was chosen

because can describe a chromatographic pattern of

samples, has a simple procedure and diverse motion

phases (Hanani, 2014).

2.2.7 Antihyperuricemic Test

From Day 15 to Day 28, all test group were induced

orally with high purine feeds of chicken liver juice

(200 g/100 ml) while the normal control was given

standard feed and 0.5 ml of Na-CMC. On Day 29

and Day 36, blood samples were taken. Uric acid

level was measured 2 hours after intraperitoneal

administration of potassium oxonate induction at 6

mg/20 g to all groups except Group I. From Day 29

to Day 36, the feed was continued to be given orally

according to the treatment group and was suspended

using Na-CMC. Serum was taken as much as 20 μl,

1000 μl of uric acid kit reagent (Human), then was

mixed in the vortex and was incubated for 5 minutes

at 37 ° C. The values of uric acid levels were read by

clinical spectrophotometer.

The following is the division of animal groups:

• Group I as a normal control (standard feed

with Na-CMC solution).

• Group II as a negative control (high purine

feed with Na-CMC solution).

• Group III as a positive control (high purine

feed with allopurinol at dose 0.8 mg/20gBW).

• Group IV as Dose 1 assay (high Purine feed

with binahong leaves subfraction at dose 1.83

mg/20gBW).

• Group V as a Dose 2 assay (high purine feed

with binahong leaves subfraction at dose of

3.60 mg/20gBW).

144

• Group VI as a Dose 3 assay (high purine feed

with binahong leaves subfraction at dose 5.40

mg/20gBW).

2.2.8 Statistical Analysis

Data was analyzed by one-way ANOVA which have

previously tested for normality and homogeneity.

The data then continued with Pos Hoc Tukey test to

know the differences between groups.

3 RESULTS AND DISCUSSION

One of the plants that can be used as herbal remedies

is the leaf of binahong (Anredera cordifolia (Ten.)

Steenis) which traditionally treats gout, heart

disease, diabetes, stroke, asthma, acne, influenza,

stiff, burn and so on (Susetya, 2012) . Binahong

contains active compounds such as saponins,

polyphenols, flavonoids and polysaccharides

(Rachmawati, 2008).

Table 1 shows the results of the extraction

process, The percentage of subfraction yields is

26.76%. The percentage of yields can show the

effectiveness in determining the appropriate method

for the process. The calculation of yield aims to find

out how much recovery of secondary metabolite

compounds in the subfraction. In the ethanol

subfraction results, 70% of the SF 3 binahong leaf

was carried out with a drying rate of 5.86 % in order

to see the quality of the obtained subfraction. The

compound may be a volatile oil residue, organic

solvent or water contained in there. The dry

shrinkage percentage of the binahong leaves was

less than 10 %. Thus, the result of drying drift is

<10%, it can be said the quality of the good

subfraction results.

The preparation of subfraction using column

chromatography resulted from 20 g ethanol fraction

obtained 5 stain which then continued checking of

flavonoid compound. There are four spot were

confirmed positive of containing flavonoids.

Flavonoids are phenolic compounds because their

color will change to purple light color if they are

added with bases or ammonia. In addition,

flavonoids contain conjugated aromatic compounds

because they exhibit strong absorption bands in UV

light (Harborne, 1987).

The results of flavonoid checking of the 5

subfraction stains. The positive results, which

contain flavonoids, are given a yellow circle (figure

1). The results of binahong leaves subfraction with

ethanol 70 % has the most flavonoid stain which

dominant among the other. The Rf values obtained

from SF 3 are 0,41; 0,87; and 0,94. Thus, SF 3 was

selected to continue testing of antihyperuricemia

activity because it has the dominant flavonoids

among other stains. Flavonoid compound are

suspected to inhibit xanthine oxidase which can

inhibit the formation of uric acid.

The next step is phytochemical screening. As

shown in Table 2, the binahong leaves contain

flavonoids compound only. Due to the process of

column chromatography with mixed motion phases

and variations in the phase comparison of motion.

The measurements of uric acid levels in white

Figure 1. Thin layer chromatography results with Rf

value (a) 0,94; (b) 0,87; (c) 0,90; (d) 0,43; (e) 0,41;

(f) 0,20.

Table 1. The results of sample preparation

No. Data Results

1. The wei

ht of sim

licia

owde

1,75 k

2. Thick extract of binahong

leaves

186,93 g

4. Fraction of binahon

leaves 46,21

5. Subfraction of binahong leaves

SF 3

5,35 g

6. Subrafraction of yield of

inahon

leaves SF 3

26,76 %

7. Subfraction of dry loss of

inahon

SF 3

5,86 %

Tabel 2. The result of phytochemical screening of

binahong leaves SF 3 subfraction yield using TLC

method.

No. Secondary Metabolite Result

1. Alkaloi

2. Flavonoi

3. Sa

onin -

4. Tanin -

5. Steroi

Note: (+) Positive, (-) Negative

The Potency of Binahong Leaves (Anredera cordifolia (Ten.) Steenis) Subfraction with Ethanol 70

145

male mice induced high purine feed in the form of

chicken liver juice can be seen in Table 3 the

significance value of homogeneity test was 0.122

(p>0.05) which show that the data is homogeneously

distributed. The Shapiro-Wilk test indicates that the

data is normally distributed. The significant values

was less than 0.05 which stated a significant

difference in the treatment.

The result of the Tuckey test showed no

significant differences between negative control,

dose 1 dan dose 2 groups which is presumably

because the dose given is too small to affect the

small activity at this dose. There was also no

significant difference between positive control group

and dose 3 group. Binahong leaves subfraction with

dose 5.40 mg/20 gBW have equal lowering uric acid

activity with allopurinol group (table 3).

The binahong leaves subfraction has a flavonoid

compounds that have antihiperuricemic activity.

Allopurinol has the same mechanism of action with

flavonoids in reducing uric acid level. It contains

oxipurinol, the main metabolite, as xanthine oxidase

inhibitors that the conversion of hypoxanthine to

xanthine, and xanthine to uric acid (Sukandar et al.,

2009)

4 CONCLUSIONS

Based on the results of this research, ethanol

subfraction of 70% of binahong leaves (Anredera

cordifolia (Ten.) Steenis) SF 3 obtained has

antihyperurisemic activity. The activity of

decreasing uric acid level in dose 3 (5.40 mg/20

gBW) is proportional to allopurinol.

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Dianati NA.. 2015. Gout and Hyperuricemia. J Majority:

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Hanani E. 2014. Analisis Fitokimia. Jakarta: Buku

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Kementerian Kesehatan RI. 2011. Infodatin Pusat Data

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RI.http://www.depkes.go.id/ Diakses 9 Februari 2017.

Lidinilla NG. 2014. Uji Aktivitas Ekstrak Etanol 70%

Daun Binahong (Anredera cordifolia (Ten.) Steenis)

terhadap Penurunan Kadar Asam Urat dalam Darah

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Tabel 3. The percentage of uric acid decline

Group Baseline ±

SD (mg/dl)*

Final Level ±

SD (mg/dl)*

Percentage

(%)

Normal

control

1,08 ± 0,06 1,07 ± 0.09 0,09

Positive

control

3,14 ± 0,27 1,18 ± 0,17 62,44

Dose 1 3,42 ± 0,55 2,48 ± 0,37 27,44

Dose 2 2,81 ± 0,35 2,03 ± 0,23 27,68

Dose 3 3,67 ± 0,23 1,59 ± 0,08 56,6

*Note: average of 5 experimental mice

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