Phytochemical Screening of Phenolic Levels from Extracted Bitter

Mustard Leaves (Brassica Juncen L. Czern.) using UV-Visible

Spectrophotometer

Fahma Shufyani

, David Ginting

, Jhon Patar Sinurat

, Tika Afriani

, Muhammad Mabrur

, Ritmah

Syah Putri

Faculty of Pharmacy, Institut Kesehatan Medistra Lubuk Pakam, Sumatera Utara, Indonesia,

Department of Pharmacy, Mohammad Natsir University, Bukittinggi 26136, Indonesia

Keywords: Bitter Mustard, Brassica Juncea, Phenolic, Folin Ciocalteu and UV-Visible Spectrophotometer

Abstract: To determine the total phenolic content of the ethyl acetate extract of bitter mustard leaf. To determine the

total phenolic levels, the Folin – Ciocalteu method with gallic acid comparison compounds using ethyl

acetate solvents using UV-Visible spectrophotometer is expected to provide information and scientific

evidence to develop new medicines from this plant. Antioxidants are inhibitors of oxidation reactions due to

free radicals that can cause damage to unsaturated fatty acids, cell wall membranes, blood vessels, DNA

bases, and lipid tissue causing disease. Phytochemical screening was using FeCl

5% to identify phenolic

compound. Total phenolic is gotten by many process such maceration and partition. Analysis of total

phenolic was doing by TLC analysis. Total phenolic was analysed on plate of thin layer chromatography

with used chloroform and methanol with 70:30 comparison. Antioxidant test was measured by UV-Visible

spectrophotometer at 516 nm wavelength. Bitter mustard leaf extract contains phenolic compounds based on

phytochemical screening results. Weight total phenolic content o 39.7252 ± 0.7326 mg GAE/g extract, the

total phenol content determined according to the Folin-Ciocalteu medthod is not an absolute level, but

principally based on the reduction capacity of the material being tested against an equivalent reduction of

gallic acid. Calibration curve measurement with a concentrations of 200, 225, 250, 275 and 300 μg/ml. All

solutions were measured at a wavelength of 739.50 nm. Phytochemical screening results show that the

extract of bitter mustard leaf (Brassica juncea L.), contains chemicals, flavonoids, tannins, saponins,

glycosides, steroids / triterpenoids, anthraquinone and polyphenols. The acetyl extract of bitter mustard leaf

has a total phenolic activity value (39.7252 ± 0.7326) mg GAE / g extract. Penol compounds are susceptible

to oxidation at higher temperature in extractions that are to long can provide opportunities for phenol

compounds to oxidize more, but measured total phenol levels can be lower.

1 INTRODUCTION

Brassica juncea L. commonly known as Indian

mustard belongs to family brassicaceae. B. juncea is

an economically important plant widely used as an

oil source, a green vegetable and also having a

medicinal value. This species has been described in

traditional remedies in the ancient literature

(Manohar et al., 2009). Indian mustard is consumed

as leafy vegetable and is a source of various

micronutrients as well as antioxidants, vitamin c and

e, β-carotenoids etc. B. juncea is believed as eco-

friendly source for various nutraceuticals or drugs

which are used to prevent and cure of wide range of

non-communicable diseases in present time (Kumar

et al., 2011). Food preparation of Indian mustard

leaves is helpful in lowering the cost for diabetic

patients suffering with comorbid anxiety disorders

(Thakur et al., 2013). Plants of genus Brassica are

also known for the production of various volatile

organic compounds like ketones, aldehydes, esters,

alcohols, terpenes and glucosinolates.

The leaves of B. juncea are utilized to produce

medicines which act as stimulants, diuretics and

expectorants (Farrell et al., 1985). Indian mustard is

also known for its therapeutically pharmacological

uses due to its active bio-constituents (Kumar et al.,

2011). Glucosinolates and isothiocyanates are

reported to be very active in B. juncea (Hill et al.,

1987; McNaughton and Marks, 2003) which act as

anti-cancerous and anti-microbial compounds

(Luciano and Holley, 2009; Okulicz, 2010; Zhang et

Shufyani, F., Ginting, D., Sinurat, J., Afriani, T., Mabrur, M. and Putri, R.

Phytochemical Screening of Phenolic Levels from Extracted Bitter Mustard Leaves (Brassica juncen L. Czern.) using UV-Visible Spectrophotometer.

DOI: 10.5220/0009973605070514

In Proceedings of the International Conference on Health Informatics and Medical Application Technology (ICHIMAT 2019), pages 507-514

ISBN: 978-989-758-460-2

507

al., 2010). Leaves of Indian mustard were also

reported to have anti-depressant effects during

diabetes (Thakur et al., 2014). The presence of

different brassinosteroids namely castasterone,

teasterone, 24-epibrassinolide and typhasterol have

been reported from B. juncea (Kanwar et al., 2015).

Indonesia has the potential to grow vegetables.

Among the various types of vegetables that can be

cultivated, mustard (Brassica juncea L.) is one of the

vegetables that has commercial value and high

prospects. Mustard greens include leaf vegetable

plants from the Cruciferae family or cabbage plants

which have high economic value because it is rich in

fiber, high in nutritional content and has medicinal

properties. Some of the results of epidemiological

studies, it is known that eating vegetables from the

genus Brassica can reduce the risk of several types

of cancer, namely breast, prostate, kidney, colon,

bladder and lung cancer. Vegetables included in the

Brassicaceae tribe are mustard greens, bitter mustard

greens, cabbage, broccoli (Sulihandari, 2013).

Antioxidants are the ability to capture free

radicals. Radicals contained in biological systems

can oxidize nucleic acids, proteins, lipids or DNA

and cause degenerative diseases. Antioxidant

components found in plants such as phenolic acids,

polyphenols and flavonoids will capture free radicals

such as peroxide, hydroperoxide or peroxyl lipids

and also inhibit oxidative mechanisms that cause

degenerative diseases (Prakash, 2001). Extraction is

needed to obtain the desired compound in the bitter

mustard greens. Selection of the right solvent can

increase extraction efficiency. Things that need to be

considered in the selection of solvents include

selectivity, toxicity, polarity, ease of evaporation and

the price of solvents. Ethyl acetate is a solvent with

low toxicity that is semi-polar so it is expected to

attract polar and nonpolar compounds from bitter

mustard leaves (Akbar, 2010). Phytochemical

screening needs to be done to determine the class of

compounds contained in the extract used. In this

study phytochemical screening was conducted to see

the class of compounds in the ethyl acetate extract of

bitter mustard leaves so that it can also be known the

ability of the ethyl acetate solvent to attract

compounds contained in bitter mustard leaves

(Akbar, 2010).

Phenolic compounds are natural compounds

which are widely used at present. Its ability as an

active biological compound gives a large role to

human interests. One of them is as an antioxidant,

for the prevention and treatment of degenerative

diseases, cancer, premature aging and immune

system disorders in the body (Apsari, 2011). This

study aims to determine the total phenolic content of

the ethyl acetate extract of bitter mustard leaves. To

determine the total phenolic content, the Folin-

Ciocalteu method with gallic acid comparison

compound using ethyl acetate solvent using UV-

Visible Spectrophotometer is expected to provide

information and scientific evidence to develop new

medicines from this plant (Andarwulan, 2012).

2 METHOD

Place of research, The extract making is carried out

in Chemical laboratory, at Pharmacy Faculty,

Institut Kesehatan Medistra Lubuk Pakam. Research

time, This research is carried out on the month (May

2019 to September 2019). Material, Fresh bitter

mustard leaves, ethyl acetate extract from bitter

mustard leaf. Gallic acid, Hydrochloric acid,

Sulfuric acid, Iron (III) chloride, Sodium carbonate,

Sodium hydroxide, Mayer reaents, Bouchardate

reagents, Liebermann reagents Burchard, Folin –

Ciocalteau reagents and Methanol. Equipment:

Beaker Glass, Macerator, Separate Funnel (Schoot

Duran), Rotary evaporator (Heidolph), Steaming

waterbath (Memmert), TLC plate, Chamber,

Incubator (Memmert) and UV-Visible

Spectrophotometer (Shimadzu. Sample Processing,

this research was carried out sequentially in

laboratory with the following research scheme.

Process started from maceration and screening test,

then continued to evaporate solvent. Solid extract is

soluted by water to remove the lipid. Then filtrated

the fraction that soluted in water. Filtrate is partitied

using ethyl acetate conducted partition using n-

hexane by separate funnel. TLC is done to analysis

of total phenolic compound and measure of

antioxidant activity. The scheme of research is

showed in Figure 2.

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

508

Figure 2. Scheme of Research

Phytochemical Screening:

This research was conducted in the laboratory of

organic chemistry of natural materials, Institute

Medical Lubuk Pakam, Faculty of pharmacy to

determine the presence of phenolic compounds in

the leaves of the Bitter Mustard plant. A preliminary

test was carried out, phytochemical screening where

10 g fresh leaves of Bitter Mustard plant that had

been blended with a blender macerated with

methanol and then filtered. The filtrate was tested by

adding 3 drops of 5% FeCl

reagent solution,

forming a black precipitate if Bitter Mustard extract

is positive contained phenolic compound (Eko BM,

2015). Sample as 1000 g of Bitter Mustard leaves

powder which had been dried and finely macerated

for ± 24 hours with methanol as much as 5 liters at

room temperature. Macerate was filtered and a

extract of Bitter Mustard leaves was obtained.

Maceration was repeated using methanol as a

solvent until the methanol extract obtained gave a

negative test result with 5% FeCl

reagent. The

methanol extract obtained was concentrated by

rotary evaporator at a temperature of 60

C with a

rotation of 80 rpm. The following is the

determination of the wavelength of gallic acid with

the addition of the Flin Ciohalteu reagent, measured

in wavelength and Operating Time using a UV-

Visible Spectrophotometer. This is shown in Figure

Figure 3: Flow Chart Determination of Maximum

Wavelength and Operating Time

The following is the determination of the

standard acid gall curve using the Folin-Ciohalteu

reagent by dissolving the raw parent sample solution

from bitter mustard leaves, where the wavelength is

measured using a UV-Visible Spectrophotometer

tool. This is shown in Figure 4.

Figure 4: Flow Chart of Determination of Raw Acid Gallic

Curve with Folin-Ciocalteu Reagent

The following is the determination of the total

phenolic content of bitter mustard ethyl acetate

extract by dissolving a bitter mustard leaf sample

soltion, which is measured in wavelength using a

UV-Visible Spectrophotometer tool as shown in

Figure 5.

Phytochemical Screening of Phenolic Levels from Extracted Bitter Mustard Leaves (Brassica juncen L. Czern.) using UV-Visible

Spectrophotometer

509

Figure 5: Flowchart Determination of Total Phenolic

Content of Bitter Mustard Extract Ethyl Acetate

Bitter mustard leaf is considered dry if it is

fragile (when squeezed it will be easily destroyed),

then dried bitter mustard leaf is made into powder by

blending, then powder which has become weighed

dry weight and obtained as much as 1 kg of powder

weight. Simplicia powder that has been weighed is

then put into a dry plastic bag that is well closed,

protected from sunlight and protected from heat. A

total of 500 g of simplex powder of bitter mustard

leaves were macerated using ethyl acetate solvent.

For the first treatment 3750 ml of ethyl acetate was

used in a tightly closed container and protected from

sunlight for 5 days, the maserat could be separated

into another container, the pulp was macerated again

with 1250 ml of ethyl acetate for 2 days, then

filtered so that it was obtained by the maserate. The

first and second maserates obtained were transferred

to another container which was tightly closed and

then evaporated in a rotary evaporator so that a thick

extract was obtained.

Determination of water content is done by the

azeotropic method (toluene distillation). The device

consists of a 500 ml round bottom flask, a container,

a ball cooler, a connecting tube and a 0.1 ml scale

receiving tube. A total of 200 ml of toluene and 2 ml

of distilled water were put into a round bottom flask,

a container was installed and cooled, then distilled

for 2 hours. The distillation is stopped and allowed

to cool, then the volume of water in the receiver tube

is read as the initial volume of water with an

accuracy of 0.05 ml. In a round bottom flask

containing saturated toluene, 5 g of driedia bitter

mustard leaf powder which has been carefully

weighed is then carefully heated for 15 minutes.

Toluent to boil, the droplet speed is set to 2 drops

per second until as distilled water, then the

distillation speed is increased to 4 drops per second,

after all the water is distilled, the inside of the cooler

is rinsed with saturated toluene. The distillation is

continued for 5 minutes, then the receiver tube is

allowed to cool at room temperature, after the water

and toluene separate completely, the volume of

water is read as the final volume with accuracy of

0.05 ml. The second difference in the volume of

water reads according to the water content contained

in the material being examined.

As much as 2.5 ml of 1000 μg gallic acid

solution was put into a 10 ml volumetric flask, then

stirred up to the mark with ethanol, then pipetted 0.2

ml put into a 25 ml flask, then added 15.8 ml of

distilled water and 1 ml of Folin-reagent Ciocalteu,

then shaken and allowed to stand for 8 minutes. To

the solution was added 3 ml of Na

solution,

shaken until homogeneous and allowed to stand for

2 hours at room temperature, then the absorbance

was measured at a wavelength of 400-800 nm. As

much as 2.5 ml of 1000 μg gallic acid solution was

put into a 10 ml volumetric flask, then stirred up to

the mark line, then pipetted 0.2 ml put into a 25 ml

flask, then added 15.8 ml of distilled water and 1 ml

of Folin-Ciocalteu reagent, then shake until

homogeneous and sit for 8 minutes. To the solution

was added 3 ml of Na

solution, then shaken

homogeneously and allowed to stand for 2 hours at

room temperature, measured its absorbance in the

span of 1-25 minutes wavelength 739.50 nm. From

the gallic acid mother liquor concentration of 1000

μg / ml pipetted 2, 2, 25, 2, 5, 2, 75 and 3 ml then

put into a 10 ml flask, then stirred to the mark line

with ethanol. From each pipette 0.2 ml was put into

a 25 ml flask and then added 15.8 ml of aquadest

and 1 ml of the Folin-Ciocalteu reagent were shaken

until homogeneous, allowed to stand for 8 minutes.

To the solution was added 3 ml of Na

solution

then shaken homogeneously, allowed to stand for 2

hours at room temperature. The absorbance was

measured at a wavelength of 739.50 nm to obtain

concentrations of 200, 225, 250, 275 and 300 μg /

ml.

A total of 100 mg of bitter mustard extract was

dissolved with 10 ml of distilled water to obtain a

concentration of 10 mg / ml. From a concentration

of 10 mg / ml pipette 5 ml put into a 10 ml flask,

then stirred up to the mark line, then pipette 0.2 ml

extract put into a 25 ml flask then added 15.8 ml of

distilled water and 1 ml of Folin-Ciocalteu reagent,

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

510

shaken homogeneously let stand for 8 minutes then

add 3 ml of Na

solution. allowed to stand for 2

hours at room temperature. The absorbance of the

extract solution was measured with a UV-Visible

Spectrophotometer.

3 RESULT AND DISCUSSION

The results of phytochemical screening of bitter

mustard seed ethanol extract. Phytochemical

screening uses 5% FeCl3 reagent where the sample

was previously dissolved with methanol solvent

repeatedly. In this case, the extract becomes a black

precipitate after decreasing 5% FeCl3 while the

previous extract is green. The screening results are

tested in a test tube. The black sediment in question

is bitter mustard leaf which contains phenolic. The

maceration process is processed on a sample of

bitter mustard leaf powder in macerator. Maceration

is treated repeatedly to maximize the extract

produced. Samples as macerated solid extracts in

methanol solvents were obtained at 300 g. This

method is carried out by inserting suitable plant

powders and solvents into a humidly closed

container at room temperature. The principle of

maceration method is based that samples soaked

using organic solvents will break down the cell walls

and membranes due to pressure differences found

outside and inside the cell so that secondary

metabolites contained in the cytoplasm will dissolve

into organic solvents. The extraction process is

stopped when a balance is reached between the

concentration of the compound in the solvent and

the concentration in the plant cell18.

After maceration, the partition was carried out

using ethyl acetate to obtain a 600 g solid extract.

The last partition was carried out using n-hexane to

partition non-polar compounds from phenolic

compounds. the actual total phenolic content was

39.7252 ± 0.7326 mg GAE / g extract, the total

phenol content determined according to the Folin-

Ciocalteu method was not an absolute level, but in

principle based on the reduction capacity of the

material being tested against an equivalent reduction

of gallic acid. Bitter mustard leaf extract from the

partition contains total phenolic because it reacts

positively to the FeCl3 reagent when we do the

filtering again. In the liquid-liquid partition process,

the two phases of solution have different solubility.

Separate funnel shocks during partition aim to

expand the contact surface area between insoluble

solvents. The solvent requirements for the partition

method have suitable polarity for the extracted

material and must be separated after shaking. Total

phenol levels are influenced by the type of solvent.

Phenol is a polar compound so that its solubility is

highest in a semi-polar solvent. Polar solvents are

able to dissolve phenols so that the levels in extracts

are high. Phenol compounds are susceptible to

oxidation at high temperatures so that they

experience degredation while extraction that is too

long can provide an opportunity for phenol

compounds to oxidize more, but the total phenol

levels measured can be even lower.

Phytochemical screening tests were conducted to

determine the class of chemical compounds

contained in bitter mustard leaves using ethyl acetate

extract. Based on phytochemical screening

conducted on ethyl acetate extract is a low toxicity

solvent that is semi polar so that it can attract polar

and non polar compounds from bitter mustard

leaves. Bitter mustard leaves contain flavonoids,

alkaloids, glycosides, tannins, saponins, steroids /

triterpenoids and anthraquinones. On examination of

flavonoids with the addition of hydrochloric acid to

the Mg powder, it gives a red color which indicates

the presence of flavonoid compounds. Alkaloide

examination with Mayer, Dragendorff and

Bouchardat reagents results in turbidity and

deposition. The examination of anthraquinone

glycosides with the addition of NaOH to the

simplicia powder gives a positive red color, while

the extract does not form red. Tannin examination

with the addition of FeCl

solution will show blue or

blackish green discoloration (Harborne, 1996).

Determination of total phenolic levels using the

spectrophotometric method of visible light with the

Folin-Ciocalteu reagent is the simplest, easiest

method, using a relatively small number of samples

and a shorter processing time. Total phenolic testing

begins with the measurement of the maximum

wavelength of gallic acid solution with a

concentration of 1000 μg / ml in ethanol using a

Visible spectrophotometer to obtain a wavelength of

739.50 nm with an absorbance of 0.340.

The color of the Folin-Ciocalteu reagent standard

solution is usually less stable so it is necessary to

find the right working time to take measurements

because the amount of absorbance in the

spectrophotometry of the visible light is strongly

influenced by the color. Determination of working

time is done by using a standard solution of the

Folin-Ciocalteu reagent accompanied by the addition

of an extract which aims to find a stable

measurement time when the sample reacts

completely with a color reagent, measured at a

wavelength of 739.50 nm. Operating Time

Phytochemical Screening of Phenolic Levels from Extracted Bitter Mustard Leaves (Brassica juncen L. Czern.) using UV-Visible

Spectrophotometer

511

measurement results obtained results in minutes 23-

25.

According to Rahmawati research, 2015.

Analysis of flavonoid and phenolic levels was done

using the Chang method, then the wavelength

optimization was performed to determine the

maximum wavelength to be used in measurements

on UV-Visible Spectrophotometer. From the

measurement results obtained a maximum

wavelength of 415 nm for flavonoids and 730 nm for

phenolics. Quantitative analysis of flavonoids was

carried out by making a series of regular standard

solution concentrations of 3.75 ppm, 5 ppm, 6.25

ppm, 7.5 ppm, 8.75 ppm and 10 ppm, from each

concentration in a 1 ml pipette, then add 3 ml of

95% ethanol 0.2 ml of aluminum chloride 10% 0.2

ml (to make a shift towards a longer wavelength,

thus changing the standard wavelength routine to

enter the Spectrophotometer of UV-Visible

wavelength range).

Determination of total phenolic levels using the

spectrophotometric method of visible light with the

Folin-Ciocalteu reagent is the simplest, easiest

method, using a relatively small number of samples

and a shorter processing time. Total phenolic testing

begins with the measurement of the maximum

wavelength of gallic acid solution concentration of

1000 μg / ml in ethanol using a Spectrophotometer

of UV-Visible to obtain a wavelength of 739.50 nm

with an absorbance of 0.340. According to Gandjar

and Rohman (2007) the blue color will produce

maximum absorbance at a maximum wavelength of

400-800 nm.

Calibration curve measurements were carried out

with different solution concentrations pipetted from

gallic acid solution at a concentration of 1000 μg /

ml. Obtained concentrations of 200, 225, 250, 275

and 300 μg / ml, put into a volumetric flask added 1

ml of Folin-Ciocalteu reagent and shaken after it is

allowed to stand for 8 minutes, each solution plus 3

ml of Na

beaten homogeneously and allowed to

stand for 2 hours at room temperature to perfect the

reaction. All solutions were measured at a

wavelength of 739.50 nm, then a calibration curve

was made between the concentration of gallic acid

(μg / m) and absorbance. Calibration curve

measurements were carried out with concentrations

of different solutions pipetted from gallic acid

solution at concentrations of 1000 μg/ml with

concentrations of 200, 225, 250, 275 and 300 μg/ml.

The results of the standard absorbance of gallic acid

by using a sample of bitter mustard leaf extract

based on the concentration and absorbance of the

regression equation measured with wavelength using

the Spectrophtometer of UV-Visible tool.

Table 1: Standard Absorbance of Gallic Acid

Sampel Konse

ntrasi

Absorb

ansi

Persamaan

Regresi

Ekstrak Daun

Sawi Pahit

0,00 0,000

Y =

0,08659 x

+ 0,00046

2,00 0,175

2,25 0,197

2,50 0,216

2,75 0,235

3,00 0,262

From this table a calibration curve is obtained as

shown in the following figure:

The following are the results of the calibration

curve for gallic acid compounds which were

measured using Spectrophtometer of UV-Visible at

a maximum wavelength of 515 nm. The regression

equation used in determining the total phenolic

compound content is Y = 0.08569 x + 0.00046 with

a correlation coefficient of 0.999940. The linearity

value shows the correlation between the

concentration and the absorbance produced. Testing

the total phenolic content is calculated by

substituting the absorbance value (y) of the sample

at the maximum wavelength into the linear

regression equation y = ax + b obtained from the

gallic acid calibration curve so that the concentration

(x) is obtained. The value of x is then substituted in

the formula for calculating total phenol levels.

Measurement of total phenol levels was carried out

by repetition 6 times and the average was taken as

presented in table 2 below.

From the results of the above study, the actual

total phenolic content was 39.7252 ± 0.7326 mg

GAE / g extract, the total phenol content determined

according to the Folin-Ciocalteu method was not an

absolute level, but in principle based on the

reduction capacity of the material tested against an

equivalent reduction of gallic acid. Total phenol

levels are influenced by the type of solvent. Phenol

is a polar compound so that its solubility is highest

in a semi-polar solvent. Polar solvents are able to

dissolve phenols so that the levels in extracts are

high. Phenol compounds are susceptible to oxidation

at high temperatures so that they experience

degredation while extraction that is too long can

provide an opportunity for phenol compounds to

oxidize more, but the measured levels of total phenol

can be lower. Testing the total phenolic content is

calculated by substituting the absorbance value (y)

of the sample at the maximum wavelength into the

ICHIMAT 2019 - International Conference on Health Informatics and Medical Application Technology

512

linear regression equation y = ax + b obtained from

the gallic acid calibration curve so that the

concentration (x) is obtained. The value of x is then

substituted in the formula for calculating total

phenol levels. Total phenol levels are influenced by

the type of solvent. Phenol is a polar compound so

that its solubility is highest in a semi-polar solvent.

Polar solvents are able to dissolve phenols so that

the levels in extracts are high. Phenol compounds

are susceptible to oxidation at high temperatures so

that they experience degredation while extraction

that is too long can provide an opportunity for

phenol compounds to oxidize more, but the

measured levels of total phenol can be lower.

4 CONCLUSIONS

After maceration and partition, a total phenolic

compound is obtained from saputangan leaves as

18.25 g. Results of thin layer chromatography

analysis of total phenolics using the chloroform :

methanol as eluent showed that total phenolic has 3

spots that have an Rf of 0.44 ; 0.29 and 0.22. The

total phenolic compound is able to act as a strong

antioxidant by having an IC

value of 15.22 ppm.

This antioxidant test was carried out using a DPPH

(2,2-diphenyl-1- picrilhidrazil) which was

measured using a UV-Visible spectrophotometer at

a wavelength of 516 nm.

ACKNOWLEDGEMENTS

This Research was supported by Institut Kesehatan

MEDISTRA Lubuk Pakam, Indonesia and

Grandmed Hospital, Indonesia

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