Identification and Determination of Total Flavonoids in Ethanol

Extract of Old and Young Angsana Leaves (Pterocarpus indicus

Willd.) Using Visible Spectrophotometry

M.A.H.F. Fernanda

, R.D. Andriani

, Z. Estulenggani

and G.G. Kusumo

Faculty of Pharmacy, Universitas Airlangga, Jl. Dharmawangsa Dalam No. 4-6, Surabaya, Indonesia

Akademi Farmasi Surabaya, Jl. Ketintang Madya No.81, Surabaya, Indonesia

Keywords: Total Flavonoids, Ethanol Extract, Angsana Leaves, Spectrophotometry.

Abstract: Angsana (Pterocarpus indicus Willd.) is a forest plant widely used as an ornamental garden and as a shade.

In many countries, Angsana is used as a traditional medicine, such as an antidiabetic drug. The compounds

that act as antidiabetes are flavonoids. The purpose of this study was to determine the total flavonoid

content of ethanol extract of old and young Angsana leaves using visible spectrophotometry. The wilstater

test was performed as a qualitative research test to show that the ethanol extract of Angsana leaves contains

flavonoid compounds. AlCl

was selected as a reagent in visible spectrophotometry to determine the

flavonoid of Angsana leaf. The result of the accuracy test was 83.67% with acceptance criterion 80-120%

and the precision test is 8.96% with acceptance criterion ≤10%. The total flavonoid levels of ethanol extract

of old and young Angsana leaves were respectively 7.53 ± 0.32% w/w and 3.31 ± 0.07 % w/w.

1 INTRODUCTION

Angsana is one type of forest plant that is widely

used as a protective tree and decoration for city

parks. Angsana is now widely cultivated. In big

cities, Angsana easily found on the highway.

Angsana trees are dense and have beautiful flowers,

so they are widely used as urban decorative plants,

especially as shade plants, noise absorber, and

pollution absorber (Bramasto et al., 2015). In many

countries, Angsana is used as a traditional medicine.

In Indonesia, the young leaves of Angsana are used

as an ulcer medicine and the rash of prickly heat. In

recent years, the Philippines has launched an

Angsana extract product in the form of herbal tea

preparations and pills used to treat leprosy,

menstrual pain, flu, rheumatoid arthritis, and

diabetes (Thomson, 2006). The Chemical content of

Angsana leaves showed positive tests of phenol,

flavonoid, saponin, triterpenoid and tannin

compounds (Junanto et al., 2008). The general

public knowledge around utilizing old Angsana

leaves is limited to shade and as animal feed. The

public has not come to know that the leaves of old

Angsana can also be used as a traditional medicine.

Until now, there has been no research that identifies

and determines the total flavonoid levels in old and

young Angsana leaves. For that, the researchers

intend to do research on the identification and

determination of flavonoid levels of old and young

Angsana leaves. Flavonoid compounds are generally

slightly soluble in water since 96% ethanol was used

as the extraction solvent. The identification was

done by wilstater test and determination of flavonoid

level by a visible spectrophotometric method with

the AlCl

reagent.

2 MATERIAL AND METHOD

2.1 Plant Material

Angsana leaves were collected from the Sidoarjo

and Gresik Regencies. Plant determination of

Angsana was done by LIPI, Purwodadi, Pasuruan.

2.2 Chemical

96% ethanol, standard quercetin (Sigma), aluminium

chloride (AlCl

), sodium acetate, aquadest, and

magnesium.

Fernanda, M., Andriani, R., Estulenggani, Z. and Kusumo, G.

Identiﬁcation and Determination of Total Flavonoids in Ethanol Extract of Old and Young Angsana Leaves (Pterocarpus indicus Willd.) Using Visible Spectrophotometry.

DOI: 10.5220/0007546605410544

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 541-544

ISBN: 978-989-758-348-3

541

2.3 Instrument

UV-Vis Spectrophotometer X-ma 1200 Human

Corp., macerator, vacuum rotary evaporator,

analytical scales, and analytic glassware.

2.4 Preparation and Extraction

Angsana leaves, both old and young, were taken at

random and then sorted and washed until clean, then

chopped and dried respectively. The dry leaves were

then milled. The preparation of the extract was

carried out by maceration by dissolving 200 g of

simplicia with 1000 mL of 96% ethanol solvent. The

result of maceration was then concentrated using

Rotary Vaccum Evaporator until a viscous extract

was obtained.

2.5 Qualitative Test

2 ml of Angsana leaves extract were taken and put

into a tube. 0.5 ml of concentrated HCl and 0.02 mg

of Magnesium were then added and mixed. The

presence of flavonoids is characterized by the

occurrence of discoloration. The reduction with

concentrated Mg and HCl produced red, yellow or

orange colors (Robinson, 1995).

2.6 Quantitative Test

2.6.1 Quercetin Standard Curve

Quercetin was weighed for as much as 50 mg and

inserted into a 50 mL measuring flask, then

dissolved with 96% ethanol. Then, it was diluted

through 20 consecutive concentrations; 40; 60; 80;

and 100 ppm. 5 ml were added in 15 ml of 96%

ethanol, 1 ml of 10% aluminum chloride, 1 ml of 1

M sodium acetate, and 28 ml of aquadest. Then, the

mixture was incubated at room temperature for 30

minutes. The blank sample production was done

without the addition of aluminum chloride. The next

stage was the measurement standard curve level

using visible spectrophotometry with a wavelength

of 439 nm (Chang et al., 2002). Then, a calibration

curve was made by connecting the absorption value

and the concentration.

2.6.2 Determination of Flavonoid Levels

Samples of 100 mg were weighed and inserted in a

100 mL measuring flask and then dissolved with

96% ethanol. Samples of 5.0 mL were each added

with 15 ml of 96% ethanol, 1 ml AlCl3, 1 ml of 1 M

sodium acetate, and 28 ml of aquadest. Then, they

were incubated at room temperature for 30 minutes.

The next stage was sample rate measurement using

visible spectrophotometry with a wavelength of 439

nm.

2.6.3 Accuracy and Precision

A sample of 50 mg was weighed and inserted in a 50

mL measuring flask and then dissolved with 96%

ethanol. A standard of 1000 ppm was made by

weighing 50 mg quercetin dissolved in 50 ml

ethanol 96%. 1 ml sample 1000 ppm was extracted

using pipette and inserted into 100 ml measuring

flask. 6 ml of 1000 ppm quercetin solution was

added until the water surface reached the limit

indicator. The flask was then shaken until the

solution was perfectly mixed. 5 ml of each sample

was extracted using pipette and added with 15 ml of

96% ethanol, 1 ml of aluminum chloride, 1 ml of 1

M sodium acetate and 28 ml of aquadest. Then, it

was incubated at room temperature for 30 minutes.

The next step was to measure the sample content

using visible spectrophotometry with a wavelength

of 439 nm (Chang et al., 2002). The procedure was

replicated 6 times (Riyanto, 2014).

3 RESULTS AND DISCUSSION

Flavonoid compounds can be separated from various

other compounds by maceration using 96% ethanol

solvent for analysis. The maceration results were

then concentrated using a vacuum rotary evaporator,

so as to produce a sample in the form of thick

extracts. Then, the extract was evaporated again

through aeration to produce dry extract. The

separated flavonoids were determined using visible

spectrophotometry. The standard comparison used

was quercetin, where quercetin is a type of flavonoid

compound that is most widely distributed in nature.

The results of determination indicate that the

sample used in this study was Angsana with

Pterocarpus indicus Willd species. Extraction

resulted in 200 g of Angsana leaf sample, which was

produced through 96% ethanol solvent with as much

as 1000 mL by maceration method, resulting in a

thick extract of each 2.4 g for old Angsana leaves

and 12.4 g for young Angsana leaves. Qualitative

test results showed positive results with change of

color from dark green to yellowish green.

Flavonoids are compounds containing two aromatic

rings with more than one hydroxyl group. Reduction

with concentrated Mg and HCl produces red, yellow,

or orange colors (Robinson, 1995).

ICPS 2018 - 2nd International Conference Postgraduate School

542

Before conducting a quantitative test, the

determination of the maximum wavelength was

done using quercetin work standard with a

concentration of 80 ppm in ethanol solvent 96% pa.

The absorbance reading was carried out at a

wavelength of 400 - 800 nm. The wavelength

produced a determination of total flavonoid of 439

nm. The wavelength was then used to measure the

uptake of the calibration curve and the samples of

Angsana leaf extract.

This study began with a verification test.

Verification testing is an analytical method used to

prove that the laboratory concerned is capable of

testing using the method with valid results (Gandjar

and Rohman, 2007).

The calibration curve obtained linear regression

equation, that is y = 0.008x - 0.002, with correlation

coefficient r = 0.999. The angsana leaf extract was

then tested by the quantitative colorimetric method.

The principle of the method of colorimetry is the

formation of a complex between aluminum chloride

and ketone groups at C-4 and hydroxy groups at

adjacent C-3 and C-5 of flavon and flavonol groups.

The compound used as a standard for the

determination of this flavonoid level is quercetin

since quercetin is a flavonoid group having keto

groups in C-4 atoms and also hydroxyl groups on

adjacent C-3 and C-5 (Azizah et al., 2014).

In the determination of total flavonoid levels, the

addition of sodium acetate was intended to detect the

presence of a 7-hydroxyl group (Mabry et al., 1970),

while the 30-minute incubation treatment carried out

was intended to allow the reaction to run perfectly,

thus providing maximum color intensity. The

determination of total flavonoid content from old

and young Angsana leaf extract resulted in Angsana

content of 7.53% ± 0.32% w/w (Table 1) and 3.31 ±

0.07% w/w (Table 2), respectively. Based on the

results obtained between old and young angsana

leaves, it was found that the old angsana leaves

contain more flavonoids. Therefore, the use of old

angsana leaves is more recommended than young

angsana, especially when traditionally used by the

community.

Table 1: Quantitative test result of old angsana leaves.

No The

weight

of the

sample

(g)

Absor

bance

Flavonoids in

the sample

(mg/100 mL)

%w/w

1 0.1005 0.307 7.725 7.687

2 0.1004 0.313 7.844 7.844

3 0.1004 0.279 7.025 6.997

4 0.1006 0.301 7.575 7.530

5 0.1007 0.303 7.625 7.572

7.53

0.32

Table 2: Quantitative test result of young angsana leaves.

No The

weight

of the

sample

(g)

Absor

bance

Flavonoids in

the sample

(mg/100 mL)

%w/w

1 0.1001 0.114 2.9009 2.898

2 0.1004 0.125 3.1769 3.164

3 0.1003 0.136 3.4525 3.442

4 0.1004 0.138 3.5031 3.489

5 0.1004 0.141 3.5784 3.564

x3.31

SD 0.07

Previously, precision test and accuracy test with

a recovery of 83.67% and RSD of 8,96% have been

done (Table 3). The acceptance criteria used in the

study are 80-120% for accuracy, and from the

achievement of ≤ 20% precision it can be concluded

that the precision and accuracy test has met the

requirements (Anonim, 2004).

Table 3: Test Result of Accuracy and Precision.

The

weight o

the

sample

(g)

Sample

(Abs)

Conc.

Sample

(ppm)

Sample+

Quercetin

(Abs)

Conc.

Sample +

Quercetin

(ppm)

Accuracy

( % )

1. 0.0503 0.068 8.7327 0.471 59.2974 84.275

2. 0.0500 0.075 9.6110 0.415 52.2710 71.100

3. 0.0500 0.042 5.4705 0.486 61.1794 92.848

4. 0.0500 0.059 7.6035 0.442 55.6587 80.092

5. 0.0506 0.071 9.1092 0.491 61.8086 87.832

6. 0.0500 0.077 9.8620 0.492 61.3920 85.883

x 83.67

SD 7.45

RSD (Precision) 8.96

4 CONCLUSIONS

Based on the results, it can be concluded that the

extract of old and young Angsana leaves contains

flavonoid compound with total levels of flavonoids

of ethanol extract being 7.53 ± 0.32% w/w and 3.31

± 0.07% w/w, respectively.

Identiﬁcation and Determination of Total Flavonoids in Ethanol Extract of Old and Young Angsana Leaves (Pterocarpus indicus Willd.)

Using Visible Spectrophotometry

543

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