Antioxidants Activity of the Kecombrang Flower (Etlingera elatior)

Extract by using 1,1-diphenyl-2-picrilhidrazyl (DPPH) Method

Maulidna

1,3

, Basuki Wirjosentono

, Tamrin

and Lamek Marpaung

Postgraduate Chemistry Study Program, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara,

Jl. Bioteknologi No. 1 Kampus USU, Medan, Indonesia

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan

Politeknik Teknologi Kimia Industri, Medan, Indonesia

Keywords: Antioxidant, Flower Extract Kecombrang, DPPH, Secondary Metabolite, Alkaloids.

Abstract: Kecombrang (Etlingera elatior) is a zingiberaceae plant, which has long been known as one of the vegetables

and used by the community as a nutritious food to preserve food because of the active substances contained

in it, such as saponins, flavanoids, and polyphenols. In this research, antioxidants activity of kecombrang

flower extract was measured by using 1,1-diphenyl-2-picrilhidrazil (DPPH) method. The kecombrang flower

extract has several secondary metabolite compounds, such as alkaloid, terpenoid, steroid, phenolic, flavonoid,

tanin with very small antioxidant activity, which is showed by inhibition percentage. As many as 100 ppm

concentration of the kecombrang flower extract, ie, methanol extract 3.21%, ethyl acetate extract 5.08 %, and

n-hexane extract 30.29%. So it can be concluded that the antioxidant activity of flower kecombrang (Etlingera

elatior) is less active.

1 INTRODUCTION

Human health is very dependent on the environment

nowadays. Besides, the environment which is full of

pollutants, will be able to poison the body either

through air or food. One of the causes of

environmental pollution is pesticide residue. It has

been reported that pesticide residues can cause the

diseases because of the presence of radical

compounds that can oxidize cells (Zakaria et al,

1996). Excessive cell oxidation process will lead to

many diseases, such as cancer, diabetes, heart disease

(Fajriah et al, 2007) atherosclerosis, cataracts and

premature aging (Langseth et al, 2000). The body

itself produces antioxidants that can reduce the

negative effects of free radical reactions. As long as

the balance between free radicals and endogenous

antioxidants is maintained, the adverse effects of free

radicals can be neutralized (Subarnas et al, 2001).

Various diseases caused by radical compounds are

growing, this makes the research continue to be done

as an effort to be able to find substances as drugs that

can play an active role in preventing and overcoming

radical compounds in the body reported that there are

two types of drugs that can be used as an alternative

treatment of synthetic drugs and traditional medicine

in the world of drugs. Besides, choosing a drug should

consider as the side effects of drug performance.

Drugs from natural ingredients have relatively fewer

side effects compared to synthetic drugs (Utami et al,

2008). Generally, Indonesian prefer to take the

natural-made medicines in the effort to prevent and

treat an attack of disease by drinking water extract

from certain plants or by attaching the extract to the

sick body part (Mohd Jaafar et al, 2008).

Free radical compounds can be overcome by a free

radical prophylactic called antioxidants. The

antioxidant is a component capable of inhibiting

nucleic acid, lipid oxidation by initiation or

propagation of chain oxidation reactions.

Antioxidants can protect the body from various

degenerative diseases in accordance with the main

function of antioxidants namely, neutralizing free

radical compounds (Winarsi et al, 2007), reducing

agents, free radical damping and metal pro-oxidant

complex (Poerawinata M, et al, 2007). The isolated

antioxidant compounds contained in high plants are

ß-carotene, vitamin C, vitamin E, flavonoids,

curcuminoids and polyphenol compounds

(Alamendah's et al, 2013).

Based on the above explanation, this study aims to

conduct the research on kecombrang plant, which is

Maulidna, ., Wirjosentono, B., Tamrin, . and Marpaung, L.

Antioxidants Activity of the Kecombrang Flower (Etlingera elatior) Extract by using 1.1-diphenyl-2-picrilhidrazyl (DPPH) Method.

DOI: 10.5220/0008869301970203

In Proceedings of the 1st International Conference on Chemical Science and Technology Innovation (ICOCSTI 2019), pages 197-203

ISBN: 978-989-758-415-2

197

believed to have antioxidant activity as concluded by

Tan et al.. Regarding to the activity of chewing as an

antioxidant, testing of antioxidant kecombrang can be

done by using of the DPPH method. The DPPH

method is low-cost and easy-to-prepare method

where the DPPH acted as free radical will react with

antioxidant compound so that the color of test

solution changes from purple to yellow (Pratama et

al, 2015).

2 MATERIALS AND METHODS

2.1 Materials

The instruments used are UV-Vis Spectrophotometer,

vacuum rotary evaporator, separator funnel,

sonicator, incubator, analytical scales, dilution bottle,

vial bottle. The ingredients used are flower samples

of coconut, methanol solvent, Ethyl acetate solvent,

n-Hexane solvent, DPPH powder, and methanol p.a.

2.2 Method

2.2.1 Sample Preparation

The sample was obtained from one of the areas in Deli

Serdang with a weight of 2 Kg, which then dried and

graded into small size and weighed the sample

weight.

2.2.2 Maceration

Samples that have been scaled to a small size, are

inserted into glass bottles and methanol solvent added

until the sample portion is completely submerged.

Subsequent samples in glass bottles were left

submerged for 2 x 24 h. After 2 x 24 h the sample is

filtered and then the filtrate of the sample is floated in

a clean glass bottle.

2.2.3 Vacuum Rotary Evaporator

The sample filtrate has been accommodated, then

inserted into the rotavapor flask according to the

pumpkin container, and subsequently in the rotary

vapor until the filtrate becomes thickened thick and

then allowed to evaporate until the viscous filtrate,

then repeated rotary evaporator by incorporating

other filtrate.

2.2.4 Separation of Extract with Separating

Funnel

The viscous filtrate was taken slightly and diluted

with some mL of methanol slowly, then inserted into

the separating funnel and added with several mL of n-

hexane solvent, subsequently separated funnel for a

few seconds until the second solvent was evenly

mixed and then placed the separating funnel in the

stem neck stative until both solvents are perfectly

separated. Further separated layers of methanol with

a layer of n-hexane and each of them accommodated

in glass bottles.

2.2.5 Vacuum Rotary Evaporator Methanol

Extract and N-hexane Extract

Each filtrate of methanol extract and filtrate of n-

hexane extracts were regenerated until each filtrate

changed to blackish, and then allowed to dry until a

thickened extract was formed.

2.2.6 Separation of Methanol Condensed

Extract with Ethyl Acetate Solvent

The methanol condensed extract was extracted by

adding the ethyl acetate solvent until the viscous

portion of the methanol extract was insoluble in the

ethyl acetate solvent.

2.2.7 Calculation of Yield Percentage

Each extract that has been produced then calculated

the yield by using the following formula:

  





 (1)

2.3 Characterization

2.3.1 Phytochemical Screening

The three extracts have been obtained, phytochemical

screening such as the identification of alkaloids,

terpenoids, steroids, phenolics, flavonoids, saponins,

quinones, and tannins. The alkaloid test was

performed by reacting the extract with a Dragendroff

reagent and producing a brown red and orange

(Robinson et al, 1995). The terpenoid and the steroid

tests are performed by reacting the extract with

Lieberman-Burchard reagent and producing a pink or

purple color (Harborne et al, 1987). Flavonoid test

was done by dissolving the extract in water and then

added Mg powder and the added concentrated HCl

and shaken strong, positive test by red, orange, or

purple (Robinson et al, 1995). The saponin test is

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

198

done by dissolving the extract in water and then

shaking strongly for a few seconds and will cause a

stable foam, then added 1% HCl if the foam arises

with a height of 1-3 cm and persist for 15 minutes

indicating the presence of saponins (Harborne et al,

1987). Phenolic test is performed by reacting the

extract with 1% iron and 1% chloride reagent and

positive when it gives strong green, red, violet, blue

and black (Erwinsyah et al, 2016). Tannin test is done

by dissolving the extract in water and then the added

a few drops of iron reagent (III) chloride 1% positive

result when giving rise to dark blue, green (LIPI,

2017). The quinone test is performed by diluting the

extract into water and added a few drops of positive

1N NaOH by causing a red color (LIPI, 2017).

2.3.2 The Antioxidant Activity Test

The antioxidant activity is determined by free radical

damping method using the DPPH. Weighed the

DPPH powder as much as 2.4 mg and dissolved with

methanol as much as 15 mL and then placed in a dark

bottle. As many as 0.4 mM the DPPH solution was

pierced 1 mL and inserted in a 5 mL scale reaction

tube, then the added methanol pro analysis to the

boundary marker, and the tube was covered with

aluminum foil then homogenized.

Weighed each viscous the extract of 5 mg was

used an analytical scale, then dissolved into 10 mL

the methanol pro analysis, the solution is the parent

liquor. Then the parent solution was piped into the

test tube with a volume of 5 mL of 1000 μL to obtain

a concentration of 100 ppm.

A total of 3 mg of the vitamin C was weighed and

then dissolved with the methanol p.a up to 10 mL.

Furthermore, the 150, 117, 83, 50, and 17 μL pipes

were inserted into a tube of scale covered with

aluminum foil and then added 1 mL of 1 mM the

DPPH solution and added the methanol pa to 5 mL

boundary marker on the scale tube to obtain

concentration 1, 3 , 5, 7, 9 ppm.

The test solution with a concentration of 100 ppm

was incubated with 37

C for 30 minutes. Subsequent

absorption of the solution was measured at a

maximum absorption wavelength of 517 nm by using

a visible light spectrophotometer.

Formal absorption, positive control and

absorbance of the test solution measured on the UV-

Vis spectrophotometer and recorded by entering the

absorption results percentage inhibitor in the

following formula:

 





 (2)

3 RESULTS AND DISCUSSION

3.1 Results

The results of research conducted at Bioproses

Laboratory Politeknik Teknologi Kimia Industri

Medan can be seen in Table 1; 2; 3; and 4 below:

Table 1: Value of Randemen Percentage of Kecombrang

Flower (Etlingera elatior) Extract.

Extract

Weight Extract

(g)

Yield

(%)

n-hexane

10.21

0.51

Ethyl acetate

2.41

0.12

Methanol

1.45

0.07

Table 2: Phytochemical Screening Results of Kecombrang

Flower (Etlingera elatior) Extract.

Methanol

Extract

Ethyl

Acetate

Extract

hexane

Extract

Alkaloid

Terpenoid

Steroid

Phenolic

Flavonoid

Saponin

Quinon

Tannin

Table 3: Value of Inhibitor percentage of the Kecombrang

Flower Extract (Etlingera elatior) at 100 ppm

Concentration.

Extract

Concentration

Absorbance

inhibition

n-hexane

100

0.672

30.29

Ethyl

Acetate

100

0.915

5.08

Methano

100

0.933

3.21

Blank

100

0.964

Table 4: Antioxidant Activity Test Results of Vitamin C.

Concentration

(ppm)

Absorbance

inihibition

(ppm)

0.896

7.05

4.51

0.725

24.79

0.406

57.8

0.066

93.15

0.024

97.51

Antioxidants Activity of the Kecombrang Flower (Etlingera elatior) Extract by using 1.1-diphenyl-2-picrilhidrazyl (DPPH) Method

199

3.2 Discussions

3.2.1 Extraction

The samples from flowering plants were cut into

small pieces and dried, then the dried samples were

macerated using organic solvent, methanol solvent

for 2 x 24 h with 4 repeats of maceration until the

solvent became translucent until the secondary

metabolite was no longer soluble in the methanol

solvent. Maceration is a technique of immersion to

the material to be extracted. Samples that are small in

size are immersed in organic solvents for some time

and then filtered and the result is a filtrate (Sitorus et

al, 2010). The purpose of maceration so that the

secondary metabolite compounds contained in the

sample can be dissolved in the solvent so that it can

be obtained filtrate sample flowers kecombrang.

Maceration uses organic solvents, the function of

organic solvents to penetrate the cell wall of the

sample and into the cell cavity resulting in the

secondary metabolite compounds contained in the

cell to dissolve. The dissolution of secondary

metabolite compounds is due to the difference in

concentration between the secondary metabolite

compounds and the organic solvent resulting in

diffusion. After the diffusion process takes place, the

sample filtrate is accommodated into a glass for the

rotary evaporator process.

In the rotary evaporator, the filtrate concentration

process of the kecombrang flower sample is based on

the vapor pressure of the solvent influenced by the

temperature. The concentration process uses a

vacuum pump with a cooling water stream so that the

solvent present in the apparatus will decrease the

vapor pressure. When the solvent vapor pressure drop

is equal to the atmospheric pressure the solvent will

boil, so that the solvent present in the sample filtrate

will be able to evaporate faster at a temperature below

its boiling point. This process is done until the extract

obtained thick flowers kecombrang.

The obtained flower extract of the resulting

kecombrang was then extracted with a n-hexane

solvent in order for the non-polar secondary

metabolite compounds to be separated by a polar one.

The extract of thick flower extract of kecombrang

was done by using separating funnel tool so that

separation of secondary metabolite compounds with

different polarity properties will be seen clearly with

the formation of 2 layers where the top layer is n-

hexane solvent while the bottom layer is methanol

solvent. Each flower extract that will be extracted in

a separating funnel is dissolved first with a methanol

solvent so that the extract becomes more dilute and at

the time of added n-hexane solvent will be easier

separation process. After separation by using

separating funnel, both layers of flower extract of

kecombrang are accommodated in glass bottles'.

Furthermore, the two flower extracts concentrated by

using a rotary evaporator. The concentrated methanol

extract of the kecombrang flower was further

extracted with ethyl acetate solvent by adding ethyl

acetate solvent in the methanol concentrated extract

until the methanol extract of the flower kecombrang

was insoluble in the ethyl acetate solvent, then the

ethyl acetate extract of the flower kecombrang was

left alone the solvent evaporated. The result of extract

process was obtained three extracts from kecombrang

interest that is methanol extract, ethyl acetate extract,

and n-hexane extract then calculated% yield,

phytochemical screening, calculation of DPPH

damping resistor.

Figure 1: Value of Randemen Percentage of Kecombrang

Flower (Etlingera elatior) Extract.

The graph above illustrates the percentage of yield of

kecombrang flower (Etlingera elatior) extract. The

extract was done yield percentage calculation with

the aim to know how many extracts will be obtained

from so many samples was used, so to conduct further

research will be easy to predict how many samples

will be extracted to get the extract according to

requirement. Randemen percentage were extracted

methanol 1.45 g with percentage of 0.07%, ethyl

acetate extract 2.41 g with randemen percentage

0.12% and n-hexane extract of 10.21 g with 0.5%

yield percentage.

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

200

3.2.2 The Phytochemical Screening

The three extracts that have been calculated

rendemen percentage phytochemical screening. The

purpose of phytochemical screening is to know the

type of secondary metabolite compounds contained in

the sample. The method was used because easier and

simpler to do it.

Figure 2: Phytochemical Screening Results of the Extract.

3.2.3 Kecombrang Flower (Etlingera elatior)

The graph above describes the results of

phytochemical screening of the kecombrang flower

extract (Etlingera elatior). Based on the Fig. 2. can be

explained that the extract of n-hexane found the

secondary metabolite content of alkaloids,

terpenoids, and tannins. Ethyl acetate extract of

secondary metabolite content in the form of alkaloids,

phenolics, flavonoids, and tannins. While the extract

methanol terpenoid content, steroids, fennolik and

tannins. The results of screening of studies that have

been performed with similar extracts from bogor

areas indicate the presence of secondary metabolites

from ethanol extracts of alkaloids, flavonoids,

saponins, tannins, steroids and terpenoids, ethyl

acetate extracts of alkaloids, flavonoids, steroid and

terpenoidal saponins, and n-hexane extract not

detected secondary metabolite content (Verawati et

al, 2014). The content of secondary metabolite of

kecombrang flower extract from Deli Serdang area

with extract of kecombrang flower from Deli Serdang

area has difference such as ethanol extract of

kecombrang flower from Deli Serdang area detected

alkaloid and falvonoid content and not found in

methanol extract of kecombrang flower from Tanah

Karo area. This difference in detectable secondary

metabolite content may be caused by the precursor of

the biosynthesis of secondary metabolite formation as

well as the texture of the soil in which the flower

derived origin (Verawati et al, 2014). (Milana et al,

2016) also reported that the formation of secondary

metabolites is strongly influenced by soil nutrients

having linear relationships such as nitrogen,

potassium, organic matter and carbon.

3.2.4 Test of Antioxidant Activity by using

the DPPH Method

The antioxidant activity test was performed to see the

bioactivity of the kecombrang flower by looking at

the percentage value of the sample inhibitory power.

To determine the inhibitory power of the sample as

antioxidant can be done by using the DPPH method.

The antioxidant activity test used the DPPH method

because it is easier to do and not expensive.

Testing of antioxidant activity using 0.4 mM

DPPH solution made by weighing DPPH powder as

much as 2.4 mg and dissolved into 15 mL methanol

pa. Further weighed the extract to be tested as much

as 5 mg and dissolved in a solution of methanol pa 10

mL and disonikasi so that the solution becomes

homogeneous. Test solution was then prepared with

concentration of 100 ppm by pipette 1 mL of DPPH

solution into three scale test tubes wrapped with

aluminum foil and added extract solution on each

tube as much as 1 mL then added methanol pa to 5

mL scale and for blank DPPH solution in 5 mL test

tube. The test tube was then detoxified for 30 minutes

to speed up the reaction between samples acting as

antioxidants with DPPH free radicals. After 30

minutes the test solution is ready for measurement by

a UV-Vis spectrophotometer to determine the

absorbance of each test solution.

Testing with UV-Vis spectrophotometer was

performed at 517 nm wavelength because at that

wavelength had the optimum absorbance ability for

antioxidant activity test. Furthermore, the

measurement of the absorbance of each test solution

in duplicate.

For methanol extract test solution obtained

absorbance 0.933, and for ethyl acetate extract

obtained absorbance of 0.915, while extract n-Hexan

obtained absorbance of 0.672, and for blank obtained

absorbance 0.964.

Antioxidants Activity of the Kecombrang Flower (Etlingera elatior) Extract by using 1.1-diphenyl-2-picrilhidrazyl (DPPH) Method

201

Figure 3: Value of Inhibiton Percentage of Kecombrang

Flower Extract (Etlingera elatior) at 100 ppm

Concentration.

After calculation can be obtained percentage of the

inhibitory of each-tipa extract. The extract of n-

hexane has an inhibitory percentage of 30.29%, ethyl

acetate extract has 5% inhibition of 5.08% and

methanol extract has an inhibitor percentage of

3.21%, so it can be said that the antioxidant activity

of the three extracts are very small. (Anisa et al, 2014)

says antioxidant activity can be expressed as IC50

(Inhibition Concentration fifty) if concentration to

free radical clearance is 50%. Vitamin C test results

have very good antioxidant activity and obtained

IC50 4.51 ppm, while the results of calculation of the

inhibitory of the three extracts are not up to 50%,

Therefore the three extracts in the test beforehand in

a concentration of 100 ppm in order to know whether

the extract has activity 50 good percentage or not

before IC50 measurement.

4 CONCLUSIONS

Based on the data of the research, it can be concluded

that the sample extract of kecombrang flowers has

some secondary metabolite compounds such as

Alkaloid, Terpenoid, Steroid, Phenolic, Flavonoid,

Tanin. In addition, the extract of kecombrang flower

sample also has very small antioxidant activity at

concentration of 100 ppm that is, methanol extract

3.21%, etil acetate extract 5.08%, and 30.29% n-

hexane extract, so it can be concluded that the

antioxidant activity of flower kecombrang (Etlingera

elatior) less active.

ACKNOWLEDGEMENTS

This work had been supported by Politeknik

Teknologi Kimia Industri Medan. The authors would

like to thank the very useful suggestion from the

editors and reviewers.

REFERENCES

Alamendah’s Blog. 2009. Tingkat pencemaran Udara di

Indonesia. https://alamendah.org/2009/09/23/tingkat-

pencemaran-udara-di indonesia/. Tanggal akses 12

November 2017.

Erwinsyah, 2016. Skrining Fitokimia dan Uji Aktivitas

Antioksidan Ekstrak Benalu yang Tumbuh pada

Tanaman Benalu Bungur (Scurrula ferruginea (Jack)

Danser) Dengan Metode DPPH (1.1-difenil-2-

pikrilhidrazil). Universitas Mulawarman, Samarinda.

Fajriah S, Akhmad Darmawan, Andini Suridowo dan Nina

Artanti. 2007. Isolasi Senyawa Antioksidan dari

Ekstrak Etil Asetat Daun Benalu (Dendrophthoe

petandra L) yang Tumbuh pada Inang Lobi-Lobi

Kawasan Puspitek, Serpong: Pusat Penelitian Kimia

Lembaga Ilmu Pengetahuan Indonesia.

Harborne, J. B. 1987. Metode Fitokimia Penuntun Cara

Modera Menganalisis Tumbuhan. Terjemahan: Kosasih

P, Soediro iwang, Bandung ITB.

Jackie et al. BMC Research Notes. 2011.Antioxidant

effects of Etlingera elatior flower extract against lead

acetate - induced perturbations in free radical

scavenging enzymes and lipid peroxidation in rats.

https://bmcresnotes.biomedcentral.com/articles/10.118

6/1756-0500-4-67. Tanggal akses 12 November 2017.

Langseth, L. 2000. Antioxidants and Their Effect on

Health. Essentials of Functional Foods. Aspen

Publication, Gainthersbur

Lipi, 2014. Bioteknologi Untuk Kehidupan Lebih Baik.

http//www.biotek.lipi.go.id. Tanggal akses 29

Novmber 2017.

Mohd Jaafar, Faridahanim et al. 2007. Analysis of Essential

Oils of Leaves, Stems, Flowers, And Rhizomes Of

Etlingera Elatior (JACK) R. M. Smith.The Malaysian

Journal of Analytical Scienses. Universiti Teknologi

MARA. Kuala Lumpur.

Naufalin Rifda and Herastuti Sri Rukmini. 2016.

Antioxidant Activity and Physicochemical Properties

of Nicolaia speciosa Flower extract.Agriculture and

Agricultural Science Procedia 9. Jendral Soedirman

University. Purwokerto.

Rifda, Naufalin. Antimicrobial Activity of Fruit Peel

Kecombrang Formula (Nicolaia speciosa HORAN) As

Natural Preservative. Program Studi Ilmu dan

Teknologi Pangan. Universitas Jenderal Soedirman.

Purwokerto

Poerawinata M. 2007 Uji Aktivitas pada Daun Pandan

(Pandanus polycephalus). skripsi. Universitas

Indonesia, Depok.

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

202

Pramata, Galih Putra. 2015. Identifikasi Senyawa Aktif

Antioksidan dalam Tanaman Selada Air (Nasturtium

officinale R. Br). Skripsi. Universitas Pancasila,

Jakarta.

Robinson, T. 1995. Kandungan Organik Tingkat Tinggi.

Bandung: Institut Teknologi Bandung.

Subarnas, A. 2001. Komponen Aktif Antioksidan dalam

Bahan Alam, Seminar Nasional dan Lokakarya

Pemahaman dan Konsep Radikal Bebas dan Peranan

Antioksidan dalam Meningkatkan Kesehatan Menuju

Indonesia Sehat 2010. Pusat Penelitian Padjadjaran: 28-

40.

Utami N., dan Robbara. M. 2008. Identifikasi Senyawa

Alkaloida dari Ekstrak Heksana daun Argratum

conyzoides. Lin. Prosiding Hasil Penelitian Unila.

Antioxidants Activity of the Kecombrang Flower (Etlingera elatior) Extract by using 1.1-diphenyl-2-picrilhidrazyl (DPPH) Method

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