Antioxidant Activity Test of Green Tea (Camellia sinensis L. Kuntze)

Ethanolic Extract using DPPH Method

Nazliniwaty

, T. Ismanelly Hanum

1,2

, and Lia Laila

Department of Pharmaceutical Technology , Faculty of Pharmacy, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Ethanol Extract, Green Tea, Antioxidant Activity, DPPH.

Abstract: Green tea (Camellia sinensis (L.) Kuntze) is believed to have efficacy as a powerful antioxidant since it

contains polyphenols that can reduce free radicals. The aim of this study was to determine the class of

chemical compounds of the green tea leaf powder, to characterize the green tea ethanolic extract and to

evaluate the antioxidant activity of the green tea ethanolic extract with DPPH method. Green tea was extracted

by maceration method using 96% of ethanol and followed by the phytochemical screening, characterization

and the antioxidant activity test by DPPH method. The ethanol extract of green tea contained flavonoids,

saponins, tannins and steroids / triterpenoids. The moisture content of green tea extract was 14.25%; the total

ash was 1.06% and the acid insoluble ash content was 0.099%.The antioxidant activity assay gave 11.83 ±

0.005 µg/ml of IC

. It can be concluded that the ethanol extract of green tea has a very strong antioxidant

activity.

1 INTRODUCTION

Antioxidant is a compound that can counteract or

mitigate the negative effects of free radicals in the

body by donating an electron to the free radical so that

its activity is inhibited (Ramadan, 2015).

Antioxidants are needed to overcome the presence of

free radicals, whereby they are not only addressed for

health, but also to take care and beautify the skin

including preventing the premature aging (Anggai et

al., 2015). Tea (Camellia sinensis) is one of the plants

that popular as a drink (Rohdiana, 2009). These plants

contain flavonoids, the potent antioxidant derived

from polyphenol compounds (Sudrajat et al., 2015).

There are a wide variety of teas known in the

community, including green tea. According to Dewi

et al, green tea has a powerful antioxidant activity

because the process of making green tea is not

fermented like the other types of tea (Dewi et al.,

2009). Based on the described facts, the researchers

were interest to do the phytochemical screening,

characterization and evaluation of the antioxidant

activity of the ethanol extract of green tea (Camellia

sinensis (L.) Kuntze).

2 METHODS

2.1 Phytochemical Screening

The phytochemical screening was done to determine

the class of chemical compounds contained in the

green tea leaves dried powder, including groups of

alkaloids, flavonoids, saponins, tannins, and steroids

/ triterpenoids (Ministry of Health Republic of

Indonesia, 1995).

2.2 Preparation of Green Tea

Ethanolic Extract (GTEE)

Green tea leaves powder was extracted by maceration

method using 96% of ethanol. The macerate was then

evaporated with a rotary evaporator at a temperature

of 40-50

C to obtain a viscous extract (Directorate

General 0f Drugs and Food Control, 1979).

The yield value was calculated by comparing the

obtained extract with the original amount of green tea

powder and stated as percentage.

752

Nazliniwaty, ., Hanum, T. and Laila, L.

Antioxidant Activity Test of Green Tea (Camellia sinensis L. Kuntze) Ethanolic Extract using DPPH Method.

DOI: 10.5220/0010087307520754

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

752-754

ISBN: 978-989-758-449-7

2.3 GTEE Characterization

The characterization of the GTEE included the

determination of moisture content using azeotropic

distillation method, the determination of total ash

according to SNI 01-2891-1992, gravimetric assay

and acid insoluble ash based on Indonesian Materia

Medica (Ministry of Health Republic of Indonesia,

1995; Indonesian National Standard, 1992).

2.4 Antioxidant Activity Test

Antioxidant activity test of GTEE was evaluated by

using 2,2-diphenyl-1-picryl-hydrazyl-hydrate

(DPPH) Free Radical Scavenger method as described

in Molenux with slight modification (Molyneux,

2004). Measurement of antioxidant activity was

performed using UV-Vis spectrophotometer.

Maximum wavelength results obtained were 516 nm.

The measurement of the antioxidant activity used

quercetine as the positive control with several

concentration levels which was then defined in the

quercetine calibration curve with regression equation

Y = 3.9734X + 3.0115.

3 RESULTS AND DISCUSSION

3.1 Phytochemical Screening Results

The results of phytochemical screening of green tea

powder can be seen in Table 1

Table 1: Phytochemical screening test results of GTEE.

Compound classification Result

Alkaloids -

Flavonoids +

Saponin +

Tannin +

Steroids / Triterpenoids +

(+) Positive: containing the compound

(-) Negative: do not contain the compound

The dried green tea leaves powder are extracted

by maceration which is a cold extraction method that

processes several times extraction with shaking or

stirring at room temperature, so that the substances

contained in the sample are relatively stable

compared to the heat extraction. The solvent used in

the extraction process was 96% of ethanol which is

relatively safe compared to the methanol and has a

nature that may extract all of the secondary

metabolites in botanical sample.

3.2 Characterization of GTEE

Approximately 500 grams of green tea powder gave

extracts as much as 97.10 g (19.42%). This result

fulfilled the Indonesian Herbal Pharmacopoeia which

stated that the yield requirements for the ethanol

extract of green tea is not less than 7.8%. The GTEE

can be seen in the Figure 1.

Figure 1: Green tea ethanolic extract (GTEE) .

Characteristics of the GTEE were dark green

colour, has bitter taste and specific odor. The results

of determination of moisture content, total ash and

acid insoluble ash content in GTEE were shown in

Table 2.

Table 2: The characteristics of GTEE.

Parameters Results (%)

(n=3)

Water content 14.25 ± 0.46

Ash content 1.06 ± 0.13

Acid insuble ash content 0.099 ± 0.013

The determination of water content was

performed in the GTEE since the water will allow the

growth of microbes and affect the quality of GTEE

during storage. The results of water content in GTEE

amounted to 14.25 ± 0.46%. Based on the water

content value, GTEE was included in the group of

condensed extract which the water content is less than

16%.

The determination of total ash content was made

to provide an overview mineral content contained in

the green tea leaves powder as well as the

contamination during the process of making the

extract. The result of the determination of total ash

content of GTEE fulfilled the requirements according

to Indonesian National Standardization (SNI 01-

2891-1992) that stated the maximum of total ash

content is 8% (Indonesian National Standard, 1992).

Antioxidant Activity Test of Green Tea (Camellia sinensis L. Kuntze) Ethanolic Extract using DPPH Method

753

3.3 Antioxidant Activity

One of the most common methods used to test the

antioxidant activity is the method using a radical

scavenger, 1,1-diphenyl-2- picrylhydrazil (DPPH).

Measurement of radical scavenger is a method of

measuring antioxidant that is simple, fast and does not

require a lot of reagents compared to other methods

that require considerable chemical reagents, analysis

time and expensive. In this method, DPPH solution

acts as a free radical that will react with antioxidant

compounds. It will bind the electrons of the

antioxidants as the test material which is

characterized by changing the purple colour into pink

or pale yellow (Molyneux, 2004).

Measurement of antioxidant activity by radical

scavenger method using DPPH in dark purple

methanol was detected at visible wavelengths of about

500-520 nm. The parameter to interpret the results of

DPPH testing was with IC

(Inhibitor Concentration)

values. IC

is the concentration of the substrate or

sample solution that can reduce DPPH activity by

50%. The smaller the IC

value means the higher the

antioxidant activity (Prakash, 2001).

values were obtained based on the calculation

of linear regression equation obtained by plotting

concentration of the test solution and DPPH damping

percentage as parameter of antioxidant activity, where

the concentration of test solution (µg/ml) as X axis

and the percentage of damping as Y axis. IC

GTEE and control values can be seen in Table 3.

Table 3: Antioxidant activity ( IC

)

GTEE values

compared to controls.

Sample IC

(µg/ml)

Green Tea Ethanolic Extract

(GTEE)

11.83 ± 0.005

.Quercetine 2.32 ± 0.006

According to Mardawati, et al. (2008) GTEE

antioxidant activity can be categorized as very strong

antioxidant. Green tea ethanolic extract has high

antioxidant activity because it contains flavonoids

compound which contains hydroxyl group that

donates hydrogen to the free radical. The compound

can neutralize the free radical by giving electrons,

therefore atom with the unpaired electron can get

electron and no longer become free radical.

4 CONCLUSION

Green tea ethanolic extract contains flavonoids,

saponins, tannins and steroids / triterpenoids

compounds. It has moisture content of 14.25 ±0.46%,

total ash of 1.06 ± 0.13% and acid insoluble ash

content of 0.099 ± 0.013%, and shows very strong

antioxidant activity.

ACKNOWLEDGMENTS

This research was funded by Research Institute of the

University of Sumatera Utara with contract No. 403 /

UN5.2.3.1 / PPMKP-TALENTA USU / 2018.

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