Identification and Analysis of Potential Antioxidants from Leaves of

Eucalyptus robusta PT. Toba Pulp Lestari, Tbk.

Chintya Cahaya

, Muhammad Taufik

, Zul Alfian

, Sovia Lenny

and Rizky Hidayati

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, Indonesia

rizkyhidayati12345@gmail.com

Keywords: Eucalyptus, Antioxidants, DPPH Method, GCMS, Cineol.

Abstract: PT Toba Pulp Lestari Tbk is a global pulp producing company that develops forest industry concessions and

stores a variety of underutilized biodiversity such as Eucalyptus robusta leaves which are widely found

around the PT Toba Pulp Lestari Tbk forest area. Eucalyptus robusta leaves produce oil that is used as

liniment, cough medicine, perfume, soap, detergent, disinfectant and pesticides. Eucalyptus robusta and

supports its antioxidant potential. Eucalyptus leaves are first described with several solvents, such as

methanol, ethanol and dichloromethane. The potential of antioxidants using the DPPH method on the 1800

Uv-Vis Spectrometer. The antioxidant activity test was carried out with the DPPH method (1.1 diphenyl

pikrilhidrazil) with a variation of sample volume 20; 30; 40; 50; 60 μL. Eucalyptus leaf extract showed

antioxidant activity with IC50 of 8,386 μg / mL. The Eucalyptus leaf extract is categorized as providing

weak antioxidant activity. Analysis of active composition that can be used in eucalyptus leaves using the

GCMS method. The main compounds contained were cineol 52%.

1 INTRODUCTION

Eucalyptus is a genus of the Myrtaceae family and

has as many as 600 species and subspecies.

Although native eucalyptus originated in Oceania,

these tall green trees spread throughout the world

and occupy an area estimated at between 16 and 19

million hectares (Bułkowskaet al., 2016).

Eucalyptus sp. Plant. (Myrtaceae) has various

species, namely E. camadulensis, E. grandis, E.

pellita, E. tereticornis, and E. torreliana. Planting

of Eucalyptus sp. Where most are found in Sumatra

(Aceh, North Sumatra, Jambi) and Kalimantan

(West Kalimantan, East Kalimantan and South

Kalimantan) (Nair, 2000).

The Eucalyptus robusta species to be carried out

in this study was produced by PT. Toba Pulp Lestari

Tbk, which is one of the HTI companies located in

Indonesia, especially the North Sumatra province

which is the HTI with the largest area in North

Sumatra, where the total area reaches 188,055 Ha.

And the commodities mainly developed are

eucalyptus plants (Eucalyptus spp). Eucalyptus

belongs to fast-growing plants or better known as

Fast Growing Species. One location in North

Sumatra that has a high biodiversity composition is

at PT Toba Pulp Lestari Tbk where TB has

developed eucalyptus plants to be used as raw

material for paper making but which is still utilized

from eucalyptus plants still in wood and its

branches lack of utilization of Eucalyptus leaves in

the area Toba Pulp Lestari which actually has the

potential to be used as raw material for making

essential oils or better known as eucalyptus oil

which can be traded as a precursor in chemical

synthesis.

The effects of etiologically free radicals have

been believed to be the cause of various chronic and

aging diseases. This is because these reactive free

radicals can attack various biomolecules.

Antioxidants can come from natural or synthetic

ingredients. Synthetic antioxidants include BHA

(butyl hydroxyanisole) and BHT (butyl

hydrozxytoluene). Antioxidants derived from

natural ingredients include vitamin E (- tocopherol)

and resveratrol. Both are natural phenolic

antioxidants (Hart et al, 2003).

Cahaya, C., Tauﬁk, M., Alﬁan, Z., Lenny, S. and Hidayati, R.

Identiﬁcation and Analysis of Potential Antioxidants from Leaves of Eucalyptus robusta PT. Toba Pulp Lestari, Tbk..

DOI: 10.5220/0008920002450248

In Proceedings of the 1st International Conference on Chemical Science and Technology Innovation (ICOCSTI 2019), pages 245-248

ISBN: 978-989-758-415-2

245

Synthetic antioxidants have a harmful effect if

consumed by humans. Synthetic antioxidants such

as BHT (Butyl Hydroxy Toluene), TBHQ (Tertiary

Butyl Hydroquinone) can increase the occurrence of

carcinogenesis in humans (Amarowicz et al., 2000)

and liver damage (Osawa & Namiki, 1981).

Therefore, natural antioxidants are more

recommended for human consumption. This fact

encourages a lot of research done to look for natural

ingredients that can be used as antioxidants

(Rohman & Riyanto, 2005).

The working principle of GC-MS is based on

differences in the polarity and molecular mass of

samples that can be evaporated. Samples in the

form of liquid or gas are directly injected into the

injector, if the sample is solid, it must be dissolved

in a solvent that can be evaporated. The flow of gas

flowing will bring the evaporated sample to enter

the column. The components in the sample will be

separated based on the partition between the mobile

phase (carrier gas) and stationary phase (column).

The result is a gas molecule which will then be

ionized in a mass spectrophotometer so that the gas

molecule will experience fragmentation in the form

of positive ions. Ions will have a specific ratio

between the mass and the charge (Fowlis, 1998).

The principles of the Gas Chromatography -

Mass Spectrometry tool are: Sample injection can

be done manually or use an automatic sample taker

through a rubber seal that can be closed again. The

sample is evaporated on the portal part of the

injection which is heated and subjected to

condensation at the top of the column. The column

can be a packed capillary column or column, which

will be discussed in more depth. The mobile phase

used to carry samples through the column is a gas -

usually nitrogen or helium. The column is closed in

an oven which can be set at a temperature between

room temperature and approximately 400 ° C. The

detector used is mass spectrometry (MS). The

University of North Sumatra sample is inserted into

the instrument source by heating it at the end of a

sensor until it evaporates, aided by very hollow in

the instrument If it is in the vapor phase, the analyte

is bombarded with electron electrons produced by

the rhenium or tungsten filaments, which are

accelerated towards a positive target with an energy

of 70 eV. Two types of systems are usually used to

separate ions based on load comparisons of their

mass (Watson, 2005).

2 MATERIALS AND METHODS

2.1 Materials

The main material for this research is Eucalyptus

robusta. The solvent used in the distillation process

is aquadest. The reagents used in the DPPH test are

2,2-diphenyl-1-pikril hidrazil (DPPH) and

methanol. The instrumentation used for sampling

samples included the 1800 shimadzu UV-Vis

spectrometer.

2.2 Preparation of Sample

Leaves are obtained from PT. Toba Pulp Lestari

which is located on Jl. Indorayon Subdistrict Dolok

Nauli, Toba Regency Samosir, North Sumatra. The

selected leaves are leaves along with stems 5-10 cm

from the top of the plant. Taking is done in the

morning at 7-9 in the morning.

The fresh leaves obtained are immediately

separated from the stem. Then the leaves are

chopped using a cutter and scissors to produce

chopped ingredients with a length of ± 0.5-1.0 cm.

Plant identification has been carried out in the

HERBARIUM MEDANENSE (MEDA) Laboratory

in the Department of Biology, University of North

Sumatra. Samples in the form of: Fresh leaves in a

single branch between 10-15 cm from the top.

2.3 Sample Extraction

Eucalyptus leaves dried for 24 hours at room

temperature. Then the sample is weighed as much

as 150 grams and inserted into a 1000 mL size

flask. Adding aquabides to taste is then connected

to a Stahl distiller, and boiled for ± 5-6 hours at ±

100 ° C to produce oil and distillation ends when

the distillate is clear. The essential oil obtained is

accommodated in the Erlenmeyer glass. The

distillate obtained is a mixture of oil and water.

Then the oil layer was added to CaCl

anhydrous to

bind water which might still be mixed with essential

oils, the oil layer was decanted and put into vial

bottles, stored in a coolant in a bottle and tightly

closed. Then extracted samples are stored in glass

bottles for further analysis.

2.4 Characterization

2.4.1 Analysis of GC-MS

The Specifications Instrument GC-MS QP 2010S

Shimadzu, using Column 5MS with type of ion

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

246

source Electron Impact, Injector Temperature:

300

C, Carrier Temperature: 50

C, Carrier: Helium,

Gas flow rate of carrier: 1.0 mL / min, Temperature

oven: 50

C for 5 minutes then 240

C for 7 minutes.,

Ionization electron: 70 ev.

The solution of each 1 μL standard sineol series

was inserted into the syringer to be injected into the

GCMS. Only the conditions adjusted to the

conditions of each piece of equipment and then

observed Mass Chromatogram data generated

interpreted data. Obtained data then in Perform

calculations to get the calibration curve and do the

determination of levels through the equation.

2.4.2 Analysis of Antioxidant Potential

Analysis of antioxidant potential in essential oils

with Ultraviolet-Visible spectrophotometry method

(UV-Vis).

3 RESULTS AND DISCUSSION

3.1 Test Antioxidant Activity

The DPPH radical capture method is based on

DPPH radical solution color changes due to the

administration of compounds that are antioxidant.

Antioxidant interactions with DPPH either electron

transfer or hydrogen radicals at DPPH will

neutralize the free radical character of DPPH. If all

the electrons in the DPPH free radical are paired,

then the color of the solution changes from dark

purple to bright yellow. This color change will be

observed in the form of a decrease in DPPH

absorbance.

Max determination was carried out by scanning

survey tests on the uv-vis spectrophotometer. This

test produces max DPPH at 517 nm. After obtaining

the max value then it is determined the reaction

time between DPPH and the test sample. This test is

carried out by reacting the DPPH with the test

material sample then observing the change in

absorbance from time to time to get a constant

absorbance reading.

Shows that the radical capture reaction by the

test material has occurred perfectly. Eucalyptus

robusta extract until the 60th minute has not seen a

constant reading, but the difference in absorbance

between times is not too large, so the reaction time

for Eucalyptus robusta extract is set to 60 minutes,

while for the test material the methanol extract

absorbance reading shows a constant value in

minutes 25th, so that the reaction time for methanol

extract is set to 25 minutes.

Even though some fluctuations in the

concentration of the test material, this radical

capture action shows an upward trend along with

the increase in concentration. Positive control of

quercetin indicates radical capture, this indicates

that the test method carried out runs correctly.

Overall the results above show that the

Eucalyptus robusta methanol extract has an IC50

value of 8,386g / mL of the test material which has

weak antioxidant effectiveness, making it less

effective as an antioxidant.

3.2 Analysis of Compound Content in

E. robusta

Qualitative and quantitative analysis of compound

content in Eucalyptus pellita using GC-MS

instrumentation. The results of GC-MS Analysis

were then carried out with data interpretation. The

Qualitative Analysis results show that there are 17

(seventeen) compounds in the form of a

chromatogram which has seventeen peaks.

The results of GC-MS analysis on Eucalyptus

robusta leaf essential oil showed that the

compounds in essential oils contained 17 (twelve)

peaks of compounds contained with a large

abundance of 52% cineol compounds from the

results of the chromatogram produced by Gas

Chromatography then broken down into

fragmentation.

4 CONCLUSIONS

The Eucalyptus robusta test material has

antioxidant activity with an IC50 value of 8,386

wherein the compound has weak antioxidant

activity and GC-MS Analysis Results on Eucalyptus

robusta leaf essential oil shows that the compound

in the essential oil has 17 (twelve) peak compounds

contained in the abundance of cineol compounds as

much as 52%.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge

Kemenristekdikti of Republik Indonesia for the

financial support via DRPM Project 2019.

Identiﬁcation and Analysis of Potential Antioxidants from Leaves of Eucalyptus robusta PT. Toba Pulp Lestari, Tbk.

247

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