Isolation and Characterization of an Antioxidant Compound from

Kayu Hitam Leaves (Diospyros celebica Bakh.F.)

Helmina Br. Sembiring

and Yuni Romasni Purba

Departement of Chemistry, Faculty of Mathematics and Sciences, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Kayu Hitam (Diospyros celebica Bakh.F.), Isolation, Characterization, Methyl Gallate, Antioxidant.

Abstract: Isolation and characterization of an antioxidant compound from kayu hitam leaves (Diospyros celebica

Bakh.F.) had been done by extraction and column chromatography method. Kayu hitam leaves powder was

extracted with methanol and methanol extract reextracted with aquadest. Aqudest extract was partitioned with

ethyl acetate and ethyl acetate extract repartitioned with n-hexane. The residues which are phenolic

compounds were separated by column chromatography (SiO2, chloroform: methanol 90:10, 80:20, 70: 30,60:

40). The isolate obtained was purified with a preparative thin layer chromatography and obtained 9.5 mg of

pure isolate in the form of yellow solid. characterization of pure isolate was determined by UV-Vis, FT-IR

and 1H-NMR spectroscopic analysis. Based on the analysis carried out it can be characterized that the pure

isolate obtained is methyl gallate. The antioxidant activity of methyl gallate was determined based on the

DPPH free radical scavenging method. The activity of the methyl gallate was classified as strong with IC50

value of 4.41 µg / mL.

1 INTRODUCTION

Kayu hitam (Diospyros celebica Bakh.F.), classified

as luxury wood species. Other names of kayu hitam

in Indonesia including eboni, toetandu, sora, kayu

lotong, kayu maitong, etc. (Prajadinata et al, 2011).

Kayu hitam is endemic to Indonesia that distributes

from Northern Sulawesi and Central Sulawesi to

Southern (Larekeng, 2016). It is durable and strong

wood, the heartwood with black and reddish brown

stripes makes the texture very beautiful and widely

used for luxury furniture, sculpture, carving, fan,

statues, decorative tools, fancy veneer, musical

instruments and ornaments (Prajadinata et al, 2011).

Sawdust from the processing of kayu hitam can

function as a fungicide. At a concentration of 5%

sawdust ethanol extract can cause a clear zone of 11

mm against the growth of Phytophthora palmivora

Butler (Alwi et al., 2010) and Minimum Bactericidal

Concentration (MBC) value of S. aureus and E. coli

were 12% and 13% respectively (Wahyuni et al.,

2018). This is due to, the sawdust extract contain

chemical compounds such as tannins, saponins and

terpenoids (Wahyuni et al., 2018). Ethanol extract of

kayu hitam also had acute toxicity with LD50 value

of 5.168 mg / kg against male mice (Mus musculus)

(Syam, 2016. The toxicity of a plant depends on

various factors, including quanti-consumed, time of

exposure, different parts of the plant, individual

chemistry, climate and soil, and genetic, species

differences and strength of secondary metabolites

(Mounanga et al, 2015).

Secondary metabolites are products of

metabolism found in plants. Secondary metabolite

compounds are divided into several parts, including

phenolic compounds (Cheynier et al, 2013).

Phenolics are characterized by having at least one

aromatic ring with one or more hydroxyl groups

attached (Crozier, et al., 2006). Phenolics are

important components of the human diet due to their

potential antioxidant activity and their capacity to

diminish oxidative stress induced tissue damage

resulted from chronic diseases (Khadem and Marles,

2010).

Antioxidants are compounds that neutralize

chemically active products of metabolism, such as

free radicals which damage the body. Sources of

natural antioxidants are primarily phenolics that may

occur in all products and parts of a plant such as fruits,

vegetables, nuts, seeds, leaves, roots, and bark (Hajaji

et al., 2010) and also in woody plants such as Toona

sureni (Ekaprasada, et al., 2009 and in the

Archidendron jiringa plants (Lubis, et al., 2018).

234

Br. Sembiring, H. and Romasni Purba, Y.

Isolation and Characterization of an Antioxidant Compound from Kayu Hitam Leaves (Diospyros celebica Bakh.F.).

DOI: 10.5220/0008919802340238

In Proceedings of the 1st International Conference on Chemical Science and Technology Innovation (ICOCSTI 2019), pages 234-238

ISBN: 978-989-758-415-2

Herein we report the isolation characterization of

an antioxidant compound obtained from kayu hitam

leaves and its antioxidant activity. Chemical structure

was determined based on spectroscophy data

interpretation and antioxidant activity based on

scavenging activity of DPPH(1,1-diphenyl-2-

picrylhydrazil) radical method and ascorbic acid was

used as positive control. Isolation and

characterization of an antioxidant compound

obtained from kayu hitam leaves never been reported.

2 MATERIALS AND METHODS

2.1 Materials

Kayu hitam leaves were collected from the front yard

of the Universitas Sumatera Utara, Medan, Sumatera

Utara, Indonesia. Identification of plant was done at

Herbarium Medanense (MEDA) Universitas

Sumatera Utara. Silica (70 – 230 mesh, E-merck) for

column chromatography, FeCl

5%, chloroform (p.a

E Merck), silica 60 F254 (E.Merck) for thin layer

chromatography, TLC Preparative 60 F254, Benzene

(p.a E Merck), Acetone (p.a Merck) methanol (p.a E

Merck) and DPPH (Sigma Aldrich). Methanol as

solvent was distilled before used.

2.2 Instrument

The

H-NMR spectrum was recorded on a Agilent

2NMR 500MHz spectrometer instrument with

CD3OD as a solvent and TMS as an internal standard

and chemical shifts are given in δ (ppm). IR spectrum

were recorded on FT-IR (Shimadzu), UV spectrum

were recorded on Spectrophotometer UV-Vis

(Hewlett Packard Agilent), solvent evaporation with

rotary evaporator (Heidolph), monitoring sample

spots with UV lights (254nm / 356nm, UVGL 58) and

measuring antioxidant activity with a UV-Vis

spectrophotometer (SP-300).

2.3 Procedure

2.3.1 Extraction and Isolation

This extraction and isolation were done based on

Megawati, et al (2015) with a slight modification. The

leaves powder of kayu hitam (1800 g) was macerated

with 8L methanol for 2 x 24 hours. The macerate is

collected, concentrated with a rotary evaporator and

dried on a water bath. Methanol extract (209.63g) was

dissolved with aquadest, the filtrate obtained were

reextracted using ethyl acetate. The solvent in the

ethyl acetate fraction is evaporated to obtained Ethyl

acetate extract. Ethyl acetate extract (32.25 g) was

dissolved with methanol and reextracted by using n-

hexane. The methanol layer was dried using a rotary

evaporator so that the dry methanol extract (12 g) was

obtained. The phenolic compounds in the methanol

extract were separated by using column

chromatography using chloroform: methanol (100:0;

90:10, 80:20, 70:30, 60:40 (%v/%v). Isolates were

collected in the vial every 10 mL and analyzed by

TLC using chloroform: methanol 90:30. Each

fraction with the same Rf value was c combined and

evaporated. Fraction 38-92 (100 mg) at Rf 0.29 was

purified by preparative TLC (Hostettmann et al.,

1995) with chloroform: ethyl acetate 50:50 (% v /%

v) and produced one band spot at the Rf 0.45. The

band spot was crushed, eluted and tested with 5%

FeCl

, evaporated to obtain pure isolates 9.5 g in the

form of yellow solid. The pure isolate was

identification by UV-Vis, FT-IR and

H-NMR

analysis and antioxidant activity test.

2.3.2 Antioxidant Activity Test

Use Antioxidant activity test for pure isolate from

kayu hitam leaves was done based on free radical

scavenging method using DPPH (1,1-diphenyl-2-

pikrylhydrazil) developed by Molyneux (2004) and

Saranya et al., (2017). Samples and ascorbic acid

were dissolved in methanol (p.a E Merck) with

concentrations of 0.5, 10, 15 and 20 µg/mL. The

inhibition percentage can be determined using

equation formula (1) as follow:

𝒊𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏 𝒑𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 =

𝒃𝒍𝒂𝒏𝒌 𝒂𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆−𝒔𝒂𝒎𝒑𝒍𝒆 𝒂𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆

𝒃𝒍𝒂𝒏𝒌 𝒂𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆

𝒙𝟏𝟎𝟎%

(1)

Isolation and Characterization of an Antioxidant Compound from Kayu Hitam Leaves (Diospyros celebica Bakh.F.)

235

3 RESULTS AND DISCUSSION

3.1 Isolation and Characterization

Kayu hitam (Figure 1A) used in this study was the

Ebenaceae family, a species of Diospyros celebica

Bakh. F. with the local name kayu hitam. Pure isolate

was isolated from kayu hitam leaves (Figure 1B) was

phenolic compound, this was evidenced by the

formation of black colloid on the addition of FeCl3

5%. The pure isolate is a yellow solid (Figure 1C).

Identification of phenolic compounds was

determined by UV-Vis, FT-IR and

H-NMR

spectroscopic analysis.

The UV-Visible (CH3OH) spectrum λ

max

290 nm

which is the length of the gallic acid group (Sujata,

2005) is shown in Figure 2.

Figure 1: A Kayu hitam plant, 1B Kayu hitam leaf, 1C Pure

isolate.

Figure 2: Spectrum UV-Visible of pure isolate.

This can be supported by the calculation of the

wavelength for UV-Visibel in theory. Main

Chromophore (246 nm), m-OH (2 X 7 nm = 14 nm),

p-OH (25 nm), so that it is obtained λ

max

285 nm.

Based on the calculation results λ

max

pure isolate

corresponds to λ

max

comparative compound that is

gallic acid (Pavia, 2001).

Figure 3: Spectrum FT-IR of pure isolate.

Figure 4:

H NMR spectrum of pure isolate.

FT-IR spectrum of pure isolated was shown in

Figure 3. The FT-IR spectrum for pure isolates

showed (KBr, ν max, cm

-1

) 3468.01 (O-H), 3311.78

(C-H), 2955.02 (C=H), 1618.29 (C = O), 1313.52 (C-

H), 1251.80 (C-O), indicated that the pure isolate has

a group commonly found in phenolic compound

(Andersen and Markham, 2006).

H NMR spectrum

of pure isolate shown in Figure 4. Based on

H NMR

spectrum (Methanol-D6, 500 MHz, (ppm)) δ 7.04

(2H, s, H-2, H-6), δ 3.81 (3H, s, OCH

), indicated that

pure isolate had two aromatic protons and three

methyl protons. The data in FT-IR and

H NMR

spectrum are similar to FT-IR and

H NMR data

reported by Ekaprasada, et al. (2009). Based on data

analysis and interpretation carried out on the UV-

Visible, FT-IR and

H-NMR spectrum and

comparative

Spectrum reported by Hisham, et al. (2011) it was

stated that pure isolates obtained from the leaves of

kayu hitam plant was simple phenolic compound,

methyl gallate with the structure shown in Figure 5.

Figure 5: Structure of Methyl Gallate.

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

236

3.2 Antioxidant Activity of Pure Isolate

Table 1 showed the percentage of inhibition and IC50

values of methyl gallate and ascorbic acid as positive

control.

Table 1: IC50 of ascorbic acid and methyl gallate.

Sample

Concentration

Inhibition

(%)

(ug/mL)

Methyl

gallate

4.41

84.77

92.38

95.43

97.71

Ascorbic

acid

4.09

85.53

92.38

96.19

97.72

IC50 value of methyl gallate had no significantly

different with ascorbic acid. It showed that methyl

gallate has proton donating ability and could

scavenge the free radical of DPPH.

4 CONCLUSIONS

Pure compound had been isolated from kayu hitam

leaves. Based on the data spectrum UV Vis, FT-IR

and 1H NMR the pure compound was methyl gallate.

Methyl gallate is an antioxidant compound with IC50

value 4.41 μg/mL.

ACKNOLEDGEMENTS

Each We would like to thank to Herbarium

medananse (MEDA), Laboratory of Natural Sciences

Chemistry Faculty of mathematical and Science and

Laboratory of Research, Faculty of Pharmacy

University of Sumatera Utara for identification of

sample, isolation and absorbance measurements in

determining antioxidant activity of pure isolate. We

would also like to thank to Lanang solakhudin and

Elvira Hermawati for the analysis of

Spectrophotometer UV Visible, FT IR and

H-NMR,

Laboratory of Organic Chemistry, ITB Bandung.

REFERENCES

Alwi, M., Ramadanil dan Puspa, D.N., 2010. Ekstrak

Serbuk Gergaji Kayu Eboni (Diospyros celebica Bakh.)

Sebagai Fungisida Terhadap Phytophthora palmivora

Butler. Jurnal Biocelebes. 4 (2),89-97. (Indonesian).

Andersen, Q.M. and Markham, K.M., 2006. Flavonoids

Chemistry, Biochemistry and Applications. Taylor and

Francis Group, LLC. CRC Press.

Chatterjee, S., Zareena Niaz, S. Gautam, Soumyakanti

Adhikari, Prased S. Variyar and Arun Sharma, 2007.

Antioxidant Activity of Some Phenolic Constituents

from Green Pepper (Piper ningrum L.) and Fresh

Nutmeg Mace (Myristica fragrans), Food Chemistry.

101, 515-523.

Crozier, A., Clifford, M.N. and Ashihara, H., 2006. Plant

Secondary Metabolites: Occurrence, Structure and Role

in the Human Diet. Blackwell Publishing Ltd. First

Published. Oxford. USA.

Ekaprasada, M.T., Nurdin, H., Ibrahim, S. and

Dachriyanus., 2009. Antioxidant Activity of Methyl

Gallate Isolated from The leaves of Toona sureni. Indo.

J. Chem. 9 (3), 457 - 460 457.

Lubis, M.Y., Siburian, R., Marpaung,L. Simanjuntak, P.

and Nasution, M.P., 2018. Methyl Gallate From Jiringa

(Archidendron jiringa) and Antioxidant Activity. Asian

J Pharm Clin Res. 11 (1), 346-350.

Khadem, S. and Marles, R.J., 2010. Review: Monocyclic

Phenolic Acids; Hydroxy- and Polyhydroxybenzoic

Acids: Occurrence and Recent Bioactivity Studies.

Molecules. 15, 7985-8005.

Megawati, Saepudin, E., Hanafi, M., Darmawan, A.

Lotulung, P.D.N., 2015. Identification and Bioactivity

Studies of Flavonoid Compounds from Macaranga

hispida (Blume) Mull. Arg. Makara J. Sci. 19(3), 96-100

Molyneux, P., 2004. The Use of the Stable Free Radical

Diphenylpicrylhydrazyl (DPPH) for Estimating

Antioxidant Activity, Songklanakarin J. Sci. Technol,

2004, 26 (2), 211-219.

Prajadinata, S., Effendi, R. and Murniati., 2011. Review of

Management and Conservation Status of Ulin

(Eusideroxylon zwageri Teijsm & Binn.), Ebony

(Diospyros celebica Bakh.) and Cempaka (Michelia

champaca Linn.) in Indonesia. Itto Project Pd 539/09

Rev.1 (F) in Cooperation with Center for Conservation

and Rehabilitation Research and Development,

Forestry Research and Development Agency Ministry

of forestry Bogor – Indonesia

Hajaji, H.E., Lachkar, N., Alaoui, K., Cherrah, Y., Farah,

A., Ennabili, A. Bali, B.E and Lachkar, M., 2010.

Antioxidant Properties and Total Phenolic Content of

Three Varieties of Carob Tree Leaves from Morocco.

Records of Natural Products. 4(4), 193-204.

Larekeng, S.H., 2016. Polymorphism of Simple Sequence

Repeat Regions of Sulawesi Ebony (Diosphyros

celebica Bakh.) in Experimental Forest of Hasanuddin

University Provenance. Agrotech Journal. 1 (1), 37-44

Mounanga, M.B., Mewono, L. and Angone, S.A., 2015.

Toxicity studies of medicinal plants used in sub-

Isolation and Characterization of an Antioxidant Compound from Kayu Hitam Leaves (Diospyros celebica Bakh.F.)

237

Saharan Africa. Journal of Ethnopharmacology. 174,

618–627.

Saranya D, Sekar J, Adaikala RG. Assessment of

antioxidant activities, phenol and flavonoid contents of

different extracts of leaves, bark and root from the

Abutilon indicum (L.) sweet. Asian J Pharm Clin Res

2017; 10:88-94.

Sembiring, H.B., Barus, T., Marpaung, L. Simanjuntak, P.,

2015.Antioxidant and Antibacterial Activity of Some

Leaves Extracts (Methanol, Ethyl Acetate and N-

Hexane) of Scurrula fusca G. Don. International

Journal of Pharm Tech Research.8(9), 24-30.

Wahyuni, Ibrahim, N. and Nugrahani, A.W., 2018. Uji

Aktivitas Antibakteri Ekstrak Serbuk Gergaji Kayu

Eboni (Diospyros celebica Bakh.) Terhadap Bakteri

Staphylococcus aureus dan Escherichia coli.

Biocelebes. 12 (1), 54-64.

Hisham,MN., Lip MJ., Noh MJ., Normah A., Nabila MF.,

2011. Identification and Isolation Of Methyl Galate As

A Polar Chemical Marker For Lobisia Pumila Benth. J.

Trop. Agric. and Fd. Sc. 39(2), 279-284.

Hostettmann, K., Hostettmann, M., Marston, A., 1995. Cara

Kromatografi Preparatif, Penggunaan Pada Senyawa

Bahan Alam. Penerbit ITB. Bandung

Pavia, D.L., Lampman, G.M., Kriz, G.S., 2009.

Introduction to Spectroscopy: A Guide for Students of

Organic Chemistry. Saunders College. Philadelphi

Syam,A,K, (2016). Uji Toksititas Akut Ekstak Etanol Daun

Kayu Hitam (Diospyros celebica b.) Terhadap mencit

(mus musculus). Fakultas Kedokteran dan Ilmu

Kesehatan Universitas Islam Negeri Alauddin

Makassar Samata-Gowa (Skripsi).

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

238