Isolation and Antioxidant Activity of Phenolic Compound from

Leaves Extract of Clidemia hirta D. Don

Sovia Lenny

and Dea Baretta Br Sembiring

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

Medan, North Sumatra 20155, Indonesia

Keywords: Antioxidant, Chromatography, Clidemia hirta, Maceration, Spectroscopy

Abstract: Phenolic compounds have been extracted and isolated to its purity from the leaves extract of Clidemia hirta

D.Don. Extraction procedure included maceration, fractionation, and chromatographic separation of

numerous compounds. Elucidation of purified compound was performed using spectroscopy technique

included UV-Vis, and H-NMR. The results showed that the phenolic compound was categorized as phenolic

acid. Antioxidant activity (IC

) of purified phenolic acid based on DPPH assay was 49.9 µg/mL.

1 INTRODUCTION

Wild plants are sources of bioactive compounds

commonly neglected due to the absence of

profounding health and farmacological information.

However, in some region of Indonesia, local people

have already utilized some wild plant species as a

part of ingredient of their traditional remedies

(Tuttolomondo et al, 2014).

Clidemia hirta (L.) D. Don is a wild plant

species native to South America and also distributed

in Australia, South Asia, Sri Lanka, India, East

Africa and other Pacific islands. This herb is widely

used in traditional medicine. In some examples in

Malaysia, the leaves of senduduk bulu are mixed

with saliva and applied as wound-dressing to prevent

bleeding. Moreover, the root decoction of Clidemia

hirta is used by local Malaysian tribes for the

treatment of fever, diarrhea, irritation and bacterial

infections. The boiled water of the leaves and roots

can also treat stomachache and heart disease. In

Brazil, this species is used to treat skin infections

(Lopez et al, 2016).

Two studies highlighted the antibacterial activity

of the leves extract by agar diffusion method,

however no report so far on the bioactive

compounds in the extract. Although the plant has

been described as wild and native plant, they are

also known as invasive shrubs used in folk medicine

to treat several bacterial infections.

Clidermia hirta has antimicrobial activity which

is potential as a source of preservatives in cosmetic

applications. (Abdellaoui et al, 2014). Clidemia hirta

has been extracted in various organic solvents, such

as ethanol, petroleum ether and chloroform which

yielded crude extracts with antiproliferative and

antioxidant activity (Narasimham et al, 2017).

One of the factors causing disease is oxidative

stress which results in cell or tissue damage.

Currently, a viable and safe alternative to synthetic

antioxidants is being developed which are known for

their ability to prevent oxidation (Marianne et al,

2017). Phenolic compounds including phenolic acids

are secondary metabolites found in plants and fungi.

These compounds are produced for protection

against UV rays, insects, viruses and bacterial

infections as well as inhibiting the growth of other

competing plants (allelopathy).

Phenolic acid can be divided into two major

groups, hydroxybenzoic acid and hydroxyinamic

acid (Haleno, 2015; Sousa, 2018). Phenolic acid is a

polyphenol compound that has bioactivity as an

antioxidant, related to the hydroxyl group attached

to the ring structure. These molecules can act in

various roles, i.e. reducing agents, hydrogen donors,

radical scavengers metal chelating superoxides,

superoxide anions and peroxynitrites (Terpinc et al.,

2011).

Other phenolic compounds, namely gallic acid,

apart from having astringent and styptic uses, also

have several bioactivities such as anti-neoplastic,

bacteriostatic, anti-melanogenic and antioxidant

properties (Kim, 2007). This research was conducted

to isolate the phenolic compounds and test the

526

Lenny, S. and Sembiring, D.

Isolation and Antioxidant Activity of Phenolic Compound from Leaves Extract of Clidemia hirta D. Don.

DOI: 10.5220/0010612300002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 526-528

ISBN: 978-989-758-556-2

antioxidant activity of the leaves extract of Clidemia

hirta (L.) D. Don.

2 MANUSCRIPT PREPARATION

2.1 Materials

Chemicals used for separation and purification

consist of methanol, ethyl acetate, n-hexane, 60 G

(70-230 mesh) silica gel, FeCl

, leaves of Clidemia

hirta (L.) D. Don. Tools in this study were

glasswares, column chromatography, TLC GF254

plate 20×20 cm, IR spectroscopy, UV spectroscopy,

H-NMR spectroscopy.

2.2 Extraction and Isolation of

Phenolic Compound

Dried powder of Clidemia hirta leaves as much as

1.7 kg was macerated using MeOH to yield crude

MeOH extract (124.64 g). The crude MeOH extract

was further fractionated using hexane and EtOAc to

yield each extract for 37.5 and 10.36 g respectively.

The EtOAc extract was purified under column

chromatography using silica gel as stationary phase

and solvent system using chloroform:MeOH

following gradient of polarity. Each fraction was

checked for its purity on thin layer chromatography

(TLC) by spraying FeCl

to indicate any phenolic

compounds. Based on this procedure, we obtained 5

fractions with Fraction-1 (510.5 mg) containing the

highest yield of compound. F1 was further purified

using preparative TLC apparatus. Purified

compound was elucidated using spectroscopy

technique such as UV-Vis, IR, and H –NMR. The

phenolic compound was described following the

description by previous reports.

2.3 Antioxidant Assay

Antioxidative properties of phenolic compound of

Clidemia hirta was assayed based on DPPH radical

scavenging test. Standard solution (DPPH) was

prepared by dissolving 9 mg of compound into 450

mL to obtain a 50 µM solution. The standard

solution was diluted to obtain various concentration

of 100, 50, 25, 12.5, 6.25, and 3.175. Antioxidant

assay was prepared by reacting 0.2 mL of sample

solution into 3.8 mL of 50µM DPPH solution.

Mixture was homogenized for 30 min in dark room.

Absorbance of the solution was checked using UV-

Vis at λ = 516 nm. Antioxidant activity of sample

and positive control was measured using following

formula:

3 RESULTS AND DISCUSSION

3.1 Spectral Analysis of Compound

Purified EtOAc fraction of the leaves extract of

Clidemia hirta (L.) D. Don was yielded as much as

7.4 mg in the form of yellowish white solids. UV-

Vis spectrum (CH

OH) showed a peak at 275 nm,

whereas 270–290 nm was determined as the

detection region for phenolic acid compound

(Vijayalakshmi, 2012). FT-IR spectra showed

absorption for hydroxyl group at 3410 cm

-1

, =C-H

group at 2962 cm

-1

, -C-H group at 2924 and 2854

-1

, carbonyl group at 1712 cm

-1

, and aromatic

C=C group at 1612 cm

-1

The

H-NMR spectrum gave a proton signal with

chemical shift at 7.0 ppm (2H, s, H-2,6) and 3.8 ppm

(3H, s, OCH

). These spectrums displayed two

signals at chemical shift δ 7,0 ppm (2H, s) which is

the signal of two aromatic protons at H-2 and H-6

while the chemical shift of δ 3,8 ppm (3H, s) was the

signal of methoxy group. The chemical shift based

H-NMR resemble the methyl gallate compound

(Ekaprasada, 2009).

3.2 Antioxidant Activity

Determination of antioxidant activity was performed

by measuring the absorbance of remaining DPPH

radical using UV-VIS instrument at 516 nm. The

antioxidant activity was expressed as the percentage

of inhibition of a sample in reacting with radical

solution. Minami et al (1998) grouped the capacity

of antioxidant activity based on IC

value. An

antioxidant compound may be classified as very

active, active, and inactive compound if the IC

value falls between <10, <100, and >100

respectively. The antioxidant activity of phenolic

acid compound from C. hirta leaves was shown in

Figure 1.

Isolation and Antioxidant Activity of Phenolic Compound from Leaves Extract of Clidemia hirta D. Don

527

Figure 1. Percentage of DPPH inhibition by various

extracts of Clidemia hirta leaves.

DPPH is a stable free radical as common

standard in determining the antioxidant properties of

a compound or crude extracts. Based on Figure 1,

the IC

of MeOH, EtOAc, and hexane extract was

50.8, 49.9, and 88.9 µg/mL. Leaves extract of C.

hirta was classified as an active antioxidant since its

< 100. Meanwhile, the EtOAc extract was

regarded as the most prominent antioxidant in this

study.

4 CONCLUSIONS

Purified compound from the leaves extract of

Clidemia hirta was identified as phenolic derivative

or phenolic acid. Antioxidant activity of EtOAc was

determined using DPPH assay showed as IC

= 49.9

µg/mL.

REFERENCES

Abdellaoui,S.E., Destandau,E., Krolikiewicz Renimel,l.,

Cancellieri,P., Toribio,A., Jeronimo, Elfakir, C., 2014,

Centrifugal partition chromatography for antibacterial

bio-guided fractionation of Clidemia hirta roots,

Separation and Purification Technology, 123, 221-228

Ekaprasada, M.T., Hazli Nurdin, H., Sanusi Ibrahim, S.,

and Dachriyanus, 2009, Antioxidant Activity of

Methyl Gallate Isolated From The Leaves of Toona

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

Heleno, S.A., Martins, A., Queiroz, M.J.R.P., Ferreira,

I.C.F.R., 2015, Bioactivity of phenolic acids :

Metabolites versus parent compounds: A review, Food

Chemistry 173:501–513

Kim, Y. J., 2007, Antimelanogenic and antioxidant

properties of gallic acid, Biological and

Pharmaceutical Bulletin, 30, 1052–1055.

Lopez T., Corbin C., Falguieres A., Doussot J.,

Montguillon J., Hagege D., Hano C., Laine E., 2016,

Secondary metabolite accumulation, antibacterial and

antioxidant properties of in vitro propagated Clidemia

hirta L. extracts are influenced by the basal culture

medium. Universite d’Orleans , France

Marianne. H, Salam. Z, Hawraa. S, Kamar. H, Nadine. K,

Hussein. K, 2017, Antioxidant activity of water-

soluble polysaccharide extracted from Eucalyptus

cultivated in Lebanon, Asian Pacific Journal of

Tropical Biomedicine, 7(2), 157-160

Minami, H., Hamaguchi, K., Kubo, M., 1998. A

benzophenone and a xanthone from garcinia

subelliptica, Phytochemistry 49 (6) : 1783-1785

Narasimham D., Bindu Y.H., Cheriyamundath S.,

Raghavan Rahul, Kumari M.K., Chandrasekhar T.,

Madassery J., 2017, Evaluation Of In Vitro

Anticancer and Antioxidant Activities From Leaf

Extracts Of Medicinal Plant Clidemia Hirta, Int J

Pharm Pharm Sci, Vol 9, Issue 4, 149-153

Sousa, M.V., Farias, S.G.G., Castro, D.P., Silva, R.B.,

Silva, D.Y.B.O., Dias, B.A.S., Silva, A.F., Santos,

G.N.L., Matos, D.C.P. and Oliveira, C.V.A., 2018

Allelopathy of the Leaf Extract of Eucalyptus Genetic

Material on the Physiological Performance of Millet

Seeds. American Journal of Plant Sciences, 9, 34-45.

Terpinc, P., Polak, T., Šegatin, N., Hanzlowsky, A., Ulrih,

N. P., Abramovicˇ, H., 2011., Antioxidant properties

of 4-vinyl derivatives of hydroxycinnamic acids, Food

Chemistry, 128, 62–68.

Tuttolomondo, T., Licata, M., Leto. C., Bonsangue,G.,

Gargano, M.L.,Venturella, G., Bella, S.L. 2014

.Popular uses of wild plant species for medicinal

purposes in the Nebrodi Regional Park (North-

Eastern Sicily,Italy).Journal of Ethnopharmacology

157(2014) 21-37

Vijayalakshmi, R., Ravindhran, R., 2012, Comparative

fingerprint and extraction yield of Diospyrus ferrea

(willd.) Bakh. root with phenol compounds (gallic

acid), as determined by uv-vis and ft-ir spectroscopy,

Asian Pacific Journal of Tropical Biomedicine, S1367-

S1371

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