Antioxidant Activity from Lygodium microphyllum Aerial Parts

Hadi Kuncoro*, Laode Rijai

Laboratorium Penelitian dan Pengembangan Kefarmasian Farmaka Tropis, Fakultas Farmasi Universitas Mulawarman,

Samarinda, Indonesia, 75117, Phone : +62541739491

Keywords: L. microphyllum, Antioxidant, DPPH, IC

Abstract: Lygodium microphyllum is an invasive plant known as another name “Old World Climbing Fern” or

considered a weed plant. Research on L. microphyllum related pharmacological activity is limited. This

study aims to evaluate antioxidant activity from Methanol extract and Fractionation using n-hexane, Ethyl

acetate and water solvent with a different polarity from L. microphyllum plant. Methods: Aerial part of L.

microphyllum was macerated with methanol solvent, the extract obtained in fractionation using solvents

with different polarity levels and was tested in invitro free radical using 1, 1-diphenyl-2-picrylhydrazyl

(DPPH) free radical. The IC50 result was 49.328 ± 0.242 ppm for methanol extract, n-hexane extract was

32.041 ± 0.770 ppm, and ethyl acetate extract 8,732 ± 0.204 ppm and water extract 93.330 ± 1.931 ppm.

The conclusion from this study reveals the results of extraction using ethyl acetate solvent showed the

highest value antioxidant activity against DPPH.

1 INTRODUCTION

Lygodium microphyllum is an invasive plant known

as “Old World Climbing Fern” or is considered a

weed plant. L. microphyllum plants develop and

growing fast in large colonies that dominate an area

and cause disruption of the ecosystem the original

flora and degrades critical ecosystem services and

habitats of rare and endangered species (Ferriter

2001; Langeland & Hutchinson, 2001; Pemberton,

1998).

L. microphyllum spread to the nearby plant and live

in an open place to get sunshine (Bower, 2010).

Some studies showed L. microphyllum have

medicinal properties including antibacterial (Wang

et al 2014), Hepatoprotector (Gnanaraj et al, 2017),

Cytotoxic activity against P-388 Murine Leukemia

cells (Kuncoro, et al 2017), and antiplasmodial

against Plasmodium falciparum (Ratri et al, 2018).

The chemical composition of L. microphyllum has

been reported including steroid and glycoside

flavonoids ( Kuncoro, et al 2017).

Reports of the antioxidant activity from other

species Lygodium in one genus have been reported.

Methanol extract from L. flexuosum has antioxidant

activity (Jeetendra & Manish, 2011).

The aim of this studies to evaluated Antioxidant

activities aerial part from L. microphyllum against

DPPH.

2 MATERIAL AND METHOD

2.1 Material

L. microphyllum aerial part was obtained from

Lempake Village, Samarinda, Kalimantan Timur.

Specimens were identified and deposit by staff at the

Dendrology laboratory, Foresty Faculty,

Mulawarman University.

2.2 Extraction Process

Aerial parts of L. microphyllum are cleaned and

dried and powdered to obtain 3.52 Kg. The dry

powder was macerated with methanol solvent at

room temperature. The methanol extract was

obtained and concentrated 526 g with a Rotary

Evaporator (Buchi

). The methanol extract obtained

was dissolved in water (1:1) and partitioned

successively with n-hexane solvent, ethyl acetate

solvent, and Aqua solvent. The resulting extract with

solvent was evaporated and obtained n-hexane

extract (59 g), ethyl acetate extract (72 g), and aqua

extract (296,5 g).

250

Kuncoro, H. and Rijai, L.

Antioxidant Activity from Lygodium microphyllum Aerial Parts.

DOI: 10.5220/0008360802500254

In Proceedings of BROMO Conference (BROMO 2018), pages 250-254

ISBN: 978-989-758-347-6

2.3 Antioxidant Assay

2.3.1 Preparation

The DPPH crystals were weighed 4 mg to be

dissolved in 100 mL of methanol in the measuring

flask for obtaining a DPPH solution with a

concentration of 0.004% or 40 ppm (part per

million) used in the test. The solution is stored in a

tightly sealed place and protected from light.

2.3.2 Concentration Series Determination

The concentration series used was 0.2,4,6,8,10 ppm..

These concentration variations were used in

antioxidant activity testing by DPPH method.

Preparation of stock solution with a concentration of

1000 ppm (10 mg dry extract diluted with methanol

to 10 mL). Furthermore, dilution to obtain the

concentration and Duplo repetition or two

repetitions and a negative control that DPPH

solution and methanol (without the addition of

extract).

2.3.3. Determination of the Maximum

Absorption Wavelength DPPH

2 mL of 0.004% DPPH solution was added with 2

mL of methanol. After being left for 30 minutes in

the dark, Absorption of the solution was measured

by UV-Vis spectrophotometer at 515 -520 nm

wavelength to obtain the maximum wavelength.

2.3.4 Antioxidant Assay

The antioxidant test was carried out through a series

of sample solutions from the methanol extract and

the three fractions with 2 repetitions using a

methanol solvent. Each solution plus 2 mL of DPPH

solution, in order to obtain a solution of a

predetermined concentration, was allowed to stand

for 30 minutes (calculated after addition of DPPH

solution), measured its absorbance at the maximum

wavelength. The absorbance data obtained is used to

determine % inhibition (damping). Through sample

concentration curve versus % inhibition, IC

extract

value can be obtained with statistical analysis using

linear regression. There is also a measure of

absorbance of blanks (methanol). The antioxidant

test indicator is the color change of DPPH. The data

in this study is the percentage of DPPH radical

reduction obtained using the formula:

% AA=

𝐴𝑏𝑠. 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠. 𝑆

𝐴𝑏𝑠. 𝐶𝑜𝑛𝑡𝑟𝑜𝑙

× 100%

Data analysis using linear regression equation using

the formula:

y = b (x) + a

Explanation:

y: % AA

x: log concentration

a: intercept

b: slope

3 RESULT AND DISCUSSION

In this study conducted testing of antioxidant

activity of aerial parts of L. microphyllum. Dry

powder aerial parts of L. microphyllum macerated

using methanol to obtain methanol extract. Methanol

extract in fractionation based on polarity level using

n-hexane, ethyl acetate, methanol and water. Each

extract obtained was tested for its antioxidant

activity using DPPH method. This method was

chosen because is simple, easy, fast and sensitive

and requires only a little extract. This method is

often used to detect the anti-radical ability of a

compound because the result proves to be accurate,

reliable, relatively fast and practical. The principle

of this method is the measurement of synthetic free

radical capture in polar organic solvents such as

ethanol or methanol at room temperature by a

compound having antioxidant activity. This process

of radical capture through the mechanism of taking

hydrogen atoms from antioxidant compounds by

free radicals so that free radicals capture an electron

from antioxidants. With the arrest of these radicals,

the diazo double bond in DPPH decreases, resulting

in decreased absorbance. The free radical used is

DPPH (2,2-diphenyl-1-picrylhydrazyl).

DPPH reacts with antioxidant compounds by

taking hydrogen atoms from antioxidant compounds

to obtain electron pairs. The compounds that can

potentially as antioxidants are from the class of

flavonoids, alkaloids, phenols, and tannins. Before

the testing of antioxidant activity, the first

determination of the maximum wavelength of DPPH

used with the wavelength range 515-520 nm. The

maximum wavelength is obtained from the

maximum absorbance of 515 nm. This wavelength

will be used in subsequent antioxidant testing. The

compound that reacts as a radical catcher will reduce

DPPH which can be observed by the color change of

DPPH to yellow from purple when the odd electron

from the DPPH radical has paired with hydrogen.

From the free radical captured compound which will

Antioxidant Activity from Lygodium microphyllum Aerial Parts

251

form the reduced DPPH-H (Molyneux, 2004) seen

in Figure 1.

Figure 1: DPPH Radical Reaction with Antioxidant

Compound (Molyneux, 2004)

All electrons in DPPH free radicals become in pairs,

then the color of the solution changes to bright

yellow from dark purple and absorbance at 515 nm

wavelength will be lost. These changes can be

measured stoichiometrically according to the

number of electrons or hydrogen atoms captured by

DPPH molecules due to the presence of antioxidants

(Gurav, 2007). The calculation used in the

determination of radical catch activity is the value of

(Inhibition Concentration 50%). This value

describes the concentration of test compounds that

can capture radicals by 50%. IC

values in this

study were obtained by using a linear regression

equation which expresses the relationship between

sample concentrations (extract test) with the symbol

(x) with radical capture activity (y) from series

replication measurement. The smaller the value of

obtained, the test sample has more effectiveness

as a better radical catcher. Molyneux (2004) states

that a substance has antioxidant properties if the IC

value is less than 200 ppm. If the value of IC

obtained ranges from 200-1000 ppm, then the

material is less active but still potentially as an

antioxidant substance.

From the test result on methanol extract, n-hexane,

ethyl acetate, and water obtained absorbance value

and% inhibition.

Table 1: Absorbance and % inhibition of antioxidant activity (DPPH) of methanol extract.

No.

ppm

Absorbance

% inhibition

0,7745

0,0000

0,7559

0,7684

2,4015

0,7876

0,7404

0,7605

4,4028

1,8076

0,7160

0,7255

7,5533

6,3267

0,7116

8,1214

0,6973

0,6958

9,9677

10,1614

Table 2: Absorbance and % inhibition of antioxidant activity (DPPH) of n-hexane extract

No.

ppm

Absorbance

% inhibition

0,8478

0,0000

0,8068

0,8241

4,8360

2,7955

0,7678

0,7776

6,0156

8,2803

0,7573

0,7549

10,6747

10,9578

0,7478

11,7952

0,7114

16,0887

Table 3: Absorbance and % inhibition of antioxidant activity (DPPH) of ethyl acetate extract

ppm

Absorbance

% inhibition

0,8820

0,0000

0,7994

0,8033

9,3651

8,9229

0,6788

0,7070

23,0386

19,7732

0,5718

0,5749

35,1701

34,8186

0,4704

0,4828

46,6667

45,2608

0,3689

0,3849

58,1746

56,3605

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

252

Table 4: Absorbance and % inhibition of antioxidant activity (DPPH) of water extract

No.

ppm

Absorbance

% inhibition

0,9879

0,0000

0,9849

0,3037

0,9624

2,5812

0,9607

0,9489

2,7533

3,9478

0,9439

0,9454

4,4539

4,3021

0,9396

0,9388

4,8892

4,9701

Table 5: Antioxidant activity (IC

)

No.

Extract

(ppm)

Methanol

49.328 ± 0.242

n-hexane

32.041 ± 0.770

Ethyl Acetate

8,732 ± 0.204

Water

93.330 ± 1.931

From data assay against DPPH obtained that the

extract of ethyl acetate has the highest IC

value

8,732 ± 0204 ppm. Based on this, the aerial part

extract of L. microphyllum has an activity of an

antioxidant with an IC

value of less than 200 ppm.

4 CONCLUSIONS

Ethyl acetate fraction of aerial parts L. microphyllum

has the highest antioxidant activity against DPPH

with an IC

value of 8,732 ± 0,204 ppm.

ACKNOWLEDGMENTS

Thanks to Fakultas Farmasi Universitas

Mulawarman Samarinda and Central Laboratory

Padjadjaran University for Antioxidant assay against

DPPH.

REFERENCES

Bower, F. O. 2010. The Fern (Filicales). Cambridge

University Press. Cambridge

Ferriter, A. 2001. Lygodium Management Plant for

Florida. Florida: Florida Exotic Pest Plant Council

Lygodium Task Force.

Gnanaraj, C., Shah, M.D., Song, T.T. and Iqbal, M., 2017.

Hepatoprotective mechanism of Lygodium

microphyllum (Cav.) R. Br. through ultrastructural

signaling prevention against carbon tetrachloride

(CCl4)-mediated oxidative stress. Biomedicine &

Pharmacotherapy, 92, pp.1010-1022.

Gurav, S., Deshkar, N., Gulkari, V., Duragkar, N., & Patil,

A. 2007. Free radical scavenging activity of Polygala

chinensis Linn. Pharmacology Line, 2, 245-253.

Hutchinson, J.T. & Langeland, K.A., 2013. Susceptibility

Of Old World Climbing Fern (Lygodium

microphyllum) Gametophytes To Metsulfuron Methyl.

Invasive Plant Science and Management 6:304-309.

Jeetendra, N. E. H. E. T. E., & Manish, B. H. A. T. I. A.

(2011). Correlation of antioxidant activity with

phenolic content and isolation of antioxidant

compound from Lygodium flexuosum (l.) Sw.

Extracts. Int J Pharm Pharm Sci, 3(2), 48-52.

Kuncoro, H., Farabi, K. and Rijai, L., 2017. Steroids and

isoquercetin from Lygodium microphyllum. Journal of

Applied Pharmaceutical Science Vol, 7(11), pp.136-

141.

Kuncoro, H., Rijai, L., Julaeha, E., & Supratman, U.,

2017, Cytotoxic Activity Against P-388 Murine

Leukemia Cell From Lygodium microphyllum HERB,

Jurnal Farmasi Galenika Volume 3 No. 1; p 13-16

Langeland, K.A. & Hutchinson, J., 2001. Natural Area

Weeds: Old World Climbing Fern (Lygodium

microphyllum). University of Florida Cooperative

Extension Service, Institute of Food and Agricultural

Sciences, EDIS.

Molyneux, P., 2004. The use of the stable free radical

diphenylpicrylhydrazyl (DPPH) for estimating

antioxidant activity. Songklanakarin J. Sci. Technol,

26(2), pp.211-219.

Pemberton, R. W. 1998. The Potential Of Biological

Control To Manage Old World Climbing Fern

(Lygodium microphyllum), An Invasive Weed In

Florida. American Fern J. 88: 176-182.

Pemberton, R. W., and Ferriter, A. P. 1998. Old World

Climbing Fern (Lygodium microphyllum), A

Dangerous Invasive Weed In Florida. American Fern

J. 88: 165-175.

Ratri, T. R., Harlia, & Widiyantoro, A., 2018,

Characterization of Antimalarial Compounds from

Lygodium microphyllum Ethyl Acetate Fraction

Antioxidant Activity from Lygodium microphyllum Aerial Parts

253

against Plasmodium falciparum, Jurnal Kimia

Khatulistiwa, 7(2): 89-95

Wang, R.L., Zheng, Z.H., Lu, J., Shao, H., Zhang, H., Su,

Y.J., & Cai, Y.F. 2014. Allelopathic potential of

invasive climbing fern Lygodium microphyllum

against native plants and antibacterial activity of

essential oils. Allelopathy Journal. 33(1), 97–106.

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