Article

LC-MS Based Metabolite Profiling Leaves Extract of Pluchea indica With

Antioxidant Activity

Afidatul Muadifah^1,*, Dara Pranidya Tilarso², Momodou Salieu Sowe³

,

6

Indra Lasmana Tarigan⁴, Khoirul Ngibad⁵, Sonia Devi Yuliantari

^1,2,6Department of Pharmacy, STIKES Karya Putra Bangsa, Tulungagung, Indonesia

³Departement of Chemistry, University of The Gambia, Gambia

⁴Department of Chemistry, Faculty of Science and Technology, Universitas Jambi, Jambi, Indonesia

⁵Department of D3 Health Analyst, Maarif Hasyim Latif University, Sidoarjo, Indonesia

Abstract

Pluchea indica, a plant known for producing secondary metabolites such as flavonoids, alkaloids, tannins, saponins,

terpenoids, and phenols, exhibits notable antioxidant activity. This study aimed to compare the antioxidant

compound profiles of P. indica leaves using LC-MS analysis and evaluate their antioxidant activities through DPPH

assays. Additionally, molecular docking was conducted to predict the binding interactions between NADPH oxidase

(receptor) and selected small-molecule ligands (compounds from P. indica). Leaf extracts of P. indica obtained

through maceration and Soxhlet extraction were analyzed via LC-MS to identify their chemical composition.

Antioxidant activity was assessed using the DPPH assay at three concentration levels: 10 ppm, 50 ppm, and 100 ppm,

with absorbance measured at 515 nm using a UV-vis spectrophotometer. Furthermore, molecular docking

simulations were performed for the top five compounds against the NADPH oxidase receptor (PDB ID: 4Z3D) to

determine their antioxidant potential. The LC-MS analysis of Soxhlet-extracted P. indica leaves identified 112

compounds, with the top five being Kaempferol 3-glucosyl-(1→2)-[glucosyl-(1→3)-rhamnoside], Quercetin 3-

glucosyl-(1→2)-rhamnoside-7-glucoside,

dimethoxyisoflavone 7-O-(6''-glucosylglucoside),

Luteolin-7-O-(6''-malonylglucoside),

and

5,7,4'-trihydroxy-6,3'-

4'-O-(6''-

5,7,4'-trihydroxy-6-methoxyisoflavone

glucosylglucoside). Antioxidant activity assays revealed IC₅₀values of 76.76 ppm and 62.58 ppm for maceration and

Soxhlet extracts, respectively. Molecular docking results highlighted 5,7,4'-trihydroxy-6-methoxyisoflavone 4'-O-(6''-

glucosylglucoside) as having the best binding affinity, with a free energy value of -5.58 kcal/mol, an inhibition

constant of 51.14 μM, and five hydrogen bonds involving GLN 105, PHE 94, VAL 96, SER 139, and ALA 235.

Keywords: : Antioxidant, LC-MS, Molecular Docking, Pluchea indica

Graphical Abstract

*

Corresponding author

Email addresses: afidatul.muadifah@stikes-kartrasa.ac.id

DOI: https://doi.org/10.22437/chp.v8i2.36869

Received October 09^th2024; Accepted December 20^th2024; Available online December 31^st2024

(https://creativecommons.org/licenses/by/4.0 )

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Chempublish Journal, 8(2) 2024, 75-89

Introduction

In the modern

leaves extracted by cold and hot extraction

identified by Liquid Chromatography-Mass

Spectrometry (LC-MS). We investigated the effect

of maceration and Soxhletation methods on the

compound content of P. indica leaves. The

maceration and soxhlet extracts of P. indica

leaves were then tested using the DPPH method

to identify potential antioxidant compounds.

Afterthan, molecular docking: compounds in

maceration and soxhletation results of P. indica

leaves towards NADPH oxisade receptors (pdb

id:4z3d) as antioxidant

era,

supported

by

the

development of technology and science, changes

in people's lifestyles have a negative impact on

health. Lifestyle changes can lead to a decline in

people's quality of life due to a decrease in the

production of antioxidant compounds in the

body, which are compounds used to neutralize

the presence of free radicals, thus triggering

oxidative stress [1]. Oxidative stress is a condition

where the number of free radicals is not

balanced with the number of antioxidants in the

body. Oxidative stress can trigger the aging

process and the onset of degenerative diseases,

one of which is cancer [2]. One of the causes of

cancer is the presence of free radicals that attack

human body cells; these free radicals are the

leading cause of cell damage so that they can

trigger cancer [3]. The existence of oxidative

stress causes the human body to lack an excess

supply of antioxidants, there by requiring the use

of exogenous antioxidants [4]. Exogenous

antioxidants are obtained from foods that enter

the body and play a crucial role in fighting excess

free radicals in the body [5].

Material and Methods

Materials and Instrumentations

P. indica leaves were collected from Krandegan

Hamlet, RT 002 RW 002, Kalidawir Village,

Kalidawir Sub-district, Tulungagung Regency,

Indonesia. The materials used were Pluchea

indica leaves as samples, chloralhydrate reagent,

10% HCl reagent, 96% ethanol (absolute), distilled

water, Mg powder, Mayer reagent, Wagner,

Dragendorff, chloroform, ammonia, sulfuric acid,

2N HCl, FeCl₃, DPPH compound (2,2-diphenyl-2-

picrylhydraziIi) is a synthetic with free radical and

pure vitamin C (ascorbic acid) which acts as a

positive control. The main materials used in

molecular docking were the five highest

compounds from LCMS results stored in pdb

format in 3D. The receptor structures (target

proteins) of IL-10 (1LQS) and IFN-γ (3BES) are also

stored in pdb format on the respective database

webservers. The tools used are an LCMS (Liquid

et al.) instrument (Shimadzu LCMS-800) and to

One plant that contains bioactive compounds

that act as antioxidants is Pluchea indica (P.

indica), which grows wild. The antioxidant

content in the leaves of P. indica can inhibit free

radical reactions by binding free radicals and

damage to cells [6]. Research antioxidant of P.

indica leaves in maceration extract using 96%

ethanol is classified as very strong, which

produces an IC₅₀value of 37.25 ppm [7].

Antioxidant activity in P. indica leaves extracted

by Soxhletation with a % inhibition value of

51.681 for saline P. indica and 88.377 for non-

saline P. indica [8]. Based on the relevance of the

research, it can be concluded that P. indica leaves

can be extracted using cold and hot methods.

The extraction carried out is by maceration and

Soxhletation. Maceration and Soxhletation are

determine

the

category

of

antioxidant

compounds using a UV-VIS spectrophotometer

instrument (N4S), which will provide data in the

form of absorbance to calculate IC₅₀. Other tools

are a set of Soxhletation apparatus, a set of

maceration tools, a set of glass tools (Pyrex), a

microscope, analytical scales (Acis), black cloth to

cover the sample during the drying process, a

blender, a lab spatula, aluminum foil, water bath

for the concentration process, mesh 60 sieves to

sift P. indica leaf simplicia, filter paper, and spirit

burner. The tools used in molecular docking are

hardware, namely Lenovo PC IdeaCentre AIO 5i

24IAH7 F0GR006RID Strorm Gray (Intel Core i7

12700H, Win11 Home, 16GB DDR4, Intel ARC

methods

that

have

differences

in

the

temperature used during the extraction process.

Maceration and Soxhletation both experience

the soaking process, but for Soxhletation, the

soaking process occurs after the condensation

process [9]. The research aims to determine the

antioxidant effect of compounds in P. indica

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Chempublish Journal, 8(2) 2024, 75-89

A370M 4GB GDDR6) and PyRx software,

ChemDraw Ultra version 22.0, Chem 3D version

22.0, AutoDockTools, Discovery Studio Visualizer

2021 and UCSF Chimera. The webserver used is

RCSB (Research Collaboratory for Structural

Bioinformatics).

carried out using a solvent of 96% ethanol, as

much as 500 ml. This extraction process was

carried out for 15 cycles, then filtered, and the

filtrate was taken to concentrate using a water

bath with a temperature of 50^oC to obtain a thick

extract [9].

Methods

Extract Characterization Test. Ethanol Free Test:

The ethanol-free test aims to determine the

presence of ethanol in an extract. Positive

ethanol-free results are characterized by test

extracts that do not smell of esters, which are

typical of ethanol [16].

Sample Preparation. Samples of Pluchea indica

leaves were picked from Krandegan Hamlet, RT

002 RW 002, Kalidawir Village, Kalidawir Sub-

district, Tulungagung Regency, Indonesia. The

sampling technique is purposive sampling, which

takes samples based on predetermined and

Analysis of Compounds Using LC-MS. Test content

compound results of extracts of the leaves of

Pluchea indica were carried out with LC-MS by

applying the method of dissolving sample-free

ethanol using methanol pa with a ratio of 1:5, i.e.,

with 2 mg of each leaf extract dissolved in 10 ml

methanol pa. Stage protein precipitation with a

considered

criteria,

namely

non-random

sampling [10]. The collected Pluchea indica leaves

were subjected to the stages of wet sorting,

washing using running water, chopping, drying

with sunlight covered with black cloth, and dry

sorting [11]. Dried Pluchea indica leaves were

mashed using a blender and then meshed using

a 60-sieve to achieve a uniform size that is not too

fine or too coarse so that the distillation liquid

can penetrate easily [12].

filter using

a

0.45 cellulose acetate filler

micrometer was then conducted. The sample

was injected with as much as 1 microlite into the

LC-MS instrument. LC (Liquid Chromatography) is

connected to a spectrometer mass Quadrupole

Time- of flight (QTOF) equipped with source

ionization Electrospray Ionization (ESI). The mass

spectrometry (MS) is a QTOF system with a

positive ionization mode. The instrument's

Electrospray Ionization (ESI) parameters were set

at a temperature capillary 350 ºC and atomizer

gas 60ML/HR, source voltage 5.0V. Full scan

mode from m/z 100-5000 was performed with a

temperature source of 100ºC. UPLC column used

Shimadzu Shim Pack FC-ODS (2 mm x 150mm,

3µm) with eluent 95% ethanol and regulated

water on rate total flow 0.5 mL/ min.

Simplicia Characteristic Test. Macroscopic Test:

Macroscopic tests were carried out through

organoleptic testing to determine the shape,

color, smell, and taste of the simplicia, which

were then compared with the literature [13].

Drying Shrinkage Test. Drying shrinkage is the

weight reduction between the fresh sample and

the dried simplicia, which describes the weight

reduction of the material in the drying process

and illustrates the simplicia lost due to the

heating process [14].

Water Content Test. Water content testing was

carried out by weighing the test sample in a

porcelain cup containing 1 g and heating it at 105

⁰C for 30 mins. After 15 mins, it was cooled, and

the powder was weighed again [12].

Antioxidant Activity Test Using DPPH Method.

Determining the Optimum Wavelength of DPPH

Solution: DPPH solution was made with a level of

50 ppm, weighing as much as 5 mg of DPPH

dissolved in 100 ml of ethanol in a volumetric

flask. DPPH solution is kept at a low temperature

with minimal light. Determination of DPPH

absorbance aims to identify how much of the

sample can be absorbed by DPPH compounds.

This determination is done by taking 4 ml of

DPPH solution, leaving it for 30 minutes, and

measuring the absorbance. The optimum

wavelength of 50 ppm DPPH stock solution was

Extraction. This study carries out two extractions:

maceration and Soxhletation. The method of

maceration was carried out by soaking 200 g of

dry leaves of P. indica L in 100 ml of 96% ethanol

for three days. The leaves were then filtered and

concentrated using a water bath at 50^oC,

obtaining a thick extract [15]. Soxhletation was

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Chempublish Journal, 8(2) 2024, 75-89

^(50−a)........................................................ (3)

determined with a UV-Vis spectrophotometer in

the 400-800 nm range [17].

IC₅₀

=

b

Ligand Structure Preparation. Ligand preparation

was carried out by converting the 2-dimensional

(2D) molecular structure of the five main

Antioxidant Activity of Pluchea indica Leaf Extract

and

determined from the IC50 value calculated using

the DPPH method with UV-Vis

Vitamin

C.

Antioxidant

activity

was

compounds

(LCMS

results)

drawn

using

a

ChemDraw Ultra version 22.0 then converted

into a 3-dimensional (3D) structure model using

the Chem3D application version 22.0 (pdb file

format). Then add hydrogen ions to the ligand

using Discovery Studio 2021 software and save in

PDF file format. Furthermore, optimize the ligand

using the AutoDockTools program, and adjust

the number of torsion bonds on the ligand and

save in pdbqt file format.

spectrophotometer. Pluchea indica leaf extract

was made in stock solution with a concentration

of

100

ppm,

then

diluted

with

three

concentration series of 10 ppm, 50 ppm, and 100

ppm. 2 mL was taken from each solution and put

into a test tube. Each test tube was added with 4

mL of DPPH solution. The mixture was incubated

in a dark room for 30 minutes. Then, the

absorbance of the solution was measured using

a UV-Vis spectrophotometer [17].

Macromolecular

Preparation.

The

three-

dimensional macromolecules NAHDP Oxidase

were downloaded from the Protein Data Bank

data site https://www.rcsb.org with PDB codes

used are 4Z3D. Macromolecules are separated

from solvents and native ligands or non-standard

residues using the UCSF Chimera application.

Native ligands and unnecessary residues are

removed by clicking the select feature then

clicking residues and selecting all nonstandard,

then selecting the actions feature, clicking

Vitamin C was made into three concentration

series, namely 1 ppm, 5 ppm, and 10 ppm.

Testing was done by pipetting 2 ml of sample

solution from various concentrations. Each

concentration was added with 4 ml of 50 ppm

DPPH in a closed test tube and left to rest for 30

mins, after which the absorption was measured.

The absorbance results were used to calculate

the percent of free radical remission and then

entered into the equation obtained from the

linear regression curve to obtain the IC₅₀value

[18].

atoms/bonds

Macromolecular (receptor) files are saved in pdb

format. Next, the macromolecules were

then

clicking

delete.

optimized using AutoDockTools by adding

hydrogen ions and Kollman charges and saved in

pdbqt file format.

Determination of Percentage of Antioxidant Activity

of Pluchea indica Leaf Extract and Vitamin C. The

IC₅₀value is calculated based on the measured

absorbance data, and the percentage of

immersion between the DPPH radical and the

sample solution can be calculated using the

equation 1, 2, and 3 [6].

Molecular Docking. The molecular docking

process was carried out using PyRx software

based on AutoDock Tools. The macromolecular

structure (receptor) and ligand that have been

optimized separately are stored in one folder.

The molecular docking process uses a grid box

and energy minimization parameters according

to the validation results. The grid box parameter

settings are carried out using the grid box

coordinates determined based on the receptor

ligand coordinates used in the docking validation

process. Furthermore, docking was carried out

using PyRx software with the AutoDock wizard

feature. The docking data displayed is in the form

of binding affinity values and amino acid residue

interactions. The docking results are stored in

pdb format.

% Inhibition = (BA-SA)/BA × 100%

............ (1)

BA: Blank absorbance; SA: Sample absorbance

The calculation of antioxidant activity was

entered into the equation 2.

y= a+bx .............................................................. (2)

with the concentration (µg/mL) as the abscissa (x-

axis) and the % antioxidant activity value as the

ordinate (y-axis) [17]. The IC₅₀calculation formula

is:

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Chempublish Journal, 8(2) 2024, 75-89

Visualization and Analysis of Docking Results. The

visualization process is carried out to see the

interactions that occur in the docking results

between the receptor and ligand. Visualization of

the docking results is carried out using Discovery

Studio Visualizer 2021 software.

result approximately 9% which is fit to good

simplicia standard.

Furthermore, the water content of simplicia also

a part of important properties, which is less than

10% to avoid fungal growth because enzymatic

reactions only occur when the water content is

less than 10%. The results obtained are 8.2%; it

can be concluded that the drying shrinkage of P.

indica leaf simplicia is by quality standards to

minimize contamination.

Results and Discussions

Simplicia Physicochemical Properties

The results of the macroscopic test of P. indica

Leaf Simplicia are dark green, have a typical smell

of P. indica, and have a strong taste. On the other

hand, we did dried shrinkage test, we found the

The results of the ethanol-free test of maceration

and Soxhletation extracts do not smell typical of

ethanol,

meaning

that

maceration

and

Soxhletation extracts from P. indica leaves do not

contain ethanol.

Figure 1. LC-MS Chromatogram of P. indica extract (maceration process) Analysis conditions: column

Shimadzu Shim Pack FC-ODS (2mm x 150mm, 3µm); flow rate 0.5 mL/min; injection volume 1 µL; solvent

ethanol 95%; column temperature 35 ºC

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Chempublish Journal, 8(2) 2024, 75-89

Figure 2. LC-MS Chromatogram Result of the P. indica soxhlet extract (Analysis conditions: column

Shimadzu Shim Pack FC-ODS (2mm x 150mm, 3µm); flow rate 0.5 mL/min; injection volume 1 µL; solvent

ethanol 95%; column temperature 35 ºC).

Table 1. LC-MS Chromatogram Result of the Pluchea indica macerate

Compound Result

Peak

number

Composition

RT (min)

(%)

Analysis

Structure

Mass Spectrum

Benzaldehyde

1

1,23

0,21302

Fumaric acid

2

1,238

0,39034

Succinic acid

3

1,246

0,47112

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Chempublish Journal, 8(2) 2024, 75-89

Compound Result

Peak

number

Composition

(%)

RT (min)

Analysis

Structure

Mass Spectrum

Benzoic acid

4

5

1,289

0,43318

0,23169

p-cymene

1,471

Table 2. LCMS Chromatogram Result of the P. indica soxhlet extract

Peak

number

RT (min)

Composition

(%)

Compound Result

Structure

Analysis

Mass Spectrum

fumaric acid

1

2

1,238

0,55963

0,43751

succinic acid

benzoic acid

1,246

3

4

1,289

1,471

0,29217

0,45756

p-cymene

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Chempublish Journal, 8(2) 2024, 75-89

Peak

number

RT (min)

Composition

(%)

Compound Result

Structure

Analysis

Mass Spectrum

α-terpinene

5

1,476

0,61809

The LC-MS results of Pluchea indica L extract

obtained by maceration extraction method (114

compounds) and soxhletation (112 compounds)

were then grouped based on the group of

secondary metabolite compounds that have

potential as antioxidants: Alkaloids, flavonoids,

tannins, saponins, terpenoids and phenols.

Furthermore, they were compared to determine

the most secondary metabolite compounds

(which have potential as antioxidants) between

the maceration or soxhletation methods.

Table 3. Grouping of secondary metabolite compounds from LC-MS results of P. indica

Secondary metabolite

compounds

Flavonoid

Composition (%)

Maceration

72.4841

9.00625

6.11827

6.99075

2.2256

Soxhletation

67.14345

8.95854

Phenol

Terpenoids

Alkaloids

13.82165

5.35637

2.53305

Tannins

Saponins

2.04458

0.2969

Antioxidant Activity of Macerated

Antioxidant Activity of Vitamin C

Extract of P. indica Leaf

40

30

20

y = 0,3107x + 26,369

80

60

40

20

0

R² = 0,8684

y = 10,788x - 3,1497

10

R² = 0,8669

0

50

100

150

0

1

2

3

4

Sample concentration (ppm)

Antioxidant Activity of Soxhlet Extract

of P. indica Leaf

80

60

40

20

0

y = 0,3093x + 30,644

R² = 0,7672

0

20

40

60

80

100

120

Sample concentration (ppm)

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Figure 3. Linear regression of the relationship the concentration of test samples and % inhibition

(Vitamin C, Pluchea indica leaf maceration extract, and Pluchea indica leaf Soxhletation extract)

The results of calculating the antioxidant activity

test with DPPH of vitamin C as a positive control

have an IC₅₀value of 4.927 ppm, indicating good

antioxidant activity. Meanwhile, our results show

that Pluchea indica leaf extract has a strong

antioxidant activity with an IC₅₀value of 76.057

ppm for maceration and 62.580 ppm for

Soxhletation.

Table 4. Antioxidant Activity of Pluchea indica extracts

Samples

Concentration

(ppm)

Abs. Average

% Inhibition

IC₅₀(ppm)

Maceration

extract

10

50

100

± 0.470

± 0,329

± 0,288

25,984

48,189

54,646

76.057

(Strong Antioxidant)

Soxhletation

extract

10

50

100

± 0,452

± 0,286

± 0,269

28,819

54,960

57,638

62.580

(Strong Antioxidant)

Vitamin C

1

5

10

± 0,602

± 0,487

± 0,465

5,197

23,307

26,772

4.927

(Very Strong Antioxidant)

Based on this explanation, maceration and

Soxhletation extractions of P. indica leaves have

similar antioxidant content (Figure 3 and Table 4).

The antioxidant activity of a sample can be

determined by looking at its IC₅₀value. IC₅₀

(inhibition concentration) value is a number that

indicates the concentration of the sample that

can reduce DPPH by 50%. A sample can be said

to be a powerful antioxidant if it has an IC₅₀value

< 50 ppm, a potent antioxidant if its IC₅₀value is

50 - 100 ppm, a moderate antioxidant if its IC₅₀

value is 100 - 150 ppm, a weak antioxidant if its

IC₅₀value is 150 - 200 ppm, and a very weak

antioxidant if its IC₅₀value is > 200 ppm²⁴. Both

extractions produced IC₅₀values > 100 ppm,

which are potent antioxidants. Extracts from the

Soxhlet extraction show an IC₅₀value of 62.580

ppm, while the macerated extracts show 76.757

ppm. The greater the IC₅₀value, the smaller the

antioxidant potential [18]. It is also shown by the

test using LC-MS that there are many compound

compositions contained in Pluchea indica leaf

extracts that have potential as antioxidants,

namely mainly flavonoids and terpenoids, which

have a relatively large composition, and there are

differences in composition between the two

extraction methods. The maceration extract has

a flavonoid composition of 72.4841% of 60

compounds, while the solution extract is

67.14345% of 57 compounds. In macerated

extracts, the terpenoid group has a composition

of 6.11827% of 25 compounds; in solution

extracts, terpenoids are 13.82165% of 34

compounds. Flavonoids and terpenoids belong

to the class of powerful antioxidants. The Soxhlet

extraction has a smaller IC₅₀value than the

macerated extract but is still in one activity

category: potent antioxidants. The smaller the

IC₅₀value, the greater the antioxidant activity and

the greater the % inhibition value of a sample

tested. IC₅₀value is calculated based on

absorbance data that has been measured from

the percentage of immersion between DPPH

radicals and sample solution [19]. To ensure that

the compounds in the P. indica extract work as

antioxidants, molecular docking was carried out

on the five main compounds resulting from

LCMS.

Molecular Docking Results of NAHDP Oxidase

Receptor (4Z3D).

A computational method, is used to predict the

binding of a macromolecule (receptor) to a small

molecule in the form of a ligand (five main

compounds of LCMS). The purpose of this

83

Chempublish Journal, 8(2) 2024, 75-89

molecular

docking

is

to

determine

the

Kaempferol 3-glucosyl-(1→2)- [glucosyl-(1→3)-

rhamnoside (1), Quercetin 3-glucosyl-(1→2)-

rhamnoside-7-glucoside (2), Luteolin-7-O-(6''-

conformation and free energy of the bond

involved in the interaction between the

macromolecule (receptor) and the ligand [22].

This docking simulation helps in studying drugs

or ligands or receptor/protein interactions, to

obtain good geometry of the ligand-receptor

complex [23]. In this research we conducted

molecular docking of five major compounds;

malonylglucoside)

(3),

5,7,4'-trihydroxy-6,3'-

dimethoxyisoflavone 7-O-(6''-glucosylglucoside)

(4), and 5,7,4'-trihydroxy-6- methoxyisoflavone

4'-O-(6''- glucosylglucoside) (5) against Oxidase

Receptor (NAHDP).

Table 5. NAHDP Oxidase Receptor (4Z3D) Docking Results

Hydrogen

Bond

Distance

(Å)

Binding

Inhibition

Constans

(μM)

Amino

Acid

Residues

Hydrogen

Bonds

Interaction

Type

Ligans

Free Energy

Category

(kkal-mol^-1)

MET

TRP

ALA

SER

GLN

SER

GLY

234

229

235

190

105

191

194

Van Der Waals

MET 141

TYR 193

MET 234

VAL 96

H-Bond

Hidrofobik

Conv.

Carbon H-Bond

Alkyl

Van Der Waals

H-Bond

TYR 193

MET 234

2.4667

3.40771

(4)

(5)

-5.42

-5.58

107.19

51.14

ALA 93

PHE

94

ALA 192

GLN

PHE

105 H-Bond

94 H-Bond

Conv. H-Bond

Conv.

Conv. H-Bond

Sulfur-X

GLN

PHE

105 2.27997

94 2.41434

VAL 96 SER H-Bond

139

235

ALA H-Bond

H-Bond

VAL 96

SER

2.24751

139 2.54893

MET 243

TYR 193

ALA 235

2.67562

Van Der Waals

Table 6. Docking visualization

Compounds

3D

Visualization Results

2D

(1)

(2)

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Compounds

(3)

Visualization Results

3D

2D

(4)

(5)

The smaller the results of the docking process

indicate that the protein ligand complex will be

more stable, so that the compound is more

patent [24]. Based on the statement of Azzahra

et al (2021) [25], the free energy of binding (∆G)

and the inhibition constant (Ki) are parameters to

determine the quality of the bond formed. The

results of molecular docking can be seen from

the value of the free energy of binding (∆G) where

the smaller the value of the free energy of

binding, the higher the affinity between the

receptor and the ligand, conversely the greater

the value of the free energy of binding, the lower

the affinity between the receptor and the ligand

[26]. The ability of a ligand to inhibit the activity

of a receptor can be analyzed through the

inhibition constant (Ki) of the ligand. The smaller

the inhibition constant (Ki) value, the stronger the

binding affinity and the less the amount of drug

needed to inhibit enzyme activity. The smaller the

Ki value, the stronger the binding of the ligand to

the protein [27]. The inhibition constant can be

stated as strong if it has a value of ≤ 100 μM and

conversely the inhibition constant is stated as

weak if it has a value of ≥ 100 μM [28].

Molecular docking was carried out on five test

compounds, namely Kaempferol 3-glucosyl-

(1→2)- [glucosyl-(1→3)-rhamnoside, Quercetin 3-

glucosyl-(1→2)-

rhamnoside-7-glucoside,

Luteolin-7-O-(6''-

trihydroxy-6,3'-dimethoxyisoflavone

malonylglucoside),

5,7,4'-

7-O-(6''-

glucosylglucoside), and

5,7,4'-trihydroxy-6-

methoxyisoflavone 4'-O-(6''- glucosylglucoside)

which will be docked to the NADPH Oxisade

receptor (PDB id: 4Z3D) which has antioxidant

activity. From the docking results obtained, the

best affinity was obtained for 5,7,4'-trihydroxy-6-

86

Chempublish Journal, 8(2) 2024, 75-89

methoxyisoflavone 4'-O-(6''- glucosylglucoside)

with a binding free energy value of -5.58 kcal/mol

with an inhibition constant value of 51.14 μM and

produced five hydrogen bonds GLN 105 PHE 94

VAL 96 SER 139 ALA 235. The use of the NADPH

Oxidase receptor is thought to have antioxidant

activity, where this molecule can work to

neutralize reactive radicals (ROS and RNS) and

can prevent cell damage. Carbonyl reductase w-

1 in humans that depends on NADPH can

contribute to the metabolism of endogenous

Author Contibutions

Conceptualization, A.M. and D.P.T.; Methodology,

A.M., D.P.T., S.D.Y., I.L.T. Validation, A.M and K.N.;

Formal Analysis, A.M., S.D.Y., Investigation, A.M

Resources, A.M., D.P.T., and S.D.Y; Data Curation,

A.M., M.S.S; Writing – Original Draft Preparation,

A.M., S.D.Y; Writing – Review & Editing, A.M., I.L.T.,

K.N., M.S.S; Visualization, A.M.; Supervision, A,M.,

D.P.T and K.N; Project Administration, A.M.;

Funding Acquisition, A.M., D.P.T

carbonyl-containing

compounds

and

Conflict of Interest

xenobiotics. Where this enzyme works by

protecting cells from cellular damage due to

oxidative stress [29].

There are no significant conflicts

Conclusions

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