Article

Comparative Study of Macerated and Soxhlet-Extracted Moringa oleifera Leaf

Extracts: LC-MS-Based Metabolomic Profiling, Antioxidant Activity, and In Silico

Target Prediction

Afidatul Muadifah^1*, Sulastri², Momodou Salieu Sowe³, Anggun Lintang Kharizma⁴, Tri

Warni⁵

^1,2,4Department of Pharmacy, STIKES Karya Putra Bangsa, Tulungagung (Indonesia)

³Departement of Chemistry, University of The Gambia

⁵Department of Chemistry, Faculty of Science and Technology, Universitas Jambi, Muara Jambi 36361, Jambi

Abstract

Moringa leaves (Moringa oleifera L.) are rich in secondary metabolites such as flavonoids, alkaloids,

tannins, saponins, and terpenoids, which function as natural antioxidants. This study aimed to analyze

the metabolite profile of M. oleifera leaf extracts obtained through two extraction techniques using LC-

MS, evaluate their antioxidant activity via the DPPH assay, and predict the interaction between NADPH

oxidase (as a receptor) and key plant-derived compounds through molecular docking. LC-MS results

indicated that the maceration method yielded 101 secondary metabolites, with flavonoid derivatives

comprising 70.99% of the extract, dominated by five key compounds including Kaempferol 3-O-

robinobioside and Luteolin-7-glucoside. In contrast, the Soxhlet method resulted in 83 identified

compounds, with a higher proportion of flavonoids (75.61%), and prominent compounds including

quercetin-3-O-glucoside and Kaempferol 3-(6G-malonylneohesperidoside). Antioxidant testing with

DPPH at concentrations of 10, 50, and 100 ppm revealed the Soxhlet extract had a stronger activity (IC₅₀

= 14.328 ppm) compared to the macerated extract (IC₅₀ = 32.092 ppm), with statistically significant

differences

(p

<

0.05).

Molecular

docking

demonstrated

that

Kaempferol

3-(6G-

malonylneohesperidoside) exhibited the strongest binding affinity to NADPH oxidase (-10.1 kcal/mol),

followed by other flavonoid derivatives. These findings underscore the antioxidant potential of M.

oleifera, particularly from Soxhlet extraction, and suggest its promising application in pharmaceutical

development as a natural antioxidant source.

Keywords: Antioxidant activity, In silico studies, metabolomic profiling,

Graphical Abstract

*

Corresponding author

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

DOI: https://doi.org/10.22437/chp.v9i1.36870

Received August 08^th2024; Accepted May 28^th2025; Available online June 01^St2025

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

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Chempublish Journal, 9(1) 2025, 74-100

Introduction

that natural materials do not decompose. Cold

extraction allows many compounds to be

extracted, but some compounds have limited

solubility in the extraction solvent at room

temperature. Meanwhile, the hot extraction

method (soxhletation) is the best method for

obtaining extensive extract results. Also, it uses

less solvent, the extraction process is fast, and it

allows for complete sample extraction because

the process is repeated. In addition, because the

biological activity is not lost when heated, this

technique can be used to search for drug-

relevant compounds [10].

The rich abundance and diversity of natural

resources in Indonesia have resulted in a lack of

understanding among the Indonesian population

regarding the optimal utilization of these natural

products, leading them to favor the consumption

of instant foods instead [1]. Unhealthy lifestyles

can cause the body to be continuously exposed

to free radical compounds [2]. Changes in the

human body are often exposed to dangerous

substances that can cause disease and

degenerative changes. Most diseases begin with

excessive oxidation reactions in the human body

[3]. Cancer, organ cell damage, cataracts, and

degenerative diseases are some examples of cell

damage caused by free radicals [4]. Other

examples are pollution, alcohol, tobacco smoke,

heavy metals, transition metals, industrial

solvents, pesticides, certain drugs, and radiation

[5].

Herein, the effect of maceration and soxhletation

extraction on the antioxidant content of Moringa

oleifera L. leaves was analyzed using LC-MS with

ethanol solvent. The antioxidant potential of the

macerated and soxhleted Moringa oleifera L. leaf

extracts was compared by DPPH test. The

antioxidant potential of the main metabolites

from the two extraction methods was evaluated

in silico through molecular docking with NADPH

Oxidase enzyme, using Apocynin A as a positive

control. The investigation of the Moringa oleifera

L species opens perspectives towards a better

understanding of its biological efficiency and its

Antioxidants are one way to prevent free radicals

in the body. Natural ingredients in plant parts

such as roots, stems, leaves, flowers, fruit, seeds,

and pollen can contain antioxidants [6]. The

moringa leaf has the potential to be an

antioxidant. Moringa leaves contain more than

forty types of natural antioxidants, making them

a valuable source of natural antioxidant plants

with secondary metabolite compounds that

contain flavonoids, tannins, alkaloids, and

saponins [7]. Similarly, reported the antioxidant

activity of a 70% ethanol extract of Moringa

leaves (Moringa oleifera L.) on DPPH; it shows that

Moringa leaves contain flavonoid compounds

that are useful as antioxidants, as proven by the

results of the phytochemical screening carried

out [8]. This was supported by results from

another study, which showed that the ethanol

extract of Moringa leaves has antioxidant activity

with an IC.50 value of 18.15 µg/mL [9].

interpretation

composition

metabolites.

based

of the

on

studied

the

chemical

secondary

Materials and Methods

Materials

The study used samples of Moringa leaves from

Pecuk Village, Pakel District, Tulungagung

Regency, Indonesia. Chemicals like aquadest,

Vitamin

C

(ascorbic

acid),

ethanol

96%

(ABSOLUTE), Mayer's reagent, Dragendorff's

reagent, Wagner's reagent, hydrochloric acid

(HCl) (EMSURE®), Zn powder, ferric chloride

(FeCl₃), chloroform, anhydrous acetic acid,

sulphuric acid (H₂SO₄) and DPPH powder (1,1-

diphenyl-2-picrylhydrazyl (HIMEDIA®), distilled

water (ABSOLUTE).

The antioxidant compounds in Moringa leaves

can be extracted using several methods.

Maceration and soxhletation extraction methods

were chosen to extract Moringa leaves because

they have many advantages over other

extraction methods. The main advantage of the

maceration extraction method is that the

procedures and equipment used are simple; the

maceration extraction method is not heated so

Preparation and Extraction

The initial step involved selecting mature Moringa

oleifera leaves, followed by wet sorting and air-

drying under shade to prevent degradation of

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A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-100

active compounds. Extraction was performed

using two methods: (1) Maceration—250 g of

powdered simplicia was soaked in 1000 mL of

96% ethanol for 72 hours with periodic agitation

mode. The analysis conditions used are 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. After get all metabolites profle then screen

antioxidant activity used DPPH method.

to

ensure

diffusion

equilibrium.

(2)

Soxhletation—50 g of powdered simplicia was

extracted using a Soxhlet apparatus with 500 mL

of 96% ethanol at 40–55ꢀ°C. Both extracts were

concentrated using a water bath at the same

temperature range until a thick extract was

obtained

Antioxidant Activity by DPPH assay

A 1 mL aliquot of 100 ppm DPPH in absolute

ethanol was mixed with Moringa oleifera

ethanolic extract and diluted to

5

mL.

Absorbance was measured between 510–520

nm, and 515 nm was selected as the optimum

wavelength. For extract preparation, 2.5 g of thick

Moringa oleifera extract was dissolved in 5 mL

ethanol to obtain a 1000 ppm stock solution.

Phytochemical screening

Phytochemical screening of the Moringa oleifera

L. ethanolic extract was conducted using

standard qualitative methods. Phenols were

detected by the addition of FeCl₃solution,

indicated by a green or blackish-blue color

change. Flavonoids were identified by reacting

the thick extract with magnesium powder and 2%

HCl, resulting in an orange-red coloration if

positive. For alkaloid detection, the extract was

mixed with 2N HCl and distilled water, heated in

a water bath, filtered, and reacted with Mayer’s

reagent; the formation of a brownish-yellow

precipitate indicated a positive result [11].

Moreover, saponins were tested by adding the

extract to cold and hot water, shaking for 10

seconds, and adding a drop of 2N HCl; the

formation of stable foam persisting for 10

minutes indicated the presence of saponins.

Tannins were confirmed by adding 1–2 drops of

1% FeCl₃to the extract, with a green or blackish-

blue color change suggesting a positive result

[11]. Terpenoids were screened by mixing the

extract with chloroform, acetic anhydride, and

concentrated sulfuric acid; the appearance of a

red or purple coloration and the formation of a

brownish ring indicated terpenoid presence [12]-

[13].

Antioxidant

activity,

including

vitamin

C

comparison, was evaluated using the DPPH

method. A 3 mL sample was combined with 2 mL

DPPH solution and 2 mL ethanol in a 10 mL

volumetric flask, incubated in the dark at room

temperature for 30 minutes, and absorbance

was measured at 515 nm. The percentage of

inhibition was calculated using Equation (1), and

IC₅₀ values were derived from the linear

regression equation (Equation 2), indicating the

concentration required to inhibit 50% of DPPH

radicals [1].

(

blank abs−sample abs

% inhibition =

⁾× 100%

(1)

(2)

blank abs

(50−a)

IC₅₀

=

b

Molecular Docking of Potential Compounds

toward NADPH Oxidase

The preparation of ligand structures involved

converting the two-dimensional (2D) molecular

structures of five major compounds identified via

LC-MS, drawn using ChemDraw Ultra 22.0, into

three-dimensional (3D) models using Chem3D

22.0, saved in PDB format. Hydrogen atoms were

added using Discovery Studio 2021, and the

ligands were saved in PDF format. Subsequently,

ligand optimization and torsion bond settings

were performed with AutoDockTools, and the

ligands were saved in pdbqt format.

Metabolites Profilling by LC-MS

The solvent-free extract was dissolved using pro-

analysis methanol to a concentration of 20 ppm

with a sample ratio 1:5. Samples were taken at 1

μL and injected into the LC-MS instrument

system. The liquid chromatography is coupled

with a mass spectrometry of a quadrupole time-

of-flight (QTOF) system with a positive ionization

The macromolecular structure of NADPH oxidase

(PDB ID: 4Z3D) was retrieved from the Protein

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Chempublish Journal, 9(1) 2025, 74-100

Data Bank (https://www.rcsb.org). UCSF Chimera

was used to remove solvents, native ligands, and

nonstandard residues. After cleanup, the

macromolecule was saved in PDB format.

poses were saved in PDB format. Visualization

and analysis of docking results were performed

using Discovery Studio Visualizer 2021, allowing

detailed

observation

of

receptor-ligand

Optimization

was

performed

using

interactions.

AutoDockTools by adding Kollman charges and

hydrogen atoms, and the receptor was saved in

pdbqt format. Molecular docking was carried out

using PyRx software based on AutoDockTools.

Optimized ligand and receptor files were placed

in a single folder. A flexible docking protocol was

employed with grid box parameters set

according to validation results. Docking was

performed using the AutoDock Wizard feature in

PyRx, and results were presented as binding

affinity values and interaction data. Final docking

Result and Discussion

Phytochemical screening was conducted to

identify the presence of secondary metabolites in

Moringa oleifera L. leaf ethanolic extracts by

observing specific colorimetric changes following

the addition of chemical reagents [14]. The

results, summarized in Table 1, indicate the

successful identification of several bioactive

compound classes.

Table 1. Moringa leaf extract phytochemical screening test results

Results

Compound

Classes

Reagents

Discolorations

Maceration

Soxhletation

Phenols

Flavonoids

Alkaloids

FeCl₃

Mg + HCl 2%

Weagner

Dagendroft

FeCl₃1%

Green

Red

Chocolate

Orange

Green

Foam

Red ring

+

-

+

Tannins

Saponins

Terpenoids

H₂O (hot) + HCl 2 N

C₄H₆O₃+ H₂SO₄

+

Notes : (+) = contains the test compound ; (-) = does’nt contain the test compound

Phenolic compounds were positively identified in

both macerated and soxhletated extracts, as

formation,

suggesting

that

heat-assisted

extraction facilitates the release of these

glycosidic compounds. Conversely, maceration

failed to extract saponins, likely due to the milder,

cold conditions. Both extraction methods yielded

positive results for terpenoids, confirmed

through the Liebermann–Burchard reaction,

indicated by a red color and occasional brown

ring formation [18,19].

evidenced by

a

blackish-green color upon

reagent addition. Flavonoids were also present in

both extracts, indicated by a red color change.

The addition of concentrated HCl facilitates

hydrolysis of O-glycosylated flavonoids into their

aglycone forms, while magnesium and acid

reduce the compounds to produce red-colored

complexes such as flavones, flavanones, and

xanthones [15]. Alkaloid presence was confirmed

using Wagner and Dragendorff reagents. Both

tests yielded positive results for alkaloids in the

extracts by showing precipitates [16]. Moreover,

Tannin compounds were also detected in both

extracts, signaled by a blackish-green coloration

upon the addition of FeCl₃, indicating the

formation of tannin–Fe³⁺ complexes [17].

Metabolites Profilling by LC-MS

Based on the results of LC-MS analysis, the

maceration method obtained the separation

results of 101 compounds (Figure 1) that have the

potential as antioxidants and have been grouped

according to the secondary metabolite group

with a total percentage composition of flavonoids

(70.9872%),

phenols

(8.09702%),

alkaloids

(3.35195%), tannins (2.0379%), and terpenoids

(0.40774%).

Furthermore, saponins were detected only in the

soxhletation extract, evidenced by stable foam

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Chempublish Journal, 9(1) 2025, 74-100

Figure 1. LCMS Chromatogram Results of the Moringa oleifera L. macerate 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

Figure 2. LCMS Chromatogram Results of the Moringa oleifera L. 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 2 presents the LC-MS-based profiling of

secondary metabolites in Moringa oleifera L. leaf

extract included quercetin-3-O-glucoside (m/z

463.37),

kaempferol-3-O-(6″-malonylglucoside)

extracts,

including

phenolics,

flavonoids,

(m/z

534.42), and kaempferol 3-

alkaloids, tannins, terpenoids, and saponins. A

total of eight major flavonoid derivatives were

identified. Peaks 72, 80, 81, and 92 appeared

consistently in both maceration and soxhletation

extracts, with retention times ranging from 22.6

to 58.1 minutes, indicating their stability across

extraction methods. Peaks unique to the soxhlet

neohesperidoside (m/z 594.52), each confirmed

via characteristic fragmentation spectra. The

maceration extract, on the other hand, revealed

the presence of luteolin-7-glucoside (peak 65,

m/z 448.10), a high molecular weight glycoside

(peak 95, m/z 756.66), and ternatin C3 (peak 98,

m/z 1021.86), a bioactive anthocyanin (Figure 2).

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Chempublish Journal, 9(1) 2025, 74-100

Peaks 72/80 and 81/92 were tentatively assigned

natural antioxidants capable of mitigating

oxidative stress caused by free radicals.

as

kaempferol

respectively, supported by their identical m/z

values (594.52 and 680.56) and shared

kaempferol

3-O-robinobioside

and

3-(6G-malonylneohesperidoside),

Statistical analysis using a paired t-test revealed

a significant difference in antioxidant activity

between the two extraction methods, with a p-

value of 0.000 (p < 0.05), confirming that the

soxhlet method yields a more potent antioxidant

extract. This is further supported by LC-MS

fragmentation behavior. The detection of these

compounds in both extraction techniques

underscores their abundance and potential

pharmacological relevance. Previous studies

have also reported similar compounds in M.

oleifera, demonstrating significant antioxidant,

anti-inflammatory, and antidiabetic properties

[12,15,20]. The findings confirm the utility of LC-

MS in identifying bioactive flavonoid glycosides

and support M. oleifera as a rich source of

functional phytochemicals.

profiling,

content—compounds

which showed

higher flavonoid

known

for their

antioxidant properties—in the soxhlet extract

(75.61%) compared to the maceration extract

(70.99%). The high flavonoid content contributes

to the superior antioxidant capacity of the

soxhlet-derived extract, reinforcing its potential

application in pharmaceutical or nutraceutical

formulations.

Antioxidant Activity

The optimum wavelength of DPPH was 515 nm

(absorbance 0.635) and used to calculate %

inhibition and IC₅₀ via linear regression.

Antioxidant activity of Moringa leaf extract and

vitamin C was evaluated based on IC₅₀; lower IC₅₀

indicates higher antioxidant potential in reducing

DPPH radicals [20]. IC₅₀can be calculated by

drawing a linear regression curve between %

inhibition with the y-axis and the concentration

series as the x-axis.

Flavonoids

phytochemicals

abundant polyphenolic compounds found in

fruits and vegetables. They possess strong

antioxidant properties, which are expressed

are

a

and

prominent

represent

class

the

of

most

through

multiple

mechanisms,

including

scavenging reactive oxygen species (ROS) and

free radicals, chelating transition metals, and

inhibiting

lipoproteins (LDL). Flavonoids can donate

hydrogen atoms to lipid radicals, forming more

stable antioxidant derivatives less prone to

further oxidation. Their antioxidant effects are

mediated by direct ROS neutralization, metal ion

chelation (as seen in quercetin’s iron-binding

the

oxidation

of

low-density

The linear regression equations obtained for

Moringa oleifera leaf extracts from maceration

and soxhletation methods were y = 0.1916x +

43.851 and y = 0.1585x + 47.729, respectively. For

vitamin C, used as a positive control, the equation

was y = 10.788x – 3.1497 (Figure 3). These

equations were used to calculate the IC₅₀ values,

defined as the concentration required to inhibit

50% of DPPH radicals. The results demonstrated

that both extraction methods yielded extracts

with very strong antioxidant activity, comparable

to that of vitamin C.

properties),

inhibition

of

ROS-generating

enzymes, and upregulation of endogenous

antioxidant enzymes. These pathways may act

synergistically to enhance protective effects. The

soxhletation extraction method, which utilizes

continuous heating and solvent recycling, is

particularly effective in isolating thermally stable

flavonoids. It also facilitates the release of low

molecular weight bioactives from complex

polymer matrices, thereby optimizing the

extraction of flavonoid compounds from plant

materials like Moringa oleifera [24].

The IC₅₀ values obtained were 32.092 ppm for the

macerated extract, 14.328 ppm for the soxhlet

extract, and 4.927 ppm for vitamin C. Based on

the classification of antioxidant potency, where

IC₅₀ < 50 ppm indicates very strong activity, all

three samples fall into this category. These

findings suggest that Moringa oleifera leaf

extracts,

particularly

those

obtained

via

soxhletation, possess significant potential as

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Chempublish Journal, 9(1) 2025, 74-100

Table 2. Phenolic, flavonoids, alkaloids, tannins, terpenoids, and saponins derivatives identified in Moringa oleifera L. extract using LC-MS

Peak numbers

RT (min)

Composition

(%)

Measured (m/z)

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

Phenolic compounds

1

3

1.238

0.31609*

0.50197**

0.26828*

0.42605**

0.33379*

0.53008**

0.24758*

0.39318**

0.88305*

1.40236**

1.36068*

2.16088**

0.81151*

1.42569**

0.96673*

1.53525**

0.10150

116.0760

122.1230

148.1610

164.1600

168.1480

170.1200

174.1520

180.1590

416.6900

194.1860

336.2960

338.1002

354.0951

430.7170

C₄H₄O₄

117.0143, 116.0110

123.0401, 122.0368

149.0558, 148.0524

165.0507, 164.0473

169.0456, 168.0423

Fumaric acid

Benzoic acid

Cinnamic acid

p-coumaric acid

Vanillic acid

M, S

S

1.289

1.582

1.839

2.799

3.042

3.091

4.643

14.409

5.043

12.255

12.276

12.421

20.056

C₇H₆O₂

5

4

7

5

10

6

12

7

13

8

14

10

41

C₉H₈O₂

C₉H₈O₃

C₈H₈O₄

C₇H₈O₅

172.0258, 171.0249, Gallic acid

170.0215

176.0571, 175.0562, Shikimic acid

174.0528

181.0456, 180.0423, Caffeic acid

180.1590

418.3721, 418.3721, γ-tocopherol

417.3688, 416.3654

C₇H₁₀O₅

C₉H₈O₄

C₂₈H₄₈O₂

C₁₀H₁₀O₄

C₁₆H₁₆O₈

C₁₆H₁₈O₈

C₁₅H₁₈O₈

C₂₉H₅₀O₂

16

11

45

33

46

34

47

35

49

0.68199*

1.08305**

0.33420*

0.53074**

0.27924*

0.44346**

0.49636*

0.78827**

0.15834

195.0613, 194.0579

Ferulic acid

M, S

S

338.0912, 338.0888, 5-O-caffeoylshikimic acid

337.0879, 336.0845

340.1069, 340.1044, 3-p-coumaroylquinic acid

339. 1035, 338.1002

356.1018, 356.0993, Chlorogenic acid

355.0984, 354.0951

432.3878, 432.3878, α-tocopherol

431.3844, 430.3811

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Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

17.58

Composition

(%)

0.11287*

0.17925**

Measured (m/z)

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

59

47

425.4230

C₁₄H₁₈NO₁₉S₂

428.0442, 427.0517, 4-hydroxybenzyl

427.0493, 427.0408, glucosinolate

426.0484, 425.0450

M, S

61

50

21.387

1.00465*

1.59548**

432.3810

C₂₁H₂₀O₁₀

434.1099, 434.1124, Vitexin

433.1090, 432.1056

M, S

Flavonoids derivatives

9

3.275

9.104

9.732

8.013

9.104

10.322

9.112

10.523

9.353

9.365

9.376

0.732

1.20730

0.39765

1.6399

480.3780

268.2680

279.1107

254.2410

268.2680

286.2390

268.2680

298.2500

270.2810

270.2400

272.2560

C₉H₆O₄

179.0300, 178.0266, Escuetin

178.1430

270.0803, 269.0769, 3-hydoxy-4'-methoxyflavone

268.0736

274.0727, 274.0752, Naringenin

273.0718, 272.0685

S

14

17

19

21

22

C₁₆H₁₂O₄

C₁₅H₁₂NO₅

C₁₅H₁₀O₄

C₁₆H₁₂O₄

C₁₅H₁₀O₅

C₁₆H₁₂O₄

C₁₆H₁₀O₆

C₁₃H₁₈O₆

C₁₅H₁₀O₅

C₁₅H₁₂O₅

S

0.13799

0.25040

1.90336

0.21303

1.36443

256.0646, 255.0613,

254.0579

Daidzein

M

S

270.0803, 269.0769,

268.0736

288.0544, 288.0520,

287.0511, 286.0477

270.0803, 269.0769,

268.0736

3-hydroxy-4'-

methoxyflavone

Kaemferol

Formononetin

M

S

300.0520, 300.0544,

299.0511, 298.0477

272.1146, 271.1137,

270.1103

2'.5-dihydroxy-6-7-

methylenedioxyisoflavone

Benzyl-β-D-

23

15

24

0.74657*

1.18561**

1.30251

M, S

M

glucopyranoside

272.0571, 272.0595,

271.0562, 270.0528

272.0571, 272.0595,

271.0562, 270.0528

274.0727, 274.0752,

273.0718, 272.0685

Apigenin

25

26

0.27419

1.55322

Genistein

Narigenin

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Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

11.404

9.968

Composition

(%)

1.29152

Measured (m/z)

302.0790

279.2920

312.2770

284.2670

316.0583

286.2390

321.3290

298.2500

624.5480

302.1940

302.0790

302.2380

312.2770

316.0583

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

26

C₁₆H₁₄O₆

304.0833, 304.0857,

303.0824, 302.0790

281.1149, 281.1174,

280.1140, 279.1107

314.0676, 314.0701,

313.0667, 312.0634

286.0727, 286.0752,

285.0718, 284.0685

318.0650, 318.0625,

317.0617, 316.0583

288.0544, 288.0520,

287.0511, 286.0477

288.0544, 288.0520,

287.0511, 286.0477

323.1255, 323.1279,

322.1246, 321.1212

300.0520, 300.0544,

299.0511, 298.0477

334.0938, 334.0963,

333.0930, 332.0896

304.0130, 304.0105,

303.0096, 302.0063

304.0833, 304.0857,

303.0824, 302.0790

304.0494, 304.0469,

303.0460, 302.0427

314.0676, 314.0701,

313.0667, 312.0634

318.0650, 318.0625,

317.0617, 316.0583

5,2',3'-trihydroxy-7-

methoxyflavanone

Niazirin

S

27

18

28

0.10490*

0.16659**

1.84376

C₁₄H₁₇NO₅

C₁₇H₁₂O₈

C₁₆H₁₂O₅

C₁₆H₁₂O₇

C₁₅H₁₀O₆

C₁₅H₁₀O₅

C₁₆H₁₉NO₅

C₁₆H₁₀O₆

C₂₈H₃₂O₁₆

C₁₄H₆O₈

M, S

S

11.561

10.011

11.61

2'-hydrox-5-methoxy-6,7-

methylenedioxyisoflavone

Biochanin A

29

0.31347

2.23146

M

5,2'3'-trihyhydroxy-6,7-

methylenedioxyisoflavone

Luteolin

S

30

20

31

10.265

10.322

12.076

10.523

12.246

11.401

11.404

11.427

11.561

11.61

0.83243*

1.32197**

1.19853

M, S

M

Kaempferol

Niazirinin

31

32

0.62378

0.85917

1.46440

S

2'-5-dihydroxy-6,7-

methylenedioxyisoflavone

3,5,3'-trihydroxy-7,2'-

dimethoxyflavanone

Ellagic acid

M

S

34

25

35

0.72424*

1,15015**

0.81325

M, S

M

C₁₆H₁₄O₆

C₁₅H₁₀O₇

C₁₇H₁₂O₈

C₁₆H₁₂O₇

5,2',3'-trihydroxy-7-

metoxyflavonone

Quercetin

36

27

37

1.36722*

2.17126**

1.16099

M, S

M

2'-hydroxy-5-methoxy-6,7-

methylenedioxyisoflavone

5,2',3'-trihidroxy-6-7-

38

1.40512

M

methylendioxyflavonone

82

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

11.617

12.991

11.901

12.001

12.002

14.444

Composition

(%)

0.83070*

1.3922**

1.75841

Measured (m/z)

316.2650

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

39

30

39

C₁₅H₁₂O₇

318.0650, 318.0625,

317.0617, 316.0583

388.1044, 388.1069,

387.1035, 386.1002

308.0807, 308.0782,

307.0773, 306.0740

318.0650, 318.0625,

317.0617, 316.0583

318.0650, 318.0625,

317.0617, 316.0583

404.1593, 404.1568,

403.1560, 402.1526

5,6,7,4'-tetrahydroxy-8-

methoxyisoflavone

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

methylemdioxyisoflavone

Leucocyanidin

M, S

S

386.3560

C₂₀H₁₈O₈

C₁₅H₁₄O₇

C₁₆H₁₂O₇

C₁₅H₁₂O₇

C₁₈H₂₆O₁₀

40

41

42

0.43559

0.24987

0.66523

1.70645

306.2700

M

S

316.2650

Rhamnetin

316.2650

Isorhamnetin

benzyl-6-O-β-D-

402.3960

xylopyranosyl

-β-D-

glucopyranoside

3,5,3'-trihydroxy-7,2'-

dimethoxyflavonone

Glucoputranjivin

44

48

12.246

12.684

0.92211

0.06654

332.3080

361.3800

C₁₇H₁₆O₇

334.0938, 334.0963,

333.0930, 332.0896

362.0495, 363.0544,

363.0459, 362.0535,

361.0501

M

C₁₀H₁₉NO₉S₂

49

36

12.713

12.93

0.52454*

0.58500**

366.2960

376.3170

C₁₅H₁₀O₉

C₁₈H₁₆O₉

368.0113, 368.0088,

368.0003, 367.0079,

366.0046

378.0861, 378.0837,

377.0828, 376.0794

Kaempferol-3-O-sulfate

M, S

M

51

0.53432

(6R,6aS,12aR)-6,9,11,11a-

tetrahydroxy-2,3-

dimethoxyrotenone

51

52

21.429

12.985

2.22057

0.06521

432.3810

376.3690

C₂₁H₂₀O₁₀

434.1099, 434.1124,

433.1090, 432.1056

378.1425, 377.1353,

378.1450, 377.1416,

376.1383

Kaempferol-3-rhamnoside

S

C₁₇H₂₀N₄O₆

Riboflavin

M

53

38

12.963

0.89774*

1.42569**

377.3930

C₁₉H₂₃NO₇

83

379.1517, 379.1542,

378.1508, 377.1475

Pyrrolemarumine 4'-O-α-L- M, S

rhamnopyranoside

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

22.173

Composition

(%)

0.69167

Measured (m/z)

448.1006

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

53

54

56

C₂₁H₂₀O₁₁

450.1048, 450.1073,

449.1039, 448.1006

388.1044, 388.1069,

387.1035, 386.1002

450.1048, 450.1073,

449.1039, 448.1006

404.1593, 404.1568,

403.1560, 402.1526

Astragalin

S

12.991

1.10725

1.76712

1.07453

386.3560

C₂₀H₁₈O₈

C₂₁H₂₀O₁₁

C₁₈H₂₆O₁₀

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

methylemedioxyisoflavone

Kaempferol-3-O-glucoside

M

S

22.623

448.3800

14.444

402.3960

Benzyl-6-O-b-D-

xylopyranosyl-b-D-

glucopyranoside

M

2-

61

62

30.728

21.429

30.865

22.166

30,869

22.623

22.628

23.977

33.046

24.001

0.37250

1.91885

2.82097

530.4391

432.3810

534.4260

466.4080

534.4260

448.3800

448.1006

462.4070

550.8860

463.3715

C₂₅H₂₂O₁₃

266.0557, 266.0569,

265.5552, 265.0536

434.1099, 434.1124,

433.1090, 432.1056

536.1052, 536.1077,

535.1043, 534.1010

448.1255, 448.1280,

447.1247, 446.1213

536.1052, 536.1077,

535.1043, 534.1010

450.1048, 450.1073,

449.1039, 448.1006

450.1048, 450.1073,

449.1039, 448.1006

464.1205, 464.1229,

463.1196, 462.1162

570.4347, 569.4314,

568.4280

biochanin

A-7-O-β-D

S

glucoside-6"-O-malonate

Kaemferol-3-rhamnoside

C₂₁H₂₀O₁₀

C₂₄H₂₂O₁₄

C₂₂H₂₂O₁₀

C₂₄H₂₂O₁₄

C₂₁H₂₀O₁₁

C₂₂H₂₂O₁₁

C₄₆H₅₆O₂

C₂₁H₁₉O₁₂

M

S

Kaempferol-3-O-(6"-

malonylglucoside)

Calycosin-7-O-β-D-

glucoside

luteolin-7-O-(6''-

malonylglucoside)

63

52

63

1.42594*

1.43779**

1.78668

M, S

S

64

2.15389

kaemferol-3-O-glucoside

M

65

55

67

57

67

2.68230*

1.77956**

1.43035*

2.27152**

0.10184

Luteolin-7-glucoside

Hirsutrin

M, S

S

Lutein

68

58

2.38175*

3.36906**

465.0949, 465.0924,

464.0916, 463.0882

Quercetin-3-O-glucoside

M, S

84

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

24.02

Composition

(%)

0.61754*

0.98071**

2.90214^*

2.34802^**

Measured (m/z)

464.3790

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

69

59

70

78

C₂₁H₂₀O₁₂

466.1022, 466.0997,

465.0988, 464.0955

596.1652, 596.1652,

596.1627, 595.1618,

594.1585

266.0557, 266.0569,

265.5552, 265.0536

536.1052, 536.1077,

535.1043, 534.1010

536.1052, 536.1077,

535.1043, 534.1010

536.1416, 536.1441,

535.1407, 534.1373

Hyperoside

M, S

33.625

594.5220

C₂₇H₃₀O₁₅

Kaempferol 3-

neohesperidoside

2-

71

72

73

20.728

30.865

30.869

30.87

0.23456

2.16621

530.4391

534.4260

534.4700

C₂₅H₂₂O₁₃

Biochanin

A-7-O-β-D-

M

glucoside-6"-O-malonate

Kaempferol-3-O-(6"-

malonyglucoside)

Luteolin-7-O-(6”-

malonyglucoside)

6,9-dihydroxy-2,310-

trimethoxy-6a,12a-

C₂₄H₂₂O₁₄

C₂₅H₂₆O₁₃

M

74

64

0.67304*

1.06884**

M, S

didehydrorotenone

glucoside

9-O-

75

66

76

31.822

36.831

0.99273*

0.74982**

1.78863

550.0959

624.1690

C₂₄H₂₂O₁₅

C₂₈H₃₂O₁₆

552.1001, 552.1026,

551.0992, 550.0959

626.1733, 626.1757,

625.1724, 624.1690

Quercetin-3-O-(6"-

malonyglucoside)

5,7,4'-trihydroxy-6-

methoxysoflavone 4'-O(6"-

glucosylglucoside)

Naringin

M, S

S

77

69

77

33.563

36.833

2.23489*

1.89575**

1.59821

580.5390

624.5480

C₂₇H₃₂O₁₄

C₂₈H₃₂O₁₆

582.1859, 582.1835,

581.1826, 580.1792

626.1733, 626.1757,

625.1724, 624.1690

M, S

S

5,7,4'-trihydroxy-6-

methoxysoflavone

7-O-

glucoside-4'-O-glucoside

79

71

79

33.043

36.876

2,04108*

2.41469**

2.39915

594.5220

626.5200

C₂₇H₃₀O₁₅

C₂₇H₃₀O₁₇

596.1652, 596.1627,

595.1618, 594.1585

629.1559, 628.1550,

628.1550, 628.1525

kaempferol 3-O-rutinoside

M, S

S

Myricetin-3-O-rutinoside

85

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

Composition

(%)

Measured (m/z)

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

80

72

34.045

2.63787^*

2.53572^**

594.5220

C₂₇H₃₀O₁₅

596.1652, 596.1652,

596.1627, 595.1618,

594.1585

Kaempferol

3-O-

M, S

robinobioside

80

40.231

1.16501

624.5480

C₂₈H₃₂O₁₆

657.1872, 656.1863,

656.1863, 656.1838

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

dimethoxhisoflavone 7-O-

S

(6"-glucosylglucoside)

81

73

81

35.51

1.95911*

2.28451**

2.52670

610.5210

680.5680

C₂₇H₃₀O₁₆

C₃₀H₃₂O₁₈

612.1601, 612.1576,

611.1567, 610.1534

683.1665, 682.1656,

682.1656, 682.1631,

681.1622, 680.1589

612.1601, 612.1601,

612.1576, 611.1567,

610.1534

699.1614, 698.1605,

698.1605, 698.1580,

697.1571, 696.1538

759.2189, 758.2155,

758.2180, 757.2146,

756.2113

Quercetin

neohesperidoside

Kaempferol

malonyineohesperidoside)

3-O-

M, S

S

46.224

3-(6G-

82

74

35.517

46.236

46.565

36.831

36.833

36.375

1.00453*

1.01657**

610.5210

696.5670

756.6630

624.5480

624.1690

626.5200

C₂₇H₃₀O₁₆

C₃₀H₃₂O₁₉

C₃₃H₄₀O₂₀

C₂₈H₃₂O₁₆

C₂₇H₃₀O₁₇

Rutin

M, S

S

82

83

85

86

1.45227

1.77221

1.64686

1.52695

Quercetin

malonylneohesperidoside)

3-(6"-

kaempferol 3-gucosyl-(1→

2)-[glucosyl-(1→3)-

rhamnoside]

5,7,4'-trihydroxy-6-

methoxyisoflavone 4'-O-(6"-

glucosy;glucoside)

5,7,4'-trihydroxy-6-

methoxyisoflavone 7-O-4'-

O-glucoside

S

626.1733, 626.1757,

625.1724, 624.1690

M

626.1733, 626.1757,

625.1724, 624.1690

M

87

78

2.20249*

1.84431**

629.1559, 628.1550,

628.1525, 627.1517,

626.1483

Myricetin 3-

neohesperidoside/

Myricetin 3-O-rutinoside

M, S

86

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

Composition

(%)

Measured (m/z)

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

89

40.231

1.25417

654.5740

C₂₉H₃₄O₁₇

657.1872, 656.1863,

656.1838, 655.1830,

654.1796

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

dimethoxyisoflavone 7-O-

(6"-glucosylglucoside)

M

90

91

46.014

46.015

46.224

46.236

46.565

46.568

1.57274

2.33244

772.6620

680.5680

696.5670

756.6630

C₃₃H₄₀O₂₁

C₃₀H₃₂O₁₈

C₃₀H₃₂O₁₉

C₃₃H₄₀O₂₀

775.2138, 774.2105,

774.2129, 773.2096,

772.2062

775.2138, 774.2105,

774.2129, 773.2096,

772.2062

683.1665, 682.1656,

682.1656, 682.1631,

681.1622

699.1614, 698.1605,

698.1580, 697.1571,

696.1538

759.2189, 758.2155,

758.2180, 757.2146,

756.2113

759.2189, 758.218,

758.213, 757.2146,

756.2113.

Quercetin 3-glucosyl-(1→2)-

rhamnoside-7-glucoside

M

Myricetin 3-(2G-

rhamnosylrutinoside)

M

92

81

2.63219^*

Kaempferol 3-(6G-

malonylneohesperidoside)

M, S

M

2.52670^**

93

94

95

1.43505

2.15710

2.76758

Quercetiin 3-(6"-

malonylneoheseridoside)

Kaempferol 3-glucosyl-

(1→2)-[glucosyl-(1→3)-

rhamnoside]

3-(((2S,3S,4R,5S,6R)-4,5-

dihydroxy-3-

M

(((2S,3S,4R,5R,6R)-3,4,5-

trihydroxy-6-

methyltetrahydro-2H-pyran-

2- yl)oxy)-6-

((((2R,3R,4S,5R,6R)-

3,4,5-trihydroxy-6-

methyltetrahydro-2H-pyran-

2- yl)oxy)methyl)tetrahydro-

2H- pyran-2-yl)oxy)-2-(3,4-

dihydroxyphenyl)-5,7-

dihydroxy-4H-chromen-4-

one

87

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

Composition

(%)

Measured (m/z)

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

98

58.151

2.33205

1021.8595

C₄₅H₄₉O₂₇

1022.2498,

1024.2532,

1023.2498,

1023.2523,

1022.2489,

1021.2456

Ternatin C3

M

Alkaloids derivatives

4

9

1.428

2.649

0.12657

0.25482

123.1110

164,1570

C₆H₅N0₂

C₅H₁₂O₅

124.0354, 123.0320

166.0727, 165.0718,

164.0685

Niacin

Rhamnose

M

11

13

2.811

9.083

0.12266

0.57727

169.1800

268.0372

C₈H₁₁NO₃

C₁₅H₈O₅

170.0772, 169.0739

270.0414, 270.0439,

269.0405, 268.0372

195.061, 194.058

Pyrodixine

Coumestrol

M

S

17

12

23

5.053

0.30224*

0.47998**

0.26358

194.1860

297.3070

C₁₀H₁₀O₄

4-hydroxymellein

M, S

S

11.007

C₁₄H₁₉NO₆

299.1279, 299.1255,

298.1246, 297.1212

2-(4-(((2S,3R,4R,5R,6S)-

3,4,5-trihydroxy-6-

methyltetrahydro-2H-

pyran-

2yl)oxy)phenyl)acetimedic

acid

24

11.013

9.989

0.12197

298.2500

282.2510

297.3070

C₁₆H₁₀O₆

C₁₆H₁₀O₅

C₁₄H₁₉NO₆

300.0520, 300.0544,

299.0511, 298.0477

284.0571, 284.0595,

283.0562, 282.0528

299.1279, 299.1255,

298.1246, 297.1212

8-methoxycoumestrol

S

28

19

33

0.12753*

0.20253**

0.16597

9-O-methylcoumestrol

M, S

M

11.007

2-(4-(((2S,3R,4R,5R,6S)-3,4,5-

trihydroxy-6-

methyltetrahydro-2H-pyran-

2-yl)oxy)phenyl)acetimidic

acid

88

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

12.93

Composition

(%)

0.84855

Measured (m/z)

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

(6R,2aS,12aR)-6,9,11,11a-

tetrahydroxy-2-3-

dimethoxyrotenone

Niazinirin

37

376.3170

C₁₈H₂₃O₉

378.0861, 378.0837,

377.0828, 376.0794

S

43

50

12.076

12.916

13.071

0.39278

0.80533

321.3290

375.4070

391.4060

C₁₆H₁₉NO₆

323.1255, 323.1279,

322.1246, 321.1212

378.0649, 376.0652,

377.0700, 377.0616

M

C₁₁H₂₁NO₉S₂

Glucocochlearin

Glucoconringiin

M

55

40

0.07882*

0.12517**

C₁₁H₂₁NO₁₀S₂394.0598, 392.0601,

393.0649, 393.0565,

M, S

392.0640, 391.0607

-

57

44

15.509

0.12791*

0.20314**

408.4165

C₁₄H₁₈NO₉S₂

410.0496, 411.0420,

409.0422, 410.0471,

410.0386, 409.0462,

408.0428

Benzyl glucosinolate

Niaziminin B

M, S

58

75

15.63

0.22133

0.27314

411.4690

613.6020

C₁₉H₂₅NO₂S

413.1394, 413.1419,

413.1310, 412.1385,

411.1352

M

S

35.556

C₂₂H₃₁NO₁₅S₂614.1129, 616.1127,

615.1202, 615.1178,

615.1093, 614.1169,

613.1135

4-(4'-O-acetyl-α-L-

rhamnopyranosyloxyl)

benzylglukosinolate

76

68

33.06

0.62599*

0.99413**

571.5650

C₂₀H₂₉NO₁₄S₂572.1023, 574.1021,

573.1097, 573.1072,

573.0987, 572.1063,

571.1029

4-(α-L-

rhamnopyranosyloxy)-

benzylglucosinolate

M, S

Tannis derivatives

70

60

83

24.710

0.69410*

1.10229**

1.34380

480.3780

610.5240

C₂₁H₂₀O₁₃

C₃₀H₂₆O₁₄

482.0971, 482.0946,

481.0937, 480.0904

612.1390, 612.1390,

612.1365, 611.1356,

610.1323

Isomyricitrin

M, S

M

35.553

Prodelphinidin B1

89

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-100

Peak numbers

RT (min)

Composition

(%)

Measured (m/z)

Formulas

MS/MS Fragments

Proposed Metabolites

Extracts

Terpenoids derivatives

45

17.047

19.319

31.102

0.52445

426.6850

426.3862

536.8880

C₂₉H₄₆O₂

C₃₀H₅₀O

C₄₀H₅₆

428.3565, 428.3565,

427.3531, 426.3498

428.3929, 428.3929,

427.3895, 426.3862

538.4449, 537.4416,

536.4382

Stigmast-4-en-3,6-dione

β-amyrin

S

60

48

65

0.40774*

0.64753**

0.21192

M, S

S

β-carotene

Saponins derivatives

43

15.048

0.36183

0.20177

400.6910

480.3780

C₂₈H₄₈O

402.3772, 402.3772,

401.3739, 400.3705

416.3929, 416.3929,

415.3895, 414.3862

Campesterol

S

46

17.163

C₂₁H₂₀O₁₃

β-sitosterol

Note: RT, retention time; M, maceration; S, soxhletation ; *, maceration; **, soxhletation

Linear curve of moringa leaf extract from maceration

Linear curve of moringa leaf extract from soxhletation

Vitamin C linearity curve

80

60

40

30

20

10

0

80

60

y = 10,788x - 3,1497

R² = 0,8669

40

y = 0,1585x + 47,729

R² = 0,943

40

20

0

y = 0,1916x + 43,851

R² = 0,9945

20

0

1 ppm

5 ppm

10 ppm

0

20

40

Sample concentration (ppm)

60

80

100

120

0

20

40

Sample concentration (ppm)

60

80

100

120

Sample concentration (ppm)

Figure 3. The curve of the relationship between active ingredient concentration and % inhibition. (A) Antioxidant activity of Moringa oleifera L. Macerate. (B) The

antioxidant activity of the extract obtained from the soxhletation of Moringa oleifera L was examined. (C) antioxidant activity of vitamin C

90

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-99

The results of this study are in accordance with

several other studies which states that the

that a similar inhibition mechanism might be

followed

by

the

identified

3-(((2S,3S,4R,5S,6R)-4,5-

compounds.

soxhlet

method

is

slightly

better

than

Compound

1

is

maceration. Including research to evaluate the

effect of maceration and soxhletation extraction

techniques on the antioxidant activity and total

phenolic content of Bouea macrophylla Griff

Plant [25]. Compare the maceration and

soxhletation

extraction

methods

on

the

antioxidant activity and total phenolic content of

jackfruit leaves [26]. Determine the effect of

maceration and soxhletation extraction methods

on the levels of flavonoids in the leaves of the

anting-anting [27].

Evaluation of Molecular Docking

Table 3. Binding affinity of NADPH Oxidase

enzyme (2CDU) with the control ligand of the

identified active compound from the maceration

method.

Molecular docking was performed on five

compounds of maceration method and five

compounds of soxhlet method identified using

LC-MS to identify potential binding modes of the

compounds that could justify their inhibitory

activity. Table 3 and Table 4 shows the binding

affinity of the control ligand, the detected

bioactive compound against NADPH Oxidase

enzyme. The control docking procedure was

performed using co-crystallized control ligands

to validate the docking parameters. In this study,

the complex with more negative values (i.e.,

stronger binding affinity) was considered as the

best docked complex. The re-docked NADPH

Oxidase was found to bind to 2CDU in a manner

identical to its crystallographic configuration. The

mean square deviation (RMSD) value of the re-

docked NADPH Oxidase was found to be 0 Å,

indicating that the selected docking parameters

were able to reproduce the crystallized

conformation. The docking parameters were

considered acceptable if the RMSD value of the

re-docked ligand, with respect to the crystallized

one, was less than 1.9 Å [28]. Control docking

showed that the control ligand exhibited a

binding affinity of -9.8 kcal.mol to the enzyme.

The superimposed 3D docking visualization

(Figure 4 and Figure 5) depicts the predicted

binding sites of the five identified compounds

and the control ligand in the enzymatic protein.

According to the figure, all the compounds are

predicted to bind to the A domain of the enzyme,

where the catalytic site is located. The same site

is also occupied by the control ligand indicating

Compounds

Binding Affinity,

kcal/mol

Control Ligand

-9.8

-9.0

-8.8

-9.6

-10.1

-6.9

1

2

3

4

5

Table 4. Binding affinity of NADPH Oxidase

enzyme (2CDU) with the control ligand of the

identified active compound from the soxhlet

method.

Compounds

Binding Affinity,

kcal/mol

Control Ligand

-9.8

-8.8

-9.6

-9.2

-9.6

-10.1

1

2

3

4

5

Table 5 shows the binding interactions of

compounds 1, 2, 3, 4 and 5. Along with H-bonding

interactions, other interactions like pi-sigma, pi-

alkyl, pi-donor, t-shaped pi-pi and stracked

amide-pi are also involved in the docked

complexes. In the ligand complex against NADPH

oxidase, the control ligand showed interactions

involving hydrogen bonds with residues ASP179,

SER157, GLY244, TYR188, GLY156, ILE160, and

TYR159, as well as charge–charge and pi

91

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-99

interactions with LYS187, ILE243, and VAL214.

Compound 1 interacted through hydrogen bonds

with CSX42, ILE160, SER328, SER326, and TYR188,

followed by pi-donor and pi–pi T-shaped

ALA300, and LEU299. Compound

4

forms

hydrogen bonds with LYS213, ILE243, TYR159,

CYS242, TYR188, SER326, GLY158, and GLY244,

and pi-alkyl and pi-sigma interactions with ILE297

and PRO298. Compound 5 forms a number of

hydrogen bonds with ALA300, CSX42, ASP282,

LYS187, ARG246, and THR118, as well as pi-

interactions. Compound

2

formed strong

hydrogen bonds with ASP179, GLY158, and

TYR188, and showed pi-donor, pi–pi T-shaped, pi-

alkyl, and amide–pi stacked interactions with

residues PRO298 and ILE243. Compound 3 forms

hydrogen bonds with GLY161, ILE160, CSX42,

TYR188, PRO298, and CYS242, and interacts

through pi-alkyl and pi-sigma bonds with TYR159,

donor,

pi-cation,

and

amide-pi

stacked

interactions with TYR188, ILE243, and LYS213,

which overall indicate a strong binding affinity to

the active site of NADPH oxidase.

Figure 4. (A) Superimposed 3D diagram of control ligand, the docked compounds. (B) 2D binding

interactions of the docked compounds from the maceration method with 2CDU.

Table 5. Data for the molecular docking of compounds 1, 2, 3, 4 and 5 from maceration method in 2CDU.

Interacting

Compound Structure

Amino Acid

Residues

LYS213

Bonding Types

Bonding Distance (Å)

Pi-Alkyl

4.15; 4.76

3.04; 3.20

3.24; 3.29

NADPH Oksidase

Ligand Control

ASP179

Hydrogen Bond

92

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Chempublish Journal, 9(1) 2025, 74-99

Interacting

Amino Acid

Residues

SER157

Compound Structure

Bonding Types

Bonding Distance (Å)

Hydrogen Bond

Charge-Charge

Hydrogen Bond

Pi-Sigma

3.23

3.42

2.55

5.47

3.65

3.38

3.06

3.39

4.97

5.22

2.72

3.13

2.20

2.28

3.02

2.49

3.27

4.65

5.64

3.77

3.19

4.52

2.47

2.83

2.56

2.86

3.50

4.03

5.14

4.76

5.38

3.07

3.31

5.34

2.88

3.05

3.21

4.23

2.67

4.19

2.92

5.33

3.31

3.07

2.62

GLY244

TYR188

LYS187

GLY156

ILE160

TYR159

ILE243

Pi-Aklyl

Pi-Alkyl

VAL214

Hydrogen Bond

Pi-Donor

Pi-Pi T-shaped

Hydrogen Bond

Pi-Alkyl

Hydrogen Bond

Pi-Donor

3-(((2S,3S,4R,5S,6R)-4,5-

dihydroxy-3- (((2S,3S,4R,5R,6R)-3,4,5-

trihydroxy-6- methyltetrahydro-2H-

pyran-2- yl)oxy)-6-((((2R,3R,4S,5R,6R)-

3,4,5-trihydroxy-6-methyltetrahydro-

2H-pyran-2-

yl)oxy)methyl)tetrahydro-2H- pyran-

2-yl)oxy)-2-(3,4- dihydroxyphenyl)-

5,7- dihydroxy-4H-chromen-4-one

Compound 1

CSX42

ILE160

TYR188

SER328

SER326

PRO298

ASP179

GLY158

luteolin-7-glucoside

TYR188

Hydrogen Bond

Compound 2

Pi-Donor

Hydrogen Bond

Pi-Pi T-shaped

Pi-Alkyl

Amide Pi-Stacked

Hydrogen Bond

Pi-Alkyl

Hydrogen Bond

Pi-Alkyl

Hydrogen Bond

Pi-Alkyl

PRO298

ILE243

GLY161

ILE160

CSX42

TYR188

Kaempferol 3-O-robinobioside

Compound 3

PRO298

TYR159

CYS242

ALA300

LEU299

LYS213

ILE243

Hydrogen Bond

Pi-Alkyl

Pi-Sigma

Hydrogen Bond

Kaempferol 3-(6G-

malonylneohesperidoside)

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Chempublish Journal, 9(1) 2025, 74-99

Interacting

Amino Acid

Residues

Compound Structure

Bonding Types

Bonding Distance (Å)

Compound 4

Hydrogen Bond

Pi-Alkyl

Hydrogen Bond

Pi-Donor

Pi-Alkyl

Hydrogen Bond

2.80

4.80

2.16

3.10

3.83

4.37

2.21

3.51

3.56

2.73

5.28

3.95

3.02

3.09

2.37

3.10

2.76

3.37

4.84

2.89

4.07

3.72

3.60

4.07

4.18

5.10

TYR159

CYS242

TYR188

SER326

GLY158

Hydrogen Bond

GLY244

ILE297

PRO298

ALA300

CSX42

ASP282

LYS187

ARG246

THR118

PRO298

Hydrogen Bond

Pi-Alkyl

Pi-Sigma

Ternatin C3

Compound 5

Hydrogen Bond

Pi-Alkyl

Pi-Donor

TYR188

ILE243

LYS213

Hydrogen Bond

Pi-Sigma

Amide-Pi

Stracked

Amide-Pi

Stracked

Pi-Cation

Pi-Donor

Pi-Alkyl

Table 6 shows the binding interactions of

compounds 1, 2, 3, 4 and 5. Along with H-bonding

interactions, other interactions like pi-sigma, pi-

alkyl, pi-donor, t-shaped pi-pi and stracked

amide-pi are also involved in the docked

complexes. Compounds 1 to 5 showed strong

interactions with the enzyme target through

various bond types, such as hydrogen bonds, Pi-

Pi, Pi-Alkyl, Pi-Sigma, and Amide-Pi Stacked. The

most frequently interacting amino acid residues

included TYR188, CSX42, TYR159, ILE160, and

PRO298. In general, the hydrogen bond type was

dominant, with the bond distance ranging from

1.98 to 5.95 Å. Compounds 1 and 4 formed many

hydrogen and Pi-Pi bonds, while compounds 3

and 5 showed additional Pi-Donor and Amide-Pi

Stacked interactions that strengthened the

affinity to the target. These results indicated that

all compounds had strong interaction potential

through the contribution of various active

residues and diverse bond types.

94

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Chempublish Journal, 9(1) 2025, 74-99

Figure 5. (A) Superimposed 3D diagram of control ligand, the docked compounds. (B) 2D binding

interactions of the docked compounds from the soxhlet method with 2CDU.

Table 6. Data for the molecular docking of compounds 1, 2, 3, 4 and 5 from soxhlet method in 2CDU.

Compound Structure

Interacting Amino

Acid Residues

Bonding Distance

(Å)

Bonding Type

Hydrogen Bond

Pi-Pi Stacked

Hydrogen Bond

Pi-Pi T-shaped

Pi-Sigma

2.34

2.90

3.31

3.17

4.22

3.36

2.15

5.62

3.61

5.23

3.07

3.31

5.34

2.88

3.05

3.21

CSX42

TYR159

quercetin-3-O-glucoside

ILE160

TYR188

Compound 1

LEU299

PRO298

GLY161

Pi-Alkyl

Hydrogen Bond

Pi-Alkyl

Hydrogen Bond

Pi-Alkyl

ILE160

CSX42

kaempferol 3-

neohesperidoside

Compound 2

TYR188

PRO298

4.23

2.67

Hydrogen Bond

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Compound Structure

Interacting Amino

Acid Residues

TYR159

Bonding Distance

Bonding Type

(Å)

Pi-Alkyl

Hydrogen Bond

Pi-Alkyl

4.19

2.92

5.33

3.31

2.98

3.07

3.32

4.02

CYS242

ALA300

LEU299

GLY158

Pi-Sigma

Hydrogen Bond

Pi-Donor

Hydrogen Bond

Pi-Pi T-shaped

Amide-Pi Stacked

Pi-Sigma

Hydrogen Bond

Pi-Alkyl

Hydrogen Bond

Pi-Alkyl

TYR188

kaempferol-3-O-(6"-

malonylglucoside)

Compound 3

CSX42

3.13

1.98

5.95

5.03

3.88

3.07

3.31

5.34

2.88

3.05

3.21

4.23

2.67

4.19

2.92

5.33

3.31

3.07

2.62

2.80

4.80

2.16

3.10

3.83

4.37

2.21

3.51

3.56

2.73

5.28

3.95

PHE245

ILE243

ILE160

GLY161

ILE160

CSX42

kaempferol 3-O-

robinobioside

Compound 4

TYR188

PRO298

TYR159

CYS242

ALA300

LEU299

LYS213

ILE243

Hydrogen Bond

Pi-Alkyl

Hydrogen Bond

Pi-Alkyl

Pi-Sigma

Hydrogen Bond

Pi-Alkyl

Hydrogen Bond

Pi-Donor

kaempferol 3-(6G-

malonylneohesperidoside)

Compound 5

TYR159

CYS242

TYR188

Pi-Alkyl

Hydrogen Bond

SER326

GLY158

Hydrogen Bond

GLY244

ILE297

PRO298

Hydrogen Bond

Pi-Alkyl

Pi-Sigma

The similarity of interactions and bond types are

the main reasons for the closeness of binding

affinity and free energy between each ligand and

control (Apocynin A), indicating the good

antioxidant ability of the tested metabolite

compounds. According to the previous studies,

other ligands with different chemical structures

have shown antioxidant activity due to the

similarity of interactions and active sites with the

current study [29,30]. For example, similar to the

interacting residues in Figure 4 and Figure 5,

Lountos et al. [30] showed that residues LYS213,

96

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-99

(3,4-

dihydroxyphenyl)-5,7-

dihydroxy-4H-

luteolin-7-

ILE243, TYR159, CYS242, TYR188, ILE160, and

GLY158 of NADPH receptor interact with all

ligand analogs which is consistent with our

findings.

chromen-4-one

(-9.0 kcal/mol),

glucoside and quercetin-3-O-glucoside (-8.8

kcal/mol), and ternatin C3 (-6.9 kcal/mol),

compared to the control (Apocynin A). These

results prove the significant antioxidant capacity

of the studied plants, which can be considered

for further analysis in their use as possible

antioxidant agents in the pharmaceutical

industry.

Conclusions

The Soxhlet method showed superiority over the

maceration method, where there was

a

significant difference with a sig value (2 tailed):

0.000 (<0.05). LC-MS analysis showed that the

macerated Moringa leaf extract had 101

Acknowledgement

secondary

metabolite

compounds,

which

showed that the total composition of the most

effective flavonoid compound derivatives as

antioxidants in the macerated extract was

70.9872% with five main compounds, namely 3-

(((2S,3S,4R,5S,6R)-4,5-dihydroxy-3-

(((2S,3S,4R,5R,6R)-3,4,5-trihydroxy-6-

methyltetrahydro-2H-pyran-2-yl)oxy)-6-

((((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-

The authors express their sincere gratitude to

STIKES Karya Putra Bangsa Tulungagung for the

internal research grant provided to support this

study. Appreciation is also extended to

Universitas Jambi, particularly to Indra Lasmana

Tarigan, M.Sc., for conducting the in silico study.

The authors further acknowledge Gambia

University

for

the

valuable

research

methyltetrahydro-2H-pyran-2-

collaboration.

yl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-2-

(3,4-

dihydroxyphenyl)-5,7-

dihydroxy-4H-

Author Contributions

chromen-4-one; luteolin-7-glucose; kaempferol

3-O-robinobioside;

kaempferol

There

3-(6G-

83

A.L.K performed maceration

and

soxhlet

malonilneohesperidoside).

are

extraction, A.M, S, and M.S.S were involved in LC-

MS based secondary metabolite profile analysis,

A.M. performed antioxidant assay, processed

experimental data, performed analysis, drafted

the manuscript, and designed figures. A.M. and

T.W performed and evaluated In Silico Target

Prediction. A.L.K helped in interpreting the

results and working on the manuscript. All

authors discussed the results and commented

on the manuscript.

secondary metabolite compounds in the soxhlet

extract which shows that the total composition of

flavonoid compound derivatives is 75.60657%

with the 5 highest compounds: quercetin-3-O-

glucose;

kaempferol

3-

Neohesperidoside;

kaempferol-3-O-(6"-malonylglucoside);

kaempferol 3-O-robinobioside; and kaempferol

3-(6G-malonylneohesperidoside), which the total

composition of the soxhletation method is

higher. Supported by the antioxidant compound

content test with DPPH, the macerated extract

has an IC.50 value of 32.092 ppm, while the

soxhlet extract has an IC.50 value of 14.328 ppm.

In the same context, molecular docking of the

main compounds identified on NADPH oxidase

showed a higher binding affinity for Kaempferol

Conflict of Interest

The authors declare no conflict of interest.

References

3-(6G-malonylneohesperidoside)

(-10.1

3-O-

3-

[1]

Nofanda, Dwi IWI. Skrining Fitokimia Dan

Pengaruh Metode Ekstraksi Terhadap Uji

Aktivitas Antioksidan Ekstrak Etanol Daun Kelor

(Moringa oleifera L.) dengan Metode DPPH. Diss.

Universitas dr. SOEBANDI. 2022.

kcal/mol),

followed

and

by

Kaempferol

kaempferol

robinobioside

neohesperidoside (-9.6 kcal/mol), kaempferol-3-

O-(6"-malonylglucoside) (-9.2 kcal/mol), 3-

(((2S,3S,4R,5S,6R)-4,5-dihydroxy-3-

(((2S,3S,4R,5R,6R)-3,4,5-trihydroxy-6-

methyltetrahydro-2H-pyran-2-yl)oxy)-6-

((((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-

methyltetrahydro-2H-pyran-2-

[2]

[3]

Fakriah, E. Kurniasih, Adriana, and Rusydi.

Sosialisasi Bahaya Radikal Bebas dan Fungsi

Antioksidan Alami Bagi Kesehatan. J. Vokasi.

2019;3(1): 1-7. doi: 10.30811/vokasi.v3i1.960.

Simanjuntak EJ, Zulham Z. Superoksida

Dismutase (SOD) dan radikal bebas. Jurnal

yl)oxy)methyl)tetrahydro-2H- pyran-2-yl)oxy)-2-

97

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-99

Keperawatan dan Fisioterapi (Jkf). 2020;2(2):124-

129. doi: 10.35451/jkf.v2i2.342.

fitokimia ekstrak etanol kulit batang kelor

(Moringa oleifera). Indonesia Medicus Veterinus.

2015;4(1):71-9.

[4]

[5]

Pham-Huy LA, He H, Pham-Huy C. Free radicals,

antioxidants in disease and health. International

journal of biomedical science: IJBS. 2008;4(2):89.

Giovanni Martemucci, Ciro Costagliola, Michele

Mariano , Luca D’andrea, Pasquale Napolitano

and Angela Gabriella D’Alessandro. Free Radical

Properties, Source and Targets, Antioxidant

Consumption and Health. Oxygen. 2022;2:48-78.

doi: https://doi.org/10.3390/oxygen2020006.

Hadidi M, Orellana-Palacios JC, Aghababaei F,

Gonzalez-Serrano DJ, Moreno A, Lorenzo JM.

Plant by-product antioxidants: Control of

protein-lipid oxidation in meat and meat

products. Lwt. 2022;169:114003.

Chukwuebuka E. Moringa oleifera “the mother’s

best friend”. International Journal of Nutrition

and Food Sciences. 2015;4(6):624-30. doi:

10.11648/j.ijnfs.20150406.14.

Kamal SE, Aris M. Aktivitas antioksidan ekstrak

etanol 70% daun kelor (Moringa oleifera Lam.)

Terhadap DPPH. Jurnal Pro-Life. 2021;8(2):168-

77.

[16] Marliana SD, Suryanti V, Suyono S. Skrining

fitokimia dan analisis kromatografi lapis tipis

komponen kimia buah labu siam (Sechium edule

Jacq. Swartz.) dalam ekstrak etanol. Biofarmasi.

2005;3(1):26-31.

[17] Listiana L, Wahlanto P, Ramadhani SS, Ismail R.

Penetapan

Mangkokan (Nothopanax scutellarium Merr)

Perasan dan Rebusan Dengan

Spektrofotometer UV-Vis. Pharmacy Genius.

Kadar

Tanin

Dalam

Daun

[6]

2022;1(1):62-73.

doi:

10.56359/pharmgen.v1i01.152.

[18] Fransiska AN, Masyrofah D, Marlian H, Sakina IV,

Tyasna PS. Identifikasi senyawa terpenoid dan

steroid pada beberapa tanaman menggunakan

[7]

[8]

[9]

pelarut

n-heksan.

Jurnal

Health

Sains.

2021;2(6):733-41. DOI: 10.36387/Jiis.V4i1.285.

[19] Masadi YI, Lestari T, Dewi IK. Identifikasi

Kualitatif Senyawa Terpenoid Ekstrak N-

Heksana Sediaan Losion Daun Jeruk Purut

(Citrus hystrix DC). Jurnal Kebidanan Dan

Kesehatan Tradisional. 2018;3(1).

[20] R Kamal SE, Aris M. Aktivitas antioksidan ekstrak

etanol 70% daun kelor (Moringa oleifera Lam.)

Terhadap DPPH. Jurnal Pro-Life. 2021;8(2):168-

77.

Sreelatha S, Padma PR. Antioxidant activity and

total phenolic content of Moringa oleifera leaves

in two stages of maturity. Plant foods for human

nutrition. 2009;64:303-11. doi: 10.1007/s11130-

009-0141-0.

[10] Faizal IA, Alifah AA. Perbandingan Metode

Maserasi Dan Soxhletasi Ekstrak Daun Sirih

Merah (Piper crocatum Ruiz & Pav) Terhadap

Efektivitas Bakteri Staphylococcus epidermidis.

Lumbung Farmasi: Jurnal Ilmu Kefarmasian.

2023;4(1):64-72. doi: 10.31764/lf.v4i1.10728.

[11] Meigaria KM, Mudianta IW, Martiningsih NW.

Skrining fitokimia dan uji aktivitas antioksidan

ekstrak aseton daun kelor (Moringa oleifera).

[21] Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid

antioxidants: chemistry, metabolism and

structure-activity relationships. The Journal of

nutritional biochemistry. 2002;13(10):572-84.

[22] Chu YH, Chang CL, Hsu HF. Flavonoid content of

several vegetables and their antioxidant

activity. Journal of the Science of Food and

Agriculture. 2000;80(5):561-6.

[23] Kumar S, Pandey AK. Chemistry and biological

activities of flavonoids: an overview. The

scientific world journal. 2013;2013(1):162750.

[24] Rosita JM, Taufiqurrahman I, Edyson E.

Perbedaan Total Flavonoid Antara Metode

Maserasi Dengan Sokletasi Pada Ekstrak Daun

Binjai (Mangifera caesia)(Studi pendahuluan

terhadap proses pembuatan sediaan obat

penyembuhan luka). Dentin. 2019;1(1).

Wahana

Matematika,

Matematika

dan

Sains:

Pembelajarannya.

Jurnal

Sains,

2016;10(2):1-1.

doi:

https://doi.org/10.23887/wms.v10i2.12659.

[12] A. Saputra, F. Arfi, and M. Yulian. Literature

Review: Analisis Fitokimia dan Manfaat Ekstrak

Daun Kelor (Moringa oleifera). Amina. 2020;2(3):

114–119.

[13] Azalia D, Rachmawati I, Zahira S, Andriyani F,

Sanini TM, Supriyatin S, Aulya NR. Uji Kualitatif

Senyawa Aktif Flavonoid Dan Terpenoid Pada

Beberapa Jenis Tumbuhan Fabaceae Dan

Apocynaceae Di Kawasan Tngpp Bodogol.

Bioma: Jurnal Biologi Makassar. 2023;8(1):32-43.

[14] Muthmainnah B. Skrining fitokimia senyawa

metabolit sekunder dari ekstrak etanol buah

delima (Punica granatum L.) dengan metode uji

warna. Media Farmasi. 2017;13 (2): 23–28.

[25] Rudiana T, Nurbayti S, Ashari TH, Zhorif SA,

Suryani N. Comparison of Maceration and

Soxhletation Methods on the Antioxidant

Activity of the Bouea macrophylla Griff Plant.

Jurnal Kimia Valensi. 2023;9(2):244-52.

[26] Larasati RA, Utami N, Lindawati NY. The Effect of

Maceration Method and Soxhletation Method

on Total Phenolic Content and Antioxidant

Activity of Kirinyuh (Chromolaena odorata (L.)

RM King & H. Rob) LEAF. Medical Sains: Jurnal

Ilmiah Kefarmasian. 2023;8(3):1207-14.

[15] Ikalinus R, Widyastuti SK, Setiasih NL. Skrining

98

A.Muadifah et al.

Chempublish Journal, 9(1) 2025, 74-99

[27] Karisma DI, Indriasari C. The Effect of

Ngibad K, Yuliantari SD. LC-MS Based

Metabolite Profiling Leaves Extract of Pluchea

indica With Antioxidant Activity. Chempublish

Maceration

and

Soxhletation

Extraction

Methods on The Flavonoid Content of Anting-

anting Leaves Extracts (Acalypha indica L.) Using

Uv-Vis Spectrophotometry. Strada Journal of

Pharmacy. 2023;5(2):73-9.

Journal.

2024;8(2):75-89.

doi:

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

[30] Lountos GT, Jiang R, Wellborn WB, Thaler TL,

Bommarius AS, Orville AM. The crystal structure

[28] Biehn SE, Lindert S. Accurate protein structure

prediction with hydroxyl radical protein

footprinting data. Nature communications.

2021;12(1):341.

of

NAD(P)H

oxidase

from

Lactobacillus

sanfranciscensis: insights into the conversion of

O₂into two water molecules by the

flavoenzyme. Biochemistry. 2006;45(32):9648-

59.

doi:https://doi.org/10.1038/s41467-020-20549-

7

[29] Muadifah A, Tilarso DP, Sowe MS, Tarigan IL,

99