Materials Today: Proceedings 72 (2023) 2796–2802

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The total phenolic and flavonoid contents of Aloe vera and Morinda
citrifolia extracts as antibacterial material against Pseudomonas
aeruginosa
Ahmad Royani a,b,⇑, Muhammad Hanafi c, Heddy Julistiono d, Azwar Manaf a,⇑
a
Postgraduate Program of Materials Science Study, Department of Physics, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, Depok 16424, Indonesia
b
Research Center for Metallurgy, National Research and Innovation Agency, Puspiptek Area, Tangerang Selatan 15314, Indonesia
c
Research Center for Raw Material of Medicine and Traditional Medicine, National Research and Innovation Agency, Puspiptek Area, Tangerang Selatan 15314, Indonesia
d
Research Center for Applied Microbiology, National Research and Innovation Agency, Jl. Raya Bogor Km. 46 Cibinong Science Center, Cibinong 16911, Indonesia

a r t i c l e i n f o a b s t r a c t

Article history: Pseudomonas aeruginosa is a pioneer bacterium initiating the biofilm formation process so that it is cor-
Available online 8 July 2022 rosive to some metals in the marine environment. One of the efforts to prevent and control corrosion due
to environmentally friendly biofilms is using green biocides. The extraction of Aloe vera and Morinda citri-
Keywords: folia with methanol were carried out to obtain antibacterial material. The extract of those plants with
Aloe vera multilevel maceration method. The study aimed to identify the total phenolic and flavonoid contents
Antibacterial in A. vera and M. citrifolia crude extracts and compare the two antibacterial extract activities. The total
Flavonoid
phenolic and flavonoid contents were analyzed using the Folin-Ciocalteu and the aluminum chloride col-
Morinda citrifolia
Phenolic
orimetric methods. The antibacterial activity against P. aeruginosa was conducted using the microdilution
P. aeruginosa method to determine the minimum inhibitory concentration (MIC). The structure of the bioactive com-
ponents of both A. vera and M. citrifolia extracts were identified using a liquid chromatography-mass
spectrometer (LC/MS-MS). The MIC of A. vera and M. citrifolia for P. aeruginosa bacteria is about
6144 lg/mL, while the MIC of antibiotic Enrofloxacin is 16 lg/mL. The mean of total phenolic and flavo-
noid contents for the methanolic extracts of A. vera and M. citrifolia were 1.39% and 4.03% (phenolic), and
0.26% and 0.28% (flavonoids), respectively. The chemical structure of A. vera consists of 200 -O-
Feruloylaloesin, Kaempferol-3-O-rutinoside, and Natsudaidain. At the same time, the chemical structure
of M. citrifolia consists of 7-Hydroxy-Methoxycoumarin and Cirsiumaldehyde. A. vera and M. citrifolia
crude extracts had lower inhibition than antibacterial control (Enrofloxacin) for P. aeruginosa. The lower
bacteria inhibition was due to the low active materials (phenolic and flavonoid contents). So, the purifi-
cation methods of crude extracts to obtain active materials with high antibacterial activity were needed.
Ó 2023 Elsevier Ltd. All rights reserved.
Selection and peer-review under responsibility of 2nd International Conference on Sustainable Materials,
Manufacturing and Renewable Technologies 2022 (i-SMART 2022). This is an open access article under the
CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction
P. aeruginosa is a highly corrosive bacterium to some metals in
the marine environment [1]. These bacteria include rod-shaped
gram-negative bacteria that are motile because they move quickly
from one place to another [2]. These bacteria have been recognized
as pioneers of initiation in biofilm formation [2]. P. aeruginosa has
been studied and reported to increase the corrosion rate of stain-
⇑ Corresponding authors.
E-mail addresses: ahmad.royani@brin.go.id (A. Royani), azwar@sci.ui.ac.id (A.
Manaf).
less steel, mild steel, and many other alloys in marine environ-
ments [3].
Biocides are one critical point in controlling and preventing cor-
rosion caused by biofilm bacteria such as P. aeruginosa [4]. Biocides
for bacterial control are the best technical approaches because they
are practical and do not damage metals [5]. However, most bio-
cides cause many environmental problems due to their toxicity.
Therefore, an alternative biocide that is environmentally friendly
is needed. Several active ingredients from plant extracts have been
reported for biocorrosion control.
On the other hand, the diversity of Indonesian herbal plants
makes them potential candidates for new antibacterial materials.

https://doi.org/10.1016/j.matpr.2022.06.466
2214-7853/Ó 2023 Elsevier Ltd. All rights reserved.
Selection and peer-review under responsibility of 2nd International Conference on Sustainable Materials, Manufacturing and Renewable Technologies 2022 (i-SMART 2022).
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A. Royani, M. Hanafi, H. Julistiono et al.
One of the herbal plants is A. vera and M. citrifolia. A. vera is one of
the medicinal plants efficacious in curing various diseases [6]. This
plant also has good properties for health because it contains
antibacterial [7]. A. vera has also been shown to kill and inhibit
bacterial growth [8]. As an antibacterial plant, A. vera contains
active substances such as saponins, tannins, and flavonoids [9].
Saponins are alkaloids that can damage bacteria’s acids (DNA and
RNA). Tannin, as an antibacterial, it is working by inactivating
the adhesins of bacteria. Flavonoids can cause lysis and inhibit cell
wall formation. The above mechanism causes A. vera to kill or inhi-
bit the formation of bacteria. A. vera is a anti bactericidal agent for
P. aeruginosa and P. acemannan. It can prevent bacterial cells from
adhering to monolayer cultures. A. vera has been shown to inhibit
sulfate-reducing bacteria and Iron oxidizing bacteria [8].
M. citrifolia is a widely used plant in traditional medicine for
various diseases. M. citrifolia contains various bioactive compounds
such as capric acid, caproic acid, caprylic acid, and other bioactive
compounds. It is suspected that these compounds are active as
antimicrobial compounds [10] and P. aeruginosa bacteria [11]. This
activity is partly due to its antioxidant activity in M. citrifolia with
its flavonoid and phenolic compounds [12].
These various active chemical compounds make it exciting to
test the antibacterial properties of A. vera and M. citrifolia extracts
on biofilm-forming bacteria. In this study, the maceration method
for extracting those plants was carried out using methanol. The
work aimed to obtain a new material as an antibacterial P. aerug-
inosa. This study aimed to identify the total phenolic and flavonoid
content in A. vera and M. citrifolia extracts and compare the
antibacterial activity of P. aeruginosa from the two extracts. The
identification of bioactive compounds from A. vera and M. citrifolia
extracts using a liquid chromatography-mass spectrometer (LCMS-
MS) is also discussed.
2. Materials and methods
The materials used in this work were A. vera and M. citrifolia,
which were obtained from the Spice and Medicinal Research Cen-
ter (BALITTRO). Other supporting materials are methanol, bacteria,
artificial seawater (Marine Art SF-1), Nutrient broth media (Brain
Heart Infusion), Enrofloxacin, MTT solution (5 mg/mL), and Iso-
propanol solution (HCl 0.04 M).
Samples of A. vera and M. citrifolia were cleaned and washed
with running water, then cut into small pieces and mashed using
a blender. The A. vera and M. citrifolia extraction processes used
the maceration methods. Two hundred grams of wet powder from
each A. vera and M. citrifolia were put into a glass vessel and then
extracted using methanol as a solvent. Maceration was carried
out for 24 h with three repetitions for three days. After that, it
was evaporated with a rotary evaporator to obtain methanol fil-
trate, and the yield value was calculated.
The content of total phenolic compounds in A. vera and M. citri-
folia crude extracts was determined by the Follin-Ciocalteu method
[13] using gallic acids as a standard. Meanwhile, the total phenolic
contents in those extracts were expressed as equivalent gallic acid
(mg. g1 extract) based on the regression equation of the calibra-
tion curve. The content of total flavonoid compounds was calcu-
lated based on the aluminum chloride colorimetric assay method
[14]. The content of total flavonoid compounds was expressed as
quercetin equivalent (mg. g1 extract) based on the regression
equation from the calibration curve.
P. aeruginosa B3 was obtained from INACC (Indonesian Culture
Collection). Slant culture bacteria were transferred to freshwater
BHI broth media and then to marine BHI broth media. MIC was
determined using the MTT assay [15]. Aliquots of 100 ll of marine
BHI containing a series of extracts or enrofloxacin concentrations
were added to each well of a 96-well plate (Iwaki). The concentra-
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Materials Today: Proceedings 72 (2023) 2796–2802
tions of extracts ranged from 0; 512 lg/ml to 6144 lg/mL, while
antibiotic ranged from 0, 8 to 32 lg/mL. Bacterial cell suspension
(2 ll) of 24 h culture was added to the appropriate wells. Micro-
plates were incubated at room temperature for 24 h. After that,
10 ll of the MTT solution (5 mg/mL) was added to the wells, fol-
lowed by 1-hour incubation in dark conditions. To each tube of
wells, 100 ll of propan-2-ol (containing 0.04 M HCl) was added.
The absorbance of the cell suspension was measured at 595 nm
using a microplate reader (Bio-Rad xMark). The MIC was defined
as the absorbance value of the NB medium. In this work, all mea-
surements of bacterial activity were repeated in triplicate.
Bioactive compounds from A. vera extracts and M. citrifolia were
analyzed using the LC/MS-MS instrument (Agilent Technologies
7890). Measurement of bioactive compounds using LC/MS-MS
with a flow rate of 0.3 mL/min for 17 min, as shown in Table 1.
3. Results and discussions
Recently, the developments of green biocide materials empha-
sized the search for environmentally friendly antibacterial materi-
als. Phenolic and flavonoid compounds have received attention for
their high antibacterial activity. Crude extracts from plants, spices,
and other types of plants with rich phenolic content are increas-
ingly in demand in the manufacture of antibacterial agents. In this
study, the total phenolic and flavonoid contents and antibacterial
activity were determined and compared for the A. vera and M. citri-
folia crude extracts. The extract yields obtained from 200 g of wet
or equivalent to 10 g of dry powder using methanol as solvent are
listed in Table 2.
From Table 2, the yield of extracts with methanol solvents for A.
vera and M. citrifolia was three times, namely 14 % and 15 %,
respectively. The yield of the extract varies depending on the part
of the plant and the type of solvent used.
For example, 2,600 g of A. vera peel extract with ethanol solvent
could only produce 139.8 g of crude extract (about 5.37 %) [16]. The
highest extract yields were obtained from the flower parts of the
Salvia Amplexicaulis plant, respectively 14.14 % (w/w) with ethanol
solvent and 12 % with methanol solvent [17]. The average yield of
12.5 % was obtained from M. citrifolia extracts using ethanol as a
solvent with a ratio of powder to solvent is 1:3 and 1:4 for
30 min [18].
3.1. Total phenolic and flavonoid contents
Phenolic group compounds with bioactive properties can be
obtained from extracts of various plant materials. Each plant and
its parts have different phenolic content. The total phenol and fla-
vonoid contents of A. vera and M. citrifolia crude extracts from the
maceration process with methanol are presented in Table 3 and
Table 4. The total phenol content was expressed as gallic acid
equivalent (mg GAE/g dry extract). A. vera methanol extract con-
tains an average total phenolic of 1.39 %, and the methanol extract
of M. citrifolia contains an average total phenolic of 4.03 %. The total
phenolic content in A. vera extract is much less than in the M. citri-
folia extract. This result is in line with research on A. vera and M.
citrifolia, which states that the phenolic content in M. citrifolia is
higher than in A. vera [19].
The total phenolic content depends on the part of the plant and
the type of solvent used. Comparative investigation of the phenol
content in different types of the part plant using various solvents
concluded that the highest concentration of phenolic compounds
was obtained in the plant extracts with the high polarity of solvent
[12]. The optimal solvent for extracting polyphenols from A. vera
peel is ethyl acetate with a polyphenol content of 7.82 %, followed
by 0.65 % ethanol, 0.64 % methanol, and 0.48 % distilled water [20].
A. Royani, M. Hanafi, H. Julistiono et al. Materials Today: Proceedings 72 (2023) 2796–2802

Table 1
Analysis conditions for bioactive content and structure with LC/MS-MS.

Time (min) Flow rate (mL.min1) Composition A (%) Composition B (%) Curve
0.00 0.300 95.0 5.0 Initial
1.00 0.300 95.0 5.0 6
11.00 0.300 0.0 100.0 6
14.00 0.300 0.0 100.0 1
17.00 0.300 95.0 5.0 1

Table 2
The yield of A. vera and M. citrifolia extracts.
Stage of extracts % Yield extract of A. vera % Yield extract of M. citrifolia
1st 6 7
2 nd 5 5
3 th 3 3
Total 14 15
The most significant concentration of phenolic contents was
obtained in part leaves in aqueous and methanolic extracts. Other
authors have analyzed the quantitative and qualitative phyto-
chemical characteristics of several plant parts. The content results
of the active compounds in leaves, stems, and flowers were
obtained with different concentrations. The content of phenolic
and flavonoid compounds in M. citrifolia extracts that can function
as natural antibacterials with the highest phenol content obtained
from ripe M. citrifolia [12].
Meanwhile, the total content of flavonoids for A. vera and M.
citrifolia extracts were 0.26 % and 0.28 %, respectively. A. vera
methanol extract contains fewer flavonoids than M. citrifolia
extract. The concentration of flavonoids in each part of the plant
varies greatly, as was found for Salvia Amplexicaulis [17]. Concen-
trations of flavonoids in extracts of A. vera and M. citrifolia were
also obtained in other studies. The flavonoid content was found
in A. vera extract in the study of P. aeruginosa and S. pyogenes bac-
teria activities [9]. The results of phytochemical testing also
showed the presence of flavonoids as secondary metabolites in
M. citrifolia extract using ethanol as a solvent [21].
3.2. Antibacterial activity
The antimicrobial activity from crude plant extract and its
potency was assessed by broth dilution methods. The minimum
inhibitory concentration (MIC) is the lowest concentration of
antimicrobial agent that kills or/and inhibits the growth of P.
aeruginosa bacteria. The minimum inhibitory concentration of
crude extract of A. vera, M. citrifolia, and antibiotic (Enrofloxacin)
was carried out by assessing the clarity or turbidity in the micro-
plate wells. The concentration of the crude extract and the antibi-
otic that looked rather clearly indicated that the concentration had
activity to inhibit P. aeruginosa bacteria. In contrast, the concentra-
tion of the extract that looked cloudy (blue) indicated that it did
not provide inhibitory activity against the P. aeruginosa bacteria.
The dilution method with a color change indicator has also been
carried out to detect cytotoxicity in volatile substances [22] and
determine Aloe vera gel’s antibacterial effect against oral patho-
gens [23]. The MIC results of the three tests can be seen in Fig. 1.
The results showed that A. vera extracts and M. citrifolia have
antibacterial to P. Aruginosa. However, those extracts have activity
inhibition but not significantly compared to antibiotic control
(Enrofloxacin). Based on data experiments in Figs. 2 to 4, it was
found that extracts of A. vera and M. citrifolia were less effective
in inhibiting the growth of P. aeruginosa bacteria. The measure-
ment of the inhibition area showed that the methanol extract of
A. vera and M. citrifolia gave a minimum inhibitory concentration
(MIC) at 6144 lg/mL of concentration to the bacterium P. aerugi-
nosa inhibition. While for antibiotic control (Enrofloxacin), the
minimum inhibitory concentration was obtained at 16 lg/mL.
The MIC test results show that the higher the concentration of
A. vera extract against P. aeruginosa bacteria, the more the number
of test bacteria inhibited its growth. It is indicated by the decrease
in the growth of P. Aruginosa bacteria at each increase in A. vera
concentration, as shown in Fig. 3. The results of this study follow
the other studies. The higher the concentration of antibacterial
substances given to the bacteria in the test, the higher the antibac-
terial activity [24].
A. vera crude extracts have the antibacterial activity for P. aerug-
inosa at 6144 lg/mL of concentration. However, the inhibitory
activity of A. vera was lower than M. citrifolia extract (Figs. 3 and
4). These are shown in the average bacterial growth in A. vera
extracts, which is higher and still looks blue than M. citrifolia at
6144 lg/mL of concentration. These results are comparable to
the lower total phenolic and flavonoid content in A. vera extract,
as shown in Tables 3 and 4. Multi-resistant P. aeruginosa is known
for its ability to withstand several types of antibiotics. Therefore, P.
aeruginosa is a biofilm-forming bacterium harmful to all metals.
The investigation of ethanolic extract of A. vera skin has low activ-
ity as an antibacterial against P. aeruginosa bacteria with a mini-
mum inhibitory concentration (MIC) of 8.5% with an inhibition
zone of 6 mm [16]. Another study stated that A. vera flesh extract
had no inhibitory activity at all concentrations, carried out four
times for P. aeruginosa and S. Pyogenes bacteria [9]. The absence
of antibacterial activity may be influenced by the type and part
of A. vera, the method used, and the type of bacteria. Different
types of A. vera affected the presence or absence of A. vera extracts
for bacteria inhibition. This study uses the whole extract, namely A.
vera extract, without separating each leaf part.

Table 3
The results of total phenol content for A. vera and M. citrifolia Extracts.

Sample Test Vol. (lL) Absorbance Total Phenol Content (%) Total Phenol Content (%)
A. vera 1 250 0.1125 1.62 1.39 ± 0.24
2 250 0.1111 1.56
1 500 0.1285 1.12
2 500 0.1359 1.26
M. citrifolia 1 250 0.1725 3.94 4.03 ± 0.07
2 250 0.1771 4.12
1 500 0.2778 4.01
2 500 0.2786 4.03

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Table 4
The results of total flavonoid content for A. vera and M. citrifolia Extracts.

Sample Test Vol. (lL) Absorbance Total flavonoid content (%) Total flavonoid content (%)
A. vera 1 250 0.0807 0.27 0.26 ± 0.03
2 250 0.0807 0.22
1 500 0.0812 0.27
2 500 0.0812 0.27
M. citrifolia 1 250 0.0809 0.38 0.28 ± 0.07
2 250 0.0806 0.22
1 500 0.0812 0.27
2 500 0.0811 0.25

Fig. 1. The result of P. aeruginosa activity with (a) A. vera extract and Enrofloxacin and (b) M. citrifolia extracts.

Fig. 3. The result of the determination of anti-P. aeruginosa with A. vera crude
Fig. 2. The result of the determination of anti-P. aeruginosa with antibiotic
extracts.
(Enrofloxacin).

Meanwhile, M. citrifolia has a more significant activity than A. pathogenic bacteria found that S. aureus was more susceptible, fol-
vera because of its antibacterial and antiviral properties [11]. The lowed by P. aeruginosa and Bacillus [11]. The study results of M.
disc diffusion method of M. citrifolia leaf extract analysis against six citrifolia leaf ethanol extract had moderate inhibition against

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S. aureus at a concentration of 75%, namely 6.86 mm [21]. Another

study on the antibacterial properties of M. citrifolia ethanol extract
against E. Faecalis using the well diffusion (disc paper) method
gave the minimum bactericidal concentration (MBC) and mini-
mum inhibitory concentration of 25 mg/mL and 12.5 mg/mL,
respectively [25].

3.3. Component and structure of active materials

The chromatogram of A. vera and M. citrifolia extracts with LC/

MS-MS are presented in Fig. 5. The highest intensity of A. vera
extract was found at a retention time of 10.93 with a counts value
of 3,110,679. While the highest intensity of M. citrifolia extract was
Fig. 4. The result of the determination of anti-P. aeruginosa with M. Citrifolia crude found at 0.5 and 11.11 of retention time with counts values of
extracts. 602,006 and 475,307, respectively, as shown in Fig. 5.

Fig. 5. Chromatogram results of A. vera and M. citrifolia extracts.

Table 5
Component name of A. vera and M. citrifolia extracts.

Extract plants Component name Identification status Observed m/z Neutral mass (Da) Observed RT (min) Detector counts
A. vera 200 -O-Feruloylaloesin Identified 571.1820 570.17373 3.63 512,899
Kaempferol-3-O-rutinoside Identified 595.1666 594.15847 2.91 741,514
Natsudaidain Identified 419.1334 418.12638 3.78 228,069
M. citrifolia 7-Hydroxy-methoxycoumarin Identified 193.0495 192.04226 3.31 116,362
Cirsiumaldehyde Identified 235.0600 234.05282 2.36 80,387

Fig. 6. Bioactive structures of A. vera crude extracts.

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A. Royani, M. Hanafi, H. Julistiono et al.
Fig. 7. Bioactive structures of M. citrifolia crude extracts.
The difference in the growth inhibitory activity of P. aeruginosa
could be caused by differences in the content and concentration of
the active compound group in the extract. The results of identifying
the active components of A. vera and M. citrifolia with LC/MS-MS are
presented in Table 5. The chemical structure of the active compo-
nents of A. vera consists of 200 -O-Feruloylaloesin, Kaempferol-3-O-
rutinoside, and Natsudaidain. Meanwhile, the active components
in the M. citrifolia consist of 7-Hydroxy-Methoxycoumarin and
Cirsiumaldehyde. The structures of the active components in A. vera
and M. citrifolia extracts are presented in Fig. 6 and Fig. 7.
4. Conclusions
A. vera and M. citrifolia crude extracts have the antibacterial
activity to inhibit the growth of P. aeruginosa bacteria. In this
work, the minimum inhibitory concentration (MIC) of A. vera
and M. citrifolia crude extracts is about 6144 lg/mL against P.
aeruginosa. The lower bacteria inhibition of those crude extracts
than antibiotics (Enrofloxacin, 16 lg/mL) was due to those
extracts’ low phenolic and flavonoid actives compounds. The mean
of total phenolic and flavonoid contents for the methanolic crude
extracts of A. vera and M. citrifolia were 1.39% and 4.03% (pheno-
lic), and 0.26% and 0.28% (flavonoids), respectively. The chemical
structure of A. vera crude extracts consists of 200 -O-
Feruloylaloesin, Kaempferol-3-O-rutinoside, and Natsudaidain. At
the same time, the M. citrifolia crude extracts consist of 7-
Hydroxy-Methoxycoumarin and Cirsiumaldehyde. Based on the
data generated in this study, the active ingredients of crude
extracts from A. vera and M. citrifolia have low potency for the
antibacterial of P. aeruginosa inhibition. So, the purification meth-
ods of crude extracts to obtain active materials with high antibac-
terial activity were needed in future research.
CRediT authorship contribution statement
Ahmad Royani: Conceptualization, Methodology, Investigation,
Data curation, Writing – original draft, Writing – review & editing.
Muhammad Hanafi: Conceptualization, Methodology, Writing –
review & editing. Heddy Julistiono: Methodology, Investigation,
Writing – review & editing. Azwar Manaf: Methodology, Concep-
tualization, Writing – review & editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
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Materials Today: Proceedings 72 (2023) 2796–2802
Acknowledgements
This work was supported by the National Research and Innova-
tion Agency through the scholarship program, managed by the
Directorate of Talent Management - BRIN. The authors also thank
the Ministry of Education, Culture, Research, and Technology for
supporting research through PDD grants with contract number:
NKB-967/UN2.RST/HKP.05.00/2022.
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