© 2024 Journal of Pharmacy & Pharmacognosy Research, 12 (1), 50-72, 2024

ISSN 0719-4250
https://jppres.com

DOI: https://doi.org/10.56499/jppres23.1714_12.1.50

Original Article

In vitro antibacterial activity and phytochemical profiling of

Indonesian Anacardium occidentale L. leaf extract and fractions
[Actividad antibacteriana in vitro y perfil fitoquímico del extracto de hoja y fracciones de Anacardium occidentale L. de
Indonesia]
Khoirun Nisa1*, Auliya Uswatun Hasanah2, Ema Damayanti1, Andri Frediansyah1, Muslih Anwar1, Annisa Khumaira2,
Nosa Septiana Anindita2
1Research Centre for Food Technology and Processing, The National Research and Innovation Agency, Jl. Jogja-Wonosari Km 31, Playen, Gunungkidul, Indonesia.
2Universitas Aisyiyah Yogyakarta, Faculty of Science and Technology, Jl. Siliwangi 63, Nogotirto, Gamping, Sleman, Yogyakarta, Indonesia.

*E-mail: [email protected]

Abstract
Context: Uncontrolled practice of antibiotics leads to bacterial resistance. Therefore, investigating the possible utilization of several medicinal plant extracts
is necessary. Traditionally, numerous medicinal plants have been widely utilized to treat some human diseases including Anacardium occidentale.
Aims: To investigate the antibacterial activity of the ethanolic extract and fractions from A. occidentale as well as characterize the phytochemical profile of A.
occidentale leaves associated with antibacterial capacity.
Methods: The antibacterial activity was determined using minimum inhibitory concentration and time kill assay, which confirmed by streak agar plate.
Phytochemical profiling of A. occidentale leaves extract and fractions were evaluated by their total phenolic contents, total flavonoid contents, terpenoid
contents, and metabolite analysis by UHPLC-HR-ESI-MS.
Results: Among the extract and all fractions, the ethyl acetate fraction had high killing efficiency against Staphylococcus aureus, Bacillus subtillis, Escherichia
coli, and Pseudomonas aeruginosa, which was confirmed by its MIC value of 0.5, 0.5, 0.5, and 0.25 mg/mL, respectively. Corresponding to its antibacterial
capacity, the ethyl acetate fraction showed the highest of phenolic (508.89 mg GAE/g dw) and flavonoid (184 mg QE/g dw) contents. Based on HRMS
investigation, the extract and fractions contain amino acids, phenolic acids, alkaloids, flavonoids, fatty acid amides, fatty acids, and organic acids as classes
of molecules. Moreover, avicularin/quercetin-3-O-arabinofuranoside, myricitrin, and gallic acid were identified as major compounds from the active fraction.
Conclusions: The ethyl acetate fraction showed the most remarkable antibacterial activity. There was a significant correlation between the antibacterial
activity of active fraction and its phenolic and flavonoid contents.
Keywords: Anacardium occidentale; antibacterial; antibiotics; phytochemical.

Resumen
Contexto: La práctica incontrolada de antibióticos conduce a la resistencia bacteriana. Por lo tanto, es necesario investigar la posible utilización de varios
extractos de plantas medicinales. Tradicionalmente, numerosas plantas medicinales se han utilizado ampliamente para tratar algunas enfermedades
humanas, entre ellas Anacardium occidentale.
Objetivos: Investigar la actividad antibacteriana del extracto etanólico y las fracciones de A. occidentale, así como caracterizar el perfil fitoquímico de las hojas
de A. occidentale asociado a la capacidad antibacteriana.
Métodos: La actividad antibacteriana se determinó utilizando la concentración inhibitoria mínima y el ensayo de tiempo de muerte, que se confirmó
mediante una placa de agar. El perfil fitoquímico del extracto y las fracciones de las hojas de A. occidentale se evaluó mediante el contenido total de fenoles,
flavonoides y terpenoides, y el análisis de metabolitos por UHPLC-HR-ESI-MS.
Resultados: Entre el extracto y todas las fracciones, la fracción de acetato de etilo tuvo una alta eficacia letal contra Staphylococcus aureus, Bacillus subtillis,
Escherichia coli y Pseudomonas aeruginosa, lo que se confirmó por su valor MIC de 0,5, 0,5, 0,5 y 0,25 mg/mL, respectivamente. En correspondencia con su
capacidad antibacteriana, la fracción de acetato de etilo mostró los mayores contenidos fenólicos (508,89 mg GAE/g extracto seco) y flavonoides (184 mg
QE/g extracto seco). Según la investigación HRMS, el extracto y las fracciones contienen aminoácidos, ácidos fenólicos, alcaloides, flavonoides, amidas de
ácidos grasos, ácidos grasos y ácidos orgánicos como clases de moléculas. Además, se identificaron avicularina/quercetina-3-O-arabinofuranosido,
miricitrina y ácido gálico como compuestos principales de la fracción activa.
Conclusiones: La fracción de acetato de etilo mostró la actividad antibacteriana más notable. Se observó una correlación significativa entre la actividad
antibacteriana de la fracción activa y su contenido en fenoles y flavonoides.

Palabras Clave: Anacardium occidentale; antibacteriano; antibióticos; fitoquímica.

ARTICLE INFO AUTHOR INFO

Received: June 12, 2023. ORCID:
Accepted: October 1, 2023. 0000-0002-4657-067X (KN) 0000-0001-5415-1726 (MA)
Available Online: November 13, 2023. 0000-0002-4017-0499 (ED) 0000-0002-2350-8996 (AK)
0000-0003-4923-2948 (AF)
Nisa et al. Antibacterial activity of Anacardium occidentale fractions

INTRODUCTION cally analysed the killing curve of bacteria for 24 h

using microdilution method in order to determine the
Infectious diseases are a big problem faced by optimum concentration and time of extract or frac-
countries. These diseases originated from pathogenic tions to kill the bacteria. An agar streak plate assess-
microorganisms such as parasites, viruses, fungi, and ment was visually conducted to confirm the time-kill
bacteria, which are one of the main causes of death assay. Moreover, the phytochemical composition of
worldwide (Dirga et al., 2021). Uncontrolled practice the extract and fractions were investigated using LC-
of antibiotics leads to bacterial resistance due to no HRMS. This investigation provides a complete me-
inhibition in the growth of bacteria by systematic tabolite profile of A. occidentale leaves, which most
practicality of antibiotics with less or minimal inhibi- likely has antibacterial property. To date, there re-
tory doses. Therefore, it requires serious handling to mains a lack of information about the phytochemical
reduce the impact of antibiotic resistance by investi- profiling and the comprehensive antibacterial activity
gating the possible utilization of several medicinal of the fractions from A. occidentale.
plant extracts. Numerous medicinal plants have tradi-
tionally been widely used to treat some human dis- MATERIAL AND METHODS
eases (Khan et al., 2013).
Anacardium occidentale L. (Anacardiaceae), common- Chemicals
ly called the cashew tree, is commonly found in tropi-
Solvents and reagents for analytical purposes,
cal regions. Different parts of this tree have many uses
such as methanol, AlCl3, KCH3COO, Na2CO3, Folin
and have been utilised as folk medicine in many
Ciocalteu, HClO4, acetic acid (glacial), and HCl, were
countries (Palombo and Semple, 2001). Cashew nuts
purchased from Merck, Germany. Quercetin, gallic
are very popular in the food industry for snack manu-
acid, ursolic acid, and vanillin standard reagents were
facturing. The fruit is used as medicine, food, flavour,
obtained from Sigma-Aldrich, United States. Nutrient
and beverages. The leaves of A. occidentale have been
Broth (Merck, Germany), Mueller-Hinton Agar, and
used traditionally to treat some diseases. Particularly,
Mueller-Hinton Broth (Himedia, India) were used as
in Indonesia, the young leaves are often consumed as
media for bacterial culture and antibacterial assay.
a vegetable in daily diet.
MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-
According to several studies, A. occidentale leaf ex- diphenyltetrazolium bromide) (Sigma-Aldrich, USA)
tracts displayed antibacterial, antifungal, anti- were used for detection of antibacterial activity,
inflammatory, antioxidant, and hypoglycemic proper- which was initially diluted in isopropyl alcohol
ties. A study on ethanolic and petroleum ether ex- (Merck, Germany) and acidified by HCl (Merck,
tracts of A. occcidentale showed that they had antimi- Germany). All samples were diluted in dimethyl sul-
crobial activity against pathogens such as Bacillus foxide (DMSO) (Merck, Germany), while ampicillin
subtillis, Staphylococcus aureus, Pseudomonas aeruginosa, (Sigma Aldrich, USA) was used as a positive control.
Escherichia coli, Candida albicans, Aspergillus niger,
Klebsiella pneumonia, Micrococcus luteus, Streptococcus Plant material
mutans, Shigella sonnei, and Salmonella typhoid fever The A. occidentale leaves were harvested from the
(Onasanwo et al., 2012). Meanwhile, n-hexane extract Yogyakarta area, Indonesia (7°55'53.4’S 110°33'35.0’E,
from the leaves and stems of A. occidentale could in- 209 m above sea level). They were collected as the
hibit Staphylococcus aureus using agar diffusion. Late- mature leaves at the upper part of the plant. The
ly, many phytochemicals have been reported con- leaves were identified and deposited (voucher speci-
tained in this plant including phenols, flavonoids, men 1860046) at Botanical Characterization Laborato-
steroids, terpenoids, essential oils, anacardic acid, ries, The National Research and Innovation Agency,
tatrol, saponins, tannins, alkaloids, flavones, flavo- Indonesia.
nols, xanthones, chalcones, catechins, and glycosides
(Anand et al., 2015; Michodjehoun-Mestres et al., Extraction and fractionation
2009; Onasanwo et al., 2012; Santos et al.,2013). How-
ever, the phytoconstituents characterization of A. Three hundred grams of A. occidentale ground
occidentale leaves associated with antibacterial capaci- leaves were macerated with ethanol fifth fold propor-
ty remains less investigated. In our present study, we tion (w/v) using an ultrasonic bath (Elmasonic S10H,
comprehensively investigated the antibacterial activi- Elma, Germany, 37 kHz, 90 W) for 1 h at room tem-
ty of the extract and its fractions from A. occidentale perature. The extraction was repeated three times.
against both Gram-positive and Gram-negative bacte- The filtrates resulting from sonication were filtered
ria using successive methods. In this way, we specifi- and evaporated with a rotary evaporator (Buchi,
Switzerland). The collected extract (9.63 g) was dis-
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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

solved using distilled water (300 mL). The dissolved LC-HRMS analysis
extract was then partitioned by adding 300 mL of n-
Untargeted metabolomics evaluation of extract
hexane, chloroform, and ethyl acetate in each stage
and each fraction were performed using a liquid
using a separating funnel. The mixture was shaken
chromatography (Thermo Scientific™ Vanquish™
and allowed to stand to form two layers. This parti-
UHPLC Binary Pump), high-resolution mass spec-
tioning was repeated until each supernatant was
trometry (Thermo Scientific™ Q Exactive™ Hybrid
clear. All obtained fractions were concentrated using
Quadrupole-Orbitrap™ High-Resolution Mass Spec-
a rotary evaporator and lyophilized.
trometer) and an autosampler device. The samples
were subjected into a column (Accucore™ Phenyl-
Total phenolic content (TPC)
Hexyl 100 mm × 2.1 mm ID × 2.6 µm) and eluted gra-
The samples and standard gallic acid solution diently with water (0.1% formic acid) and methanol
were prepared at 1 mg/mL concentration in a metha- (0.1 % formic acid) as mobile phases for 25 min in a
nol solution. The standard solution was then diluted 0.30 mL/min flow rate. The data were detected using
at 20, 40, 60, 80, 100, 125, and 150 µg/mL concentra- an Orbitrap mass spectrometer. Full MS/dd-MS2 ac-
tions. The samples or standard solutions were added quisition mode was used to collect untargeted metab-
with 3.75 mL of distilled water and 2.5 mL of Folin olites in positive and negative ionization polarities.
Ciocalteu reagent in the dark test tube. The mixture The detected metabolites were scanned in the range of
was homogenized and incubated for 8 min. After 66.7-1000 m/z using 70,000 for full MS and 17,500 for
incubation, the mixture was added with 0.75 mL of dd-MS2 resolutions. Data analysis was processed on
20% Na2CO3 solution. The mixture was incubated for Xcalibur software 4.4 version (Thermo Scientific,
2 h, and the absorbance was observed at 765 nm (Cary Bremen, Germany).
60 UV-Vis Spectrophotometer, Agilent, USA). The
total phenolic content was defined as mg GAE/g of Bacterial strains
dry weight (Nisa et al., 2019).
The antibacterial activity was tested using four
pathogenic bacterial strains, namely Staphylococcus
Total flavonoid content (TFC)
aureus FNCC 0047, Bacillus subtillis FNCC 0059, Esche-
The samples and standard quercetin solution were richia coli FNCC 0091, and Pseudomonas aeruginosa
prepared at 1 mg/mL concentration in a methanol FNCC 0070 bacteria. All the tested bacteria were pro-
solution. The standard solution was then diluted at vided from the Food and Nutrition Culture Collection
25, 50, 100, 200, 400, and 800 µg/mL concentrations. (FNCC), Universitas Gadjah Mada, Yogyakarta, In-
Each extract, fraction, and standard solution was add- donesia.
ed into a 96-well microplate, followed by adding 60
µL methanol, 10% aluminium chloride, and 10 µL of 1 Preparation of inoculums
M potassium acetate. Finally, 120 µL of distilled water
Approximately 300 μL bacterial strains were sub-
was added and then properly homogenized. After
jected to 3 mL Nutrient Broth agar media and subcul-
incubation for 30 min, the absorbance was observed at
tured overnight at 37ºC. The Optical density (OD)
a wavelength of 415 nm (Multiskan™ FC microplate
was measured using a microplate reader (Mul-
reader, Thermo Scientific, USA). The total flavonoid
tiskan™ FC microplate reader, Thermo Scientific,
content was defined as mg QE/g of dry weight (Nisa
USA) at 600 nm and calculated according to Mc Far-
et al., 2022).
land Standard 0.5 method (1.5 × 108 CFU/mL). The
inoculum suspension was further diluted to obtain a
Total terpenoid content
viable cell count of approximately 107 CFU/mL
The extract, fractions, and standard ursolic acid so- standard bacterial turbidity.
lution were prepared at 1 mg/mL concentration in a
methanol solution. The standard solution was then Antibacterial evaluations
diluted at 100, 200, 300, 400, 500, and 600 µg/mL con-
The antibacterial activity of A. occidentale was
centrations. A 0.1 mL of each sample and standard
evaluated using minimum inhibitory concentrations
solution was added with 0.2 mL of 5% vanillin and 1
(MIC) (Nisa et al., 2022) and minimum bactericidal
mL of perchloric acid. The mixture was then incubat-
concentrations (MBC) determination and time-kill
ed for 45 min at 60ºC. After incubation, the samples
assay by microdilution method (Galgano et al., 2022).
were let to stand at room temperature for 15 min.
In a 96-well microplate, 20 mg/mL stock solution of
Finally, 2.5 mL of acetic acid was added and well
extract and fractions in DMSO were diluted at various
homogenized. The absorbance was measured using
concentrations (0.125, 0.25, 0.5, 1, 2 mg/mL) in a total
UV-Vis at a wavelength of 548 nm (Cary 60 UV-Vis
volume of 100 μL Mueller Hinton Broth (MHB) medi-
Spectrophotometer, Agilent, USA).
um. Then, 100 μL of bacterial colonies were added
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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

into extract, fractions, and positive and negative con- secondary metabolite contents. Table 1 showed that
trols. Ampicillin was served as a positive control, and the ethyl acetate fraction of A. occidentale had the
DMSO was used as a negative control. All tests were highest value of phenolic content (508.89 ± 5.0 mg
performed in triplicate. The samples were incubated GAE/g dw) followed by aqueous fraction (314.64 ±
at 37ºC for 24 h. The time kill assay was evaluated by 3.7 mg GAE/g dw) and the crude ethanol extract
monitoring the OD using a microplate reader (Mul- (249.25 ± 1.6 mg GAE/g dw) (p<0.01). Same as phe-
tiskan™ FC microplate reader, Thermo Scientific, nolic contents, ethyl acetate fraction also exhibited the
USA) (λ = 600 nm) with 2 h as intervals time of obser- highest flavonoid contents (184 ± 3.6 mg QE/g dw)
vation. The OD data of each antibacterial concentra- followed by an aqueous fraction as well (p<0.01).
tion was calculated as ΔOD (interval incubation time While in the terpenoid contents evaluation, the etha-
– initial incubation time). The ΔOD was plotted as nol extract and all fractions displayed a similar value
bacterial killing times curves, which were processed (p<0.01).
using the GraphPad Prism 9.5.1 (May et al., 2000).
These results show that the phenolic compounds
After 24 h of incubation for MBC determination, one
in A. occidentale are mostly semipolar compounds that
loop of the mixtures treatment in each level of concen-
are easy to dissolve in semipolar solvents such as
tration was inoculated on Mueller Hinton Agar
ethyl acetate. According to Chaves et al. (2020), ethyl
(MHA) media and further incubated for 24 h. Finally,
acetate solvent extraction can bind flavonoids with
15 µL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
low polarity, such as isoflavones, flavanones, methyl
dimethyltetrazolium bromide] was added into the
flavones, and flavonols. In contrast, all solvents seem
samples, followed by incubation for 1 h at 37ºC. The
to have a similar capacity to attract nonpolar com-
colour (purple-yellow) alteration was observed visu-
pounds based on their terpenoid contents. Terpenoids
ally in each well group. The lowest concentration,
are one of the largest groups of secondary metabolites
which indicated the absence of bacterial growth (yel-
derived from natural sources. They are considered
low colour), was considered the MIC value. The MBC
volatile, colourless, and fragrant liquids. Terpenoids
value was expressed as the lowest concentration
are incompatible with water but easily soluble in or-
without bacterial growth on the agar plate.
ganic solvents, such as petroleum ether, chloroform,
or ethanol (Fan et al., 2023).
Statistical analysis
The obtained phytochemical data were expressed Determination of minimum inhibitory
as the mean ± SD (standard derivation) of triplicate concentration (MIC)
experiments and examined by one-way analysis of
Antibacterial activity of A. occidentale was investi-
variance (ANOVA) and Duncan's post-hoc test. The
gated by MIC and MBC determinations and time-kill
significance of data was statistically defined to be
assay confirmed by streak agar plate evaluation.
p<0.01.
The MIC of the A. occidentale ethanol extract and
fractions were performed by microplate dillution
RESULTS AND DISCUSSION
method to evaluate their bacteriostatic properties. The
MIC is determined as the lowest concentration of the
Extraction
A. occidentale extract or fractions, in which no micro-
Comparing the chemical composition of ethanol bial growth was visually observed after incubation for
extract and all fractions, the ethyl acetate fraction was 24 h, as presented in Table 2.
observed to have more strongly positive results in

Table 1. The yields, phenolic, flavonoid, terpenoid contents of extract and fractions.

Yields Phenolic content Flavonoid content Terpenoid content

Samples
(g) (mg GAE/g dw) (mg QE/g dw) (mg UAE/g dw)

Ethanol extract 9.63 249.25 ± 1.6c 52.89 ± 4.1c 673.11 ± 0.8a

n-hexane fraction 0.56 58.89 ± 0.6e 84.04 ± 2.3bc 177.36 ± 5.2a

Chloroform fraction 0.64 90.80 ± 0.2d 79.11 ± 1.5bc 164.94 ± 7.8a

Ethyl acetate fraction 0.49 508.89 ± 5.0a 184..00 ± 3.6a 159.72 ± 5.9a
Aqueous fraction 4.89 314.64 ± 3.7b 118.00 ± 14.6b 244.00 ± 2.1a
The same column marked with a different letter shows that the values are significantly different by Duncan's post-hoc test (p<0.01).

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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

Table 2. Minimum inhibitory concentration of extract and fractions.

MIC value (mg/mL)

Samples
E. coli B. subtillis S. aureus P. aeruginosa

Ethanol extract ≤1.0 ≤1.0 ≤0.25 ≤0.5

n-hexane fraction ≤1.0 ≤1.0 ≤1.0 ≤1.0

Chloroform fraction ≤2.0 ≤1.0 ≤1.0 ≤2.0

Ethyl acetate fraction ≤0.5 ≤0.5 ≤0.5 ≤0.25

Aqueous fraction ≤2.0 ≤1.0 ≤0.5 ≤0.5

Ampicillin ≤0.001 ≤0.001 ≤0.001 ≤0.001

A. occidentale extract could inhibit the bacteria aqueous fraction could kill E. coli in the lower concen-
started from a concentration of 0.25 mg/mL to 1.0 tration (1 mg/mL), but needs a longer time (24 h).
mg/mL with selective to S. aureus and P. aeruginosa Compared with extract as well as other fractions,
yet less effective against E. coli and B. subtillis. The ethyl acetate fraction showed a very strong ability to
results are in accordance with those reported by kill E. coli until the lowest tested concentrations (0.25
Anand et al. (2015), Kaewpiboon et al. (2012), and mg/mL) in two hours of observation.
Santos et al. (2013). The ethyl acetate fraction sup-
Antibacterial evaluation toward B. subtillis exhibit-
pressed the bacterial growth from 0.25 mg/mL to 0.5
ed that at a concentration of 1 mg/mL, either ethanol
mg/mL. Among other fractions, the ethyl acetate
extract or chloroform fraction were able to kill in 6 h
fraction was the most effective against all tested bac-
and 4 h, respectively. The n-hexane fraction required
teria, followed by the aqueous fraction. S. aureus and
a minimum concentration of 2 mg/mL to kill B. subtil-
P. aeruginosa exhibited the highest susceptibility to A.
lis in 8 h, similar to the aqueous fraction. While at a
occidentale aqueous fraction. E. coli, as a Gram-
low concentration (0.5 mg/mL), the ethyl acetate frac-
negative bacteria, was apparently more resistant to
tion could kill the bacteria longer (24 h). As an antimi-
the extract and most of the fractions. However, it was
crobial agent, less concentration is taken to minimize
sensitive only to ethyl acetate fraction. Gram-negative
the use of higher doses in drug administrations to
bacteria resist because they possess a wall structure
prevent their toxicity effects (Sun et al., 2019). Toward
consisting of many layers of peptidoglycan and di-
B. subtillis, the ethyl acetate fraction presented a very
verge in chemical conformation. Hence, it is quite
strong capacity to kill bacteria at all ranges of tested
complicated than Gram-positives even though the
concentrations.
thicker types only present a single form of macro
molecule (Gonçalves and Gobbo, 2012). However, in From Fig. 1, either extract or fractions afforded to
contrast, Gram-negative bacteria P. aeruginosa has kill microbial growth of P. aeruginosa. The ethanol
sensitiveness rather than the extract and fractions extract and ethyl acetate fraction were observed to
except for the chloroform fraction. The investigation possess bactericidal activity to P. aeruginosa at low
by those three assays of extract and fractions suggest- concentrations (0.125 mg/mL) in 18 h and 2 h, respec-
ed that A. occidentale can be used as an antibiotic agent tively. Moreover, the aqueous fraction exhibited good
to treat such pathogenic bacteria causing several dis- bactericidal activity at a high concentration (0.5
eases. E. coli and P. aeruginosa are the two common mg/mL) in 2 h of growth observation. In contrast, n-
sources for producing toxins that cause human gas- hexane (2 mg/mL) and chloroform fractions (1
troenteritis diseases. While S. aureus is the organism mg/mL) were able to kill bacteria at this concentra-
that contributed to pneumonia, skin infections, oral tion in 2 h. Within all fractions, ethyl acetate fraction
cavity, and bacteremia. has the highest bactericidal activity against P. aeru-
ginosa.
Time-kill assay evaluation The ethanol extract and all fractions displayed
good antibacterial activity by killing S. aureus micro-
The minimum bactericidal concentration (MBC)
bial growth. The ethanol extract and n-hexane fraction
and time effectiveness to kill the four tested patho-
could kill the bacteria at a minimum 1 mg/mL con-
genic bacteria are presented in Fig. 1.
centration in 2 h and 8 h, respectively. While the chlo-
The Fig. 1 displayed that the ethanol extract could roform and aqueous fraction required a 2 mg/mL
kill E. coli in 18 h at a concentration of 2 mg/mL while concentration to kill bacteria in 4 h and 2 h, respec-
at the same concentration, ethanol extract, n-hexane tively. The ethyl acetate fraction showed a very strong
fraction, and chloroform fraction were able to kill E.
coli in the 20 h, 2 h, and 6 h, respectively. Moreover,
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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

Figure 1. Time-kill assay and streak agar plate of A. occidentale against S. aureus, P. aeruginosa, B. subtillis, and E. coli.
Zero growth of bacteria on agar plate indicates bactericidal concentration of active fraction (ethyl acetate fraction).

ability to kill S. aureus even in 2 h at 0.5 mg/mL of damage the cytoplasmic membrane, causing leakage
concentrations. of important metabolites that inactivate the microbial
enzyme system, while the cytoplasmic membrane and
According to those results, the ethyl acetate frac-
precipitate cell proteins are destroyed by increasing
tion was the most active against all tested bacteria.
its concentrations (Suliani et al., 2016).
Flavonoids and phenolic compounds might attribute
it as the main constituents in ethyl acetate fraction.
Streak agar plate
Several studies explained that ethyl acetate fraction
derived from plant extract contains high flavonoid The lowest MIC value of A. occidentale extract and
and phenolic content with antimicrobial activity. As fractions was confirmed by the Minimum Bactericidal
reported in Vernonia amygdalina Delile leaves, its ethyl Concentration (MBC) evaluation using a streak agar
acetate fraction had improved the microbial activity plate to show potentially bactericidal capacity against
of tetracycline against P. aeruginosa and MRSA (Satria the tested pathogenic bacteria (Fig. 1). After incuba-
et al., 2023). Flavonoids have good antimicrobial ac- tion for 24 h, the lowest concentration from an antimi-
tivity and have recently been paid much attention crobial agent, which works out to kill bacteria up to
(Tungmunnithum et al., 2018). Several flavonoid 99.9% expresses as an MBC value. By streaking agar
compounds with antibacterial capacity, such as apig- plate method, MBC is recognized by the appearance
enin, quercetin, and luteolin, have been isolated of scratches on the plate that has been streaked and
(Basile et al., 2000; Rauha et al., 2000; Torrenegra et al., incubated for 24 h (Balouiri et al., 2016).
1989).
Fig. 1 displays that the ethyl acetate fraction of A.
Alkaloid and phenolic compounds, which are occidentale has a good bactericidal capacity against E.
abundantly obtained in plant extract, work as antimi- coli, P. aureginosa, and B. subtillis bacteria at a concen-
crobial components by disrupting the function of the tration of 1 mg/mL and, at lower concentration (0.5
cytoplasmic membrane, which may cause cell death mg/mL), was more effective against S. aureus bacte-
or hinder essential enzymes for the biosynthesis of ria. The MIC determination assay of A. occidentale
amino acids (Burt, 2004). At low concentrations, it can complied with the time-kill assay, which demonstrat-
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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

ed that ethyl acetate fraction was the most active an- stearamide, the ethyl acetate fraction contains all pu-
timicrobial against all tested pathogenic bacteria. See tative compounds in the extract (n = 31). Therefore,
also Figs. S1-S3. only the fractions of chloroform and n-hexane contain
fatty acids, specifically palmitic acid.
LC-HRMS compounds profiling
Taking a retention time range of 5 to 25 min, the
The untargeted LC-HRMS chromatogram, as dis- ethyl acetate fraction contained a large variety and
played in Fig. 2, was examined. The following analyt- abundance of compounds from semipolar to nonpolar
ical parameters comprised the metabolite profile regions. Flavonoids are identified as the main con-
analysis: the conformation of candidate molecules to tents in ethyl acetate fraction, with avicular-
the fragmentation pattern in mzCloud was used to in/quercetin-3-O-arabinofuranoside and myricitrin
select them, and their molecular mass deviation did recognized as major compounds. Due to its structure,
not exceed 2 ppm. High abundance compounds in the which includes both a polar group (-COOR) and a
aqueous, chloroform, and hexane fractions appeared nonpolar group (-CH), ethyl acetate is classified as a
between 13 and 25 min (Fig. 2). semipolar compound. It should, therefore, attract
either polar or nonpolar compounds. Like flavonoid
As shown in Table 3, the fraction from ethanolic
aglycones, a semipolar polyphenol could be dissolved
extracts of A. occidentale was analysed with an LC-
in cell walls by an ethyl acetate solvent. The solvent
HRMS system to predict the metabolite variation to
ethyl acetate can bind flavonoids with low polarity,
determine the fractionation impact. In general, the
including isoflavones, flavanones, methyl flavones,
leaf extract of A. occidentale and its fractions contain
and flavonols, during extraction (Chaves et al., 2020).
amino acids, phenolic acids, alkaloids, flavonoids,
Flavonoid compounds are present in most of the ev-
fatty acid amides, fatty acids, and organic acids as
ergreen vegetation. Most flavonoid compounds in
classes of molecules. A. occidentale is an exceptionally
plants exist as sugar-bound glycosides. The hydroxyl
flavonoid-rich herb. The flavonoids were profused
group in the flavonoid molecule (aglycone) bonds to
between 3.75 and 17.7 min of retention time (Table 3
the carbonyl group of sugar (glycone) to form flavo-
and Fig. 2). Except for the stearamide compound, all
noid glycosides (Kumar and Pandey, 2013). Moreo-
fractions contained fatty acid amide compounds.
ver, the phenolic compound, such as gallic acid, was
Moreover, excluding for L-phenylalanine, trans-3-
also abundantly contained in ethyl acetate fraction.
indoleacrylic acid, catechin gallate, quercetin-3ß-D-
See also Table S1.
glucosides, luteolin, palmitic acid, myristamide, and

Figure 2. Total ion chromatogram comparison of UHPLC-HR-ESI-MS from fractions and crude extracts of A. occidentale.
(A) crude extract; (B) aqueous fraction; (C) ethyl acetate fraction; (D) chloroform fraction; and (E) hexane fraction.

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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

Table 3. Putative compound identified from A. occidentale.

Annot. Area*
MS/MS
No. RT (min) Putative compound Class Formula Delta Mass Calc. MW
fragmentation A B C D E
[ppm]

1 1.251 L-Valine Amino acid C5H11NO2 0.75 117.07907 55.05383; 68.95245; 1.2E+08 2.1E+08 1.2E+07
72.08123; 91.05432;
118.03487

2 1.515 Shikimic acid Phenolic acid C7H10O5 -5.96 174.05179 71.01268; 81.03339; 7.4E+07 3.0E+07
93.03343;
111.04402;
137.02338;
143.03384 (negative
mode)
3 2.311 Gallic acid Phenolic acid C7H6O5 -6.43 170.02043 89.07113; 5.9E+07 3.9E+07 6.4E+08 1.0E+07
109.02844;
125.02316;
127.03877;
153.01797;
171.02852
4 2.723 L-Phenylalanine Amino acid C9H11NO2 -0.11 165.07896 79.05464; 93.07007; 1.9E+08 1.7E+08
103.05441;
107.04921;
120.08077;
131.04909
5 3.750 Gallocatechin Flavonoid C15H14O7 -1.55 306.07348 139.03871; 7.4E+08 1.7E+07 4.2E+08
181.04915;
247.06029;
307.08029

6 4.542 trans-3-Indoleacrylic acid Alkaloids C11H9NO2 -1.25 187.06309 91.05447; 1.5E+08 1.2E+08 1.7E+07
118.06522;
146.05992

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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

Table 3. Putative compound identified from A. occidentale (continued...)

Annot. Area*
MS/MS
No. RT (min) Putative compound Class Formula Delta Mass Calc. MW
fragmentation A B C D E
[ppm]

7 5.484 Catechin Flavonoid C15H14O6 -1.46 290.07861 97.02832; 2.1E+08 6.5E+06 2.9E+08
109.02833;
123.04400;
203.08059;
245.08139;
289.07147 (negative
mode)
8 6.231 Epigallocatechin gallate Flavonoid C22H18O11 -1.14 458.08439 69.03343; 1.7E+07 6.6E+07
125.02329;
169.01326;
161.02342;
269.04605 (negative
mode)

9 6.757 Myricetin 3-O-β-D- Flavonoid C21H20O13 -0.14 480.08984 153.01804; 3.8E+07 2.9E+07 1.6E+08
galactopyranoside 245.04384;
319.04398;
385.05838

10 7.251 Catechin gallate Flavonoid C22H18O10 -1.41 442.08937 151.03882; 1.1E+07

123.04403;
139.03877;
273.07544
11 7.305 Quercetin-3ß-D- Flavonoid C21H20O12 -1.10 486.07687 71.50153; 96.21252; 6.9E+07 2.5E+07
glucosides 185.04170;
255.39217;
325.03085
12 7.314 Myricitrin Flavonoid C21H20O12 -1.27 464.09548 71.04960; 1.4E+08 1.7E+08 2.4E+09 1.4E+08
129.05457;
147.06473;
245.04453;
319.04410

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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

Table 3. Putative compound identified from A. occidentale (continued...)

Annot. Area*
MS/MS
No. RT (min) Putative compound Class Formula Delta Mass Calc. MW
fragmentation A B C D E
[ppm]

13 7.322 (2r)-2-(3,4- Flavonoid C15H10O7 -2.35 302.04194 109.02831; 9.4E+07 3.5E+07 4.4E+08 2.2E+07
Dihydroxyphenyl)-3,5,7- 121.02949;
trihydroxy-2,3-dihydro- 274.04694;
4h-chromen-4-one 303.04922
14 7.367 Desmanthin-1 Flavonoid C28H24O16 -2.01 616.10520 97.02862; 2.7E+07 1.9E+08
153.01805;
297.05994;
303.04922

15 7.731 Avicularin / quercetin-3-O- Flavonoid C20H18O11 -1.53 434.08491 57.03408; 73.02885; 2.1E+08 1.3E+09 5.7E+07
arabinofuranoside 85.02875;
165.01791;
303.04938

16 8.142 Juglalin / kaempferol 3-O- Flavonoid C20H18O10 -1.05 418.08956 107.01297; 4.5E+07 8.9E+07
arabinoside 135.00787;
255.02926;
284.03247;
417.08252

17 8.288 Azelaic acid Organic acids C19H16O4 -4.31 188.10486 57.03348; 97.06476; 7.5E+06 7.5E+06
125.09613;
187.09682
18 8.362 Myricetin Flavonoid C15H10O8 -0.82 318.03731 68.99761; 8.5E+06 8.5E+06
109.02860;
137.02338;
255.02853

19 9.141 5,7-Dihydroxy-2-(4- Flavonoid C22H22O12 -0.55 478.11086 61.02888; 91.03920; 9.3E+06 9.2E+07
hydroxy-3- 271.05984;
methoxyphenyl)-6-[3,4,5- 317l06458
trihydroxy-6-
(hydroxymethyl) oxan-2-
yl]chromen-4-one

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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

Table 3. Putative compound identified from A. occidentale (continued...)

Annot. Area*
MS/MS
No. RT (min) Putative compound Class Formula Delta Mass Calc. MW
fragmentation A B C D E
[ppm]

20 9.490 Quercetin Flavonoid C15H10O7 -1.12 302.04265 65.00217; 3.4E+07 7.2E+07 7.1E+06
107.01275;
151.00266;
245.04509;
301.04503 (negative
mode)
21 10.508 Luteolin Flavonoid C15H10O6 -0.77 286.04752 63.02294; 9.6E+06 2.6E+07
107.01269;
143.04926;
285.04016 (negative
mode)
22 11.376 Palmitic acid Fatty acid C16H32O2 -1.01 273.26663 70.06553; 6.6E+06 5.7E+06 5.4E+06
106.08653;
230.24518;
274.27383
23 16.514 5,5',7,7'-Tetrahydroxy- Flavonoid C30H18O10 -1.29 538.08930 68.99787; 2.9E+08 2.6E+08 2.6E+08 1.2E+08
2,2'-bis(4-hydroxyphenyl)- 121.02843;
4H,4'H-6,6'-bichromene- 163.03906;
4,4'-dione 275.01895;
403.04379;
539.09680
24 17.770 Myristamide Flavonoid C14H29NO -2.14 227.22443 71.0851; 254.2473; 2.9E+09 2.2E+09
272.2585

25 18.235 (4E)-5-Methyl-4-[(5- NA C21H18N4O2 -2.27 358.14216 93.05757; 3.1E+07 8.9E+07 1.9E+08 1.3E+08
methyl-3-oxo-2-phenyl- 118.06523;
1H-pyrazol-4-yl) 268.10818;
methylidene]-2- 266.09219;
phenylpyrazol-3-one 359.14948

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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

Table 3. Putative compound identified from A. occidentale (continued...)

Annot. Area*
MS/MS
No. RT (min) Putative compound Class Formula Delta Mass Calc. MW
fragmentation A B C D E
[ppm]

26 19.406 bis(2-Ethylhexyl)phthalate Organic C24H38O4 -3.51 390.27701 57.07042; 71.08594; 2.9E+07 9.2E+07 1.3E+08 4.0E+07 1.3E+10
compound 149.02312;
279.15747

27 19.839 Hexadecanamide Fatty acid amide C16H33NO -1.36 255.25587 57.07046; 74.06046; 2.8E+07 5.5E+06 5.7E+06 3.4E+07 3.0E+07
116.10697;
148.67090.
258.26331
28 20.078 Oleamide Fatty acid amide C18H35NO -1.92 281.27133 69.07031; 83.08582; 2.4E+08 9.8E+07 1.7E+08 2.6E+08 4.7E+08
95.08567;
149.13232;
163.14767;
247.24138;
282.27863

29 20.142 Ostruthin Terpenoid C19H22O3 -0.12 298.15980 57.45317; 89.44093; 1.4E+07 1.3E+07 1.7E+07 2.1E+07 1.3E+07
183.01117;
239.07391;
297.15244

30 21.498 Erucamide Fatty acid amide C22H43NO -3.04 337.33344 69.07029; 83.08581; 4.5E+08 6.0E+08 5.5E+08 4.7E+08 7.4E+08
97.10133;
109.10123;
149.13225;
226.21590;
303.20365;
338.34058

31 22.660 Strearamide Fatty acid amide C18H37NO -1.12 283.28720 57.07042; 74.06034; 2.2E+07 2.4E+07 3.8E+07
116.10705;
284.29446
*(A) crude extract; (B) aqueous fraction; (C) ethyl acetate fraction; (D) chloroform fraction; and (E) n-hexane fraction.

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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

CONCLUSION against Escherichia coli and Staphylococcus aureus. Antibiotics

11(7): 979. https://doi.org/10.3390/antibiotics11070979
Ethyl acetate fraction showed remarkable antimi- Gonçalves GMS, Gobbo J (2012) Antimicrobial effect of Anacardium
crobial activity against E. coli, B. subtillis, P. aeruginosa, occidentale extract and cosmetic formulation development.
Braz Arc Biol Technol 55(6): 843–850.
and S. aureus bacteria strains. The results were in ac- https://doi.org/10.1590/S1516-89132012000600006
cordance with its phytochemical profile, which pos-
Kaewpiboon C, Lirdprapamongkol K, Srisomsap C,
sessed the highest phenolic and flavonoid contents. Winayanuwattikun P, Yongvanich T, Puwaprisirisan P, Svasti
The antibacterial capacity of the active fraction might J, Assavalapsakul W (2012) Studies of the in vitro cytotoxic,
be attributed mainly to the presence of those flavo- antioxidant, lipase inhibitory and antimicrobial activities of
selected Thai medicinal plants. BMC Complement Altern Med
noid and phenolic compounds, as confirmed in LC-
12: 217. https://doi.org/10.1186/1472-6882-12-217
HRMS profiling.
Khan UA, Rahman H, Niaz Z, Qasim M, Khan J, Tayyaba, Rehman
B (2013) Antibacterial activity of some medicinal plants
CONFLICT OF INTEREST against selected human pathogenic bacteria. Eur J Microbiol
Immunol 3(4): 272–274.
The authors declare no conflicts of interest. https://doi.org/10.1556/eujmi.3.2013.4.6
Kumar S, Pandey AK (2013) Chemistry and biological activities of
ACKNOWLEDGMENTS flavonoids: An overview. Sci World J 2013: 162750.
https://doi.org/10.1155/2013/162750
The authors thank The National Agency of Research and May J, Chan CH, King A, Williams L, French GL (2000) Time-kill
Innovation, Indonesia, for technical support. The authors studies of tea tree oils on clinical isolates. J Antimicrob
acknowledge the facilities, scientific and technical support Chemother 45(5): 639–643.
from Botanical Characterization Laboratories, National https://doi.org/10.1093/jac/45.5.639
Research and Innovation Agency through E-Layanan Sains, Michodjehoun-Mestres L, Amraoui W, Brillouet JM (2009) Isolation,
Badan Riset dan Inovasi Nasional. characterization, and determination of 1-O-trans-cinnamoyl-
β-D-glucopyranose in the epidermis and flesh of developing
cashew apple (Anacardium occidentale L.) and four of its
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_________________________________________________________________________________________________________

AUTHOR CONTRIBUTION:

Contribution Nisa K Hasanah AU Damayanti E Frediansyah A Anwar M Khumaira A Anindita NS

Concepts or ideas x x

Design x x x

Definition of intellectual content x x

Literature search x

Experimental studies x x x

Data acquisition x x x x x

Data analysis x x x x x

Statistical analysis x x

Manuscript preparation x x x x

Manuscript editing x x x

Manuscript review x x x x x x x

Citation Format: Nisa K, Hasanah AU, Damayanti E, Frediansyah A, Anwar M, Khumaira A, Anindita NS (2024) In vitro antibacterial activity and
phytochemical profiling of Indonesian Anacardium occidentale L. leaf extract and fractions. J Pharm Pharmacogn Res 12(1): 50–72.
https://doi.org/10.56499/jppres23.1714_12.1.50

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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

Supplementary data

Table S1. Putative compound chromatograms.

Putative compound Fragmentation pattern

L-Valine

Shikimic acid

Gallic acid

L-Phenylalanine

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Gallocatechin

trans-3-Indoleacrylic
acid

Catechin

Epigallocatechin
gallate

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Myricetin 3-O-β-D-
galactopyranoside

Catechin gallate

Quercetin-3ß-D-
glucosides

Myricitrin

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Nisa et al. Antibacterial activity of Anacardium occidentale fractions

(2r)-2-(3,4-
Dihydroxyphenyl)-
3,5,7-trihydroxy-2,3-
dihydro-4h-chromen-
4-one

Desmanthin-1

Avicularin /
quercetin-3-O-
arabinofuranoside

Juglalin / kaempferol
3-O-arabinoside

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Azelaic acid

Myricetin

5,7-Dihydroxy-2-(4-
hydroxy-3-
methoxyphenyl)-6-
[3,4,5-trihydroxy-6-
(hydroxymethyl)oxan-
2-yl]chromen-4-one

Quercetin

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Luteolin

Palmitic acid

5,5',7,7'-
Tetrahydroxy-2,2'-
bis(4-hydroxyphenyl)-
4H,4'H-6,6'-
bichromene-4,4'-
dione

Myristamide

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(4E)-5-Methyl-4-[(5-
methyl-3-oxo-2-
phenyl-1H-pyrazol-4-
yl) methylidene]-2-
phenylpyrazol-3-one

bis(2-
Ethylhexyl)phthalate

Hexadecanamide

Oleamide

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Ostruthin

Erucamide

Strearamide

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