Antibacterial, Antifungal, and Antioxidant Activities and Polyphenol

Content of the Edible Seeds of Archidendron bubalinum

K. Y. Teh, Y. L. Wong and N. W. Sit

Department of Allied Health Sciences, Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat, 31900 Kampar,

Perak, Malaysia

Keywords: Antibacterial, Antifungal, Antioxidant, Archidendron bubalinum, Total Phenolic Content

Abstract: This study assessed the kernel extracts of Archidendron bubalinum (family Leguminosae) seeds for

antibacterial, antifungal, and antioxidant activities as well as polyphenol content. The kernels were macerated

using hexane, ethyl acetate, ethanol, and water sequentially and produced four extracts for analysis. All the

extracts showed bactericidal effects against five of the six species of bacteria tested with minimum bactericidal

concentrations of 0.63-2.50 mg/mL, and fungicidal effects against five species of fungi with minimum

fungicidal concentrations of 0.31-2.50 mg/mL. All the extracts also exerted antioxidant activities. The hexane

extract exhibited the lowest oxygen radical absorbance capacity (ORAC) and ferric-reducing antioxidant

power (FRAP) values of 81.63 mmol Trolox equivalent/g of extract and 25.93 mmol Fe

equivalent/g of

extract, respectively. In contrast, ethyl acetate extract exhibited the highest ORAC (300.13 mmol Trolox

equivalent/g of extract) and FRAP (341.36 mmol Fe

equivalent/g of extract) values. Polyphenols were

detected in all extracts with the total phenolic content (TPC) of 3.22-20.19 mg gallic acid equivalent/g of

extract and the total flavonoid content (TFC) of 0.97-4.42 mg quercetin equivalent/g of extract. Association

analysis between ORAC/FRAP and TPC/TFC revealed significant strong positive correlations (r = 0.937-

0.995; all p<0.001). In conclusion, the edible seeds of A. bubalinum possessed antimicrobial and antioxidant

properties and could be promoted as a healthy food.

1 INTRODUCTION

Communicable diseases caused by microorganisms

such as bacteria, fungi, viruses, and parasites are one

of the major contributors to human morbidity and

mortality worldwide. In 2019, more than 26.1 billion

incident cases of communicable diseases were

reported, of which ~93% of the cases involved

respiratory and enteric systems, contributing to 4.2

million death globally (GBD 2019 Diseases and

Injuries Collaborators, 2020). Bacteria have been

recognized as a main etiological agent for human

respiratory and enteric infections. They can be

broadly classified into two groups; Gram-positive

bacteria have a thick peptidoglycan layer anchored

with teichoic acids in the cell wall without an outer

membrane while Gram-negative bacteria possess a

cytoplasmic membrane and a lipopolysaccharides-

containing outer membrane with a thin peptidoglycan

layer in between the membranes (Fisher and

https://orcid.org/0000-0002-5949-491X

Mobashery, 2020). Beta-lactams, tetracyclines,

oxazolidinones, macrolides, aminoglycosides,

sulfonamides, and quinolones are among the

antibacterial agents developed for therapeutic use.

However, the misuse and overuse of these agents as

well as the natural evolutionary ability of

microorganisms have driven the increased prevalence

of antibacterial resistance (Chokshi et al., 2019;

Christaki et al., 2020). Resistance to antibacterial

therapy has resulted in patients with more serious

illnesses, prolonged hospitalization, and treatment

failure, as well as increased costs and resource

utilization of the healthcare system (Ahmad and

Khan, 2019). It has been estimated that the total

economic cost of antibacterial resistance caused by

major human pathogens in the United States of

America was $2.9 billion (Shrestha et al., 2018).

Fungi produce a wide spectrum of infections in

humans, ranging from superficial (e.g., skin, hair,

nail, and keratitis), mucosal (e.g., oral and

Teh, K., Wong, Y. and Sit, N.

Antibacterial, Antifungal, and Antioxidant Activities and Polyphenol Content of the Edible Seeds of Archidendron bubalinum.

DOI: 10.5220/0011595600003430

In Proceedings of the 8th International Conference on Agricultural and Biological Sciences (ABS 2022), pages 40-49

ISBN: 978-989-758-607-1; ISSN: 2795-5893

vulvovaginal candidiasis), allergic (e.g.,

rhinosinusitis and severe asthma with fungal

sensitization), chronic severe (e.g., chronic

pulmonary aspergillosis), to invasive infections (e.g.,

invasive candidiasis, cryptococcosis, and

aspergillosis). People with weakened or

compromised immunity such as cancer patients or

transplant recipients are more susceptible to fungal

infections (Bongomin et al., 2017). Each year, over

300 million people are afflicted with an episode of

fungal infection, resulting in more than 1.6 million

deaths globally (Urban et al., 2021; GAFFI, 2022).

Thus, effective antifungal treatment is important to

reduce the mortality rate. Yet, only polyenes,

flucytosine, azoles, and echinocandins are available

as antifungal agents for clinical use. The emergence

of fungal resistance, drugs’ adverse effects, and

undesirable drug-drug interactions hinder the

outcomes of antifungal treatment (Revie et al., 2018).

In the human body, when the antioxidative

protection system is unable to counteract reactive

species production, oxidative stress ensues (Pisoschi

et al., 2021). Hydroxyl radical, superoxide anion,

singlet oxygen, and peroxynitrite are examples of

these reactive species produced as the byproducts of

aerobic cellular metabolism, or as the response of the

body to exposure to cigarette smoke, ultraviolet

radiation, pesticides, and ozone. Meanwhile,

superoxide dismutase, glutathione peroxidase, metal-

trapping proteins, and vitamins A, C, and E constitute

important elements of the antioxidative protection

system (Rosado-Pérez et al., 2018). The excess

reactive species modify the structures and modulate

the functions of proteins, lipids, and nucleic acids.

Damages to these biomolecules lead to non-

communicable diseases such as cancer,

cardiovascular diseases, diabetes, and

neurodegenerative disorders (Liguori et al., 2018).

Due to the concern of side effects from synthetic

antioxidants (Ito et al., 1985), natural resources such

as plants could offer an alternative source for the

growing demand for exogenous antioxidants.

Edible fruits have long been recognized as an

important source of nutrients such as proteins and

minerals for human health as well as ingredients for

medicines. Different classes of phytochemicals or

secondary metabolites such as alkaloids, terpenoids,

or polyphenols are present in fruits, which endorse

them with various biological properties, including

antimicrobial and antioxidant activities (Forni et al.,

2019). Fruit with a high amount of procyanidins such

as cranberries has been shown to reduce bacterial or

fungal infections of the urinary tract (Vostalova et al.,

2015; Sundararajan et al., 2018). Regular intakes of

flavonoids through diets have been reported to

significantly decrease the risks of cardiovascular

diseases (Wang et al., 2014). In another study, a high

intake of antioxidant-rich fruits and vegetables in

healthy adults lowers the oxidized low-density

lipoprotein level, which is considered a biomarker of

cardiovascular diseases (Bacchetti et al., 2019).

Being one of the tropical countries with mega-

biodiversity, Malaysia has at least 355 species of trees

and 165 species of non-trees bearing edible fruits or

seeds (Milow et al., 2014). More than two-thirds of

them are wild or non-exclusively planted. These fruits

and seeds present a vast opportunity for exploration

as healthy foods.

Archidendron bubalinum (Jack) I.C.Nielsen

(family Leguminosae; synonym Pithecellobium

bubalinum) is a wild evergreen tree native to

Peninsular Malaysia, Thailand, and Indonesia. It has

many vernacular names, for example, ‘kerdas’ or

“keredas” in Malaysia, ‘neing-nok’ in Thailand, and

‘kabau’ or ‘julang-jaling’ in Indonesia (Lim, 2012).

The fruit has a woody pod and contains six to eight

seeds. The seeds are creamy-white, ellipsoid to ovoid,

and laterally flattened, which turn to a shining

reddish-brown color when mature. The seeds have a

strong pungent smell like jering and stink bean but the

odor disappears on cooking (Lim, 2012). The kernels

can be eaten raw after removing the hard husk. The

husk of the seeds has been extensively researched for

various biological activities, including antibacterial,

antidiabetic, anti-uric acid, and antioxidant (Hanafi et

al., 2018; Irawan et al., 2018; Styani et al., 2018;

Riasari et al., 2019). However, the edible kernel has

relatively been less studied. Recently, Riasari and

colleagues (2021) reported that the kernel extract

exerts antihyperglycemic activity in diabetic rats. The

Temuan tribe of aborigines in Peninsular Malaysia

use the seeds for treating diabetes (Ong and Azliza,

2015).

This study evaluated the antibacterial, antifungal,

and antioxidant activities of the kernel extracts of

Archidendron bubalinum seeds. The polyphenol

content of the kernel extracts was also quantified. The

results of this study provide scientific information on

the exploration of this wild edible seed as a healthy

food.

2 METHODOLOGIES

2.1 Sample Processing

Approximately 600 grams of fresh Archidendron

bubalinum seeds were bought from a wet market in

Antibacterial, Antifungal, and Antioxidant Activities and Polyphenol Content of the Edible Seeds of Archidendron bubalinum

Bachok, Kelantan, Malaysia on 29

May 2017. A few

seeds were kept as a specimen voucher with the code

number UTAR/FSC/17/001.

The husks were removed from the seeds after

cleaning with water. The kernels were then cut into

small pieces with a knife and extracted sequentially

using hexane, ethyl acetate, ethanol, and distilled

water. The maceration was performed at 110 rpm and

ambient temperature for 24 h and the process was

repeated two times. The hexane, ethyl acetate, and

ethanol filtrates were concentrated to dryness at 40°C

using a rotary evaporator whereas the water extract

was freeze-dried. All dry extracts were kept at –20°C

pending bioassay. The percentage of yield for each

extract was calculated based on fresh weight.

2.2 Antibacterial Activity

The antibacterial activity of kernel extracts was

evaluated using a colorimetric broth microdilution

method (Chan et al., 2018) with slight modifications.

The Gram-positive bacteria used were Bacillus

cereus ATCC11778 and Staphylococcus aureus

ATCC6538. While the Gram-negative bacteria tested

were Acinetobacter baumannii ATCC19606,

Escherichia coli ATCC35218, Klebsiella

pneumoniae ATCC13883, and Pseudomonas

aeruginosa ATCC27853. All the bacterial species

were obtained from the American Type Culture

Collection (ATCC) and cultured on Mueller-Hinton

agar (MHA). An extract stock solution was prepared

by dissolving 20 mg of extract in 2 mL of a methanol-

water mixture (2:1, v/v). Subsequent dilutions were

performed using Mueller-Hinton broth (MHB) in a

96-well plate to produce eight concentrations (0.02,

0.04, 0.08, 0.16, 0.31, 0.63, 1.25, and 2.50 mg/mL)

for the assay. The bacterial inoculum was prepared at

1 × 10

cfu/mL by adjusting the absorbance value at

625 nm to 0.08-0.10 (equivalent to 0.5 McFarland

turbidity standard) and diluted to 1 × 10

cfu/mL with

MHB. After that, 50 µL of prepared bacterial

inoculum was introduced to the wells containing 50

µL of extract and incubated at 37°C for 24 h. Positive

(chloramphenicol), negative (bacterial inoculum),

blank (MHB), and technical (kernel extracts) controls

were incorporated into each plate. The microbial

growth indicator p-iodonitrotetrazolium chloride (0.4

mg/mL; 20 µL) was pipetted to each well after

incubation. After 30 min of incubation at 37°C, the

wells were observed for the formation of a pink or

purple precipitate, and the extract’s minimum

inhibitory concentration (MIC) was ascertained. For

the determination of the extract’s minimum

bactericidal concentration (MBC), 20 µL of the well’s

content that showed inhibitory activity was spread on

MHA, followed by incubation at 37°C for 24 h. MBC

is the lowest concentration of kernel extract that kills

≥99.9% of bacterial inoculum. The experiment was

conducted in triplicate.

2.3 Antifungal Activity

The antifungal activity of kernel extracts was

assessed using a colorimetric broth microdilution

method (Chan et al., 2018) with slight modifications.

Five species of fungi, comprising three yeasts

(Candida albicans ATCC90028, Candida krusei

ATCC6258, and Candida parapsilosis ATCC22019)

and two filamentous fungi (Aspergillus fumigatus

ATCC204305 and Trichophyton rubrum

ATCC28188), were tested. All the fungal species

were obtained from the ATCC. The three yeasts were

cultured on Sabouraud dextrose agar (SDA). The A.

fumigatus and T. rubrum were maintained on potato

dextrose agar (PDA) and oatmeal agar (OA),

respectively. A serial dilution of the extract stock

solution (10 mg/mL) was performed using Roswell

Park Memorial Institute (RPMI)-1640 medium to

produce a concentration range of 0.02-2.50 mg/mL

for evaluation. For the fungal inoculum preparation,

the absorbances for Candida spp. and A. fumigatus

were adjusted to 0.12-0.15 and 0.09-0.11,

respectively, at 530 nm. The inoculums were then

diluted to 1.5 × 10

cfu/mL with RPMI-1640 medium.

While for T. rubrum, the cell number was enumerated

using a hemocytometer and adjusted to 2 × 10

cfu/mL (CLSI, 2008a; 2008b). The diluted fungal

inoculum (50 µL) was then pipetted into the extract

solution and incubated at 35°C and 48 h for Candida

spp., 35°C and 72 h for A. fumigatus, and 30°C and

96 h for T. rubrum. Two antibiotics, griseofulvin for

T. rubrum and amphotericin B for others, were used

as positive controls. Blank (medium), negative

(fungal inoculum), and technical (kernel extracts)

controls were incorporated into each plate. The p-

iodonitrotetrazolium chloride (20 µL) was pipetted to

each well one day before completion of the

incubation period and the MIC of the extract was

determined after incubation. After that, the spread

plate method using SDA/PDA was used to determine

the minimum fungicidal concentration (MFC) of the

active extracts.

ABS 2022 - The International Conference on Agricultural and Biological Sciences

2.4 Antioxidant Activity

2.4.1 2, 2-Diphenyl-1-picrylhydrazyl

(DPPH) Radical Scavenging Assay

The DPPH radical scavenging assay was performed

using the method of Pavithra and Vadivukkarasi

(2015) with slight modifications. The kernel extract

(4 mg/mL) and vitamin C (0.4 mg/mL), which served

as a positive control, were diluted two-fold serially

with the methanol-water mixture in a 96-well plate to

produce final concentration ranges of 1000-8 µg/mL

and 100-0.8 µg/mL, respectively. One hundred µL of

0.2 mM DPPH radical solution was pipetted to 100

µL of kernel extract/vitamin C solution, and the plate

was maintained in the dark at ambient temperature for

30 min. DPPH radical solution and extract solution

without the addition of DPPH were used as blank and

sample blank, respectively. The absorbance value

was recorded at 517 nm. The DPPH radical

scavenging activity was expressed in percentage and

plotted against the concentration of kernel extract.

The half-maximum inhibitory concentration (IC

) of

the extract was then determined graphically.

2.4.2 Oxygen Radical Absorbance Capacity

(ORAC) Assay

The peroxyl radical scavenging activity of the kernel

extracts was evaluated using the ORAC assay (Heng

et al., 2020). Six concentrations (3.13, 6.25, 12.5, 25,

50, and 100 µg/mL) were prepared for each extract.

Fifty µL of extract and one hundred fifty µL of 0.08

µM fluorescein were pipetted into a black 96-well

plate. Sodium phosphate buffer (75 mM, pH 7.0) was

used as a blank while Trolox solutions at 0.78, 1.56,

3.13, 6.25, and 12.5 µM were used to generate a

calibration curve. Following the addition of 2,2-

azobis (2-methylpropionamidine) dihydrochloride,

the fluorescence intensity of the fluorescein at 485 nm

(λ

) and 520 nm (λ

) was monitored at 37°C every

1.5 min for 1 h. The area under the fluorescence

intensity versus time curve (AUC) was then

calculated. The ORAC value was interpolated from

the plot of the net AUC value versus the concentration

of Trolox. The equation and regression coefficient for

the plot were y = 1.7577x + 1.998 and 0.9932,

respectively. The ORAC value was expressed as

mmol Trolox equivalent/g of extract. The assay was

conducted in three independent experiments.

2.4.3 Ferric-Reducing Antioxidant Power

(FRAP) Assay

The FRAP assay was conducted using the method of

Benzie and Strain (1996) with slight modifications.

The standard curve was constructed using 0.2, 0.4,

0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mM of ferrous sulfate.

Vitamin C at 1 mg/mL served as a positive control

whereas the methanol-water mixture was deployed as

a negative control. A 270 µL of FRAP reagent was

pipetted to 30 µL of extract/standard/controls and

incubated at 37°C. After 4 min of incubation, the

absorbance was read at 593 nm. The FRAP reagent

contained 300 mM acetate buffer (pH 3.6), 20 mM

FeCl

solution, and 10 mM 2,4,6-tri(2-pyridyl)-s-

triazine solution at a ratio of 10:1:1 (v/v/v). The

equation and regression coefficient for the standard

curve were y = 2.7655x + 0.0953 and 0.9990,

respectively. The FRAP value for each extract was

interpolated from the standard curve and expressed as

mmol Fe

equivalent/g of extract.

2.5 Polyphenol Content

2.5.1 Total Phenolic Content (TPC) Assay

The Folin-Ciocalteu method was used to determine

the TPC of each kernel extract in triplicate (Herald et

al., 2012). Eight gallic acid solutions (2.5, 5, 10, 20,

40, 80, 160, and 320 µg/mL) were used to generate a

calibration curve. A 10 mg/mL of extract (25 µL),

deionized water (75 µL), and 50% Folin-Ciocalteu

reagent (25 µL) were mixed in a 96-well plate and

shaken at 70 rpm for 6 min. For the sample blank, the

50% Folin-Ciocalteu reagent was replaced with

deionized water. The methanol-water mixture was

deployed as blank. Following the addition of 100 µL

of 700 mM sodium carbonate, the plate was

maintained in the dark at ambient temperature for 90

min, prior to the absorbance measurement at 765 nm.

The actual absorbance value of sample was obtained

after subtracting its absorbance value from the

absorbance value of the sample blank. The equation

and regression coefficient for the gallic acid

calibration curve were y = 0.0065x – 0.0031 and

0.9977, respectively. The TPC of each kernel extract

was expressed as mg gallic acid equivalent/g of

extract.

2.5.2 Total Flavonoid Content (TFC) Assay

The aluminium chloride method was used to

determine the TFC of each kernel extract in triplicate

(Herald et al., 2012). A 725 mM of sodium nitrite (10

µL), 10 mg/mL of extract (25 µL), deionized water

Antibacterial, Antifungal, and Antioxidant Activities and Polyphenol Content of the Edible Seeds of Archidendron bubalinum

(100 µL), and 750 mM of aluminium chloride (15 µL)

were mixed in a 96-well plate and shaken at 70 rpm

for 6 min. Quercetin (7.8, 15.6, 31.3, 62.5, 125, and

250 µg/mL) was used for constructing a calibration

curve whereas the methanol-water mixture was used

as a blank. The aluminium chloride in the mixture

was substituted with deionized water for the sample

blank. Each well was then added with 50 µL of 1 M

sodium hydroxide, followed by 50 µL of deionized

water. The plate was maintained in the dark at

ambient temperature for 60 min before the

absorbance was read at 420 nm. The equation and

regression coefficient for the quercetin calibration

curve were y = 0.0014x + 0.0068 and 0.9979,

respectively. The TFC of each extract was expressed

as mg quercetin equivalent/g of extract.

2.6 Data Analysis

The results obtained from the ORAC, FRAP, TPC,

and TFC assays were examined for statistical

significance using one-way analysis of variance

(ANOVA). Subsequently, the significant difference

between the kernel extracts within an assay was

determined using Duncan’s multiple range test.

Correlation analysis between ORAC or FRAP and

TPC or TFC was examined using the Pearson

correlation test (Chan, 2003). The significance level

was set at p<0.05. All statistical analyses were

performed using the IBM SPSS Statistics for

Windows Version 23.0 software.

3 RESULTS

3.1 Yield of Extraction

Four extracts were obtained from the extraction of the

kernel of A. bubalinum seeds. The percentages of the

yield of hexane, ethyl acetate, ethanol, and water

extracts were 0.01%, 0.05%, 5.33%, and 3.58%

(w/w), respectively. The total yield of extraction was

8.97% (w/w).

3.2 Antibacterial Activity

The bacteriostatic and bactericidal effects of an

extract are designated by its MIC and MBC values,

respectively. All the kernel extracts showed a

bacteriostatic effect against the six species of

bacteria. The MIC values ranged from 0.31 to 2.50

mg/mL (Table 1). However, the extracts required

higher concentrations to exert their bactericidal

effects. Moreover, none of the extracts exerted the

bactericidal effect against E. coli (Table 1),

suggesting an extract concentration higher than 2.50

mg/mL might be needed to kill this species. All the

bacterial species were susceptible to chloramphenicol

(positive control) with MIC values of 4 µg/mL

against the Gram-positive bacteria and 2-64 µg/mL

against the Gram-negative bacteria.

3.3 Antifungal Activity

All the kernel extracts possessed antifungal activity

against the three species of yeasts and two species of

filamentous fungi (Table 2). Their MIC values were

the same as the MFC values, indicating the extracts

possessed direct fungicidal effects on the fungi

evaluated. The fungicidal effects were slightly

stronger on the yeasts (MFC: 0.31-1.25 mg/mL) than

that of the filamentous fungi (MFC: 0.63-2.50

mg/mL). All the fungi were susceptible to the positive

controls, amphotericin B and griseofulvin, with MIC

values of 1-2 and 0.50 µg/mL, respectively.

Table 1: Antibacterial activities of the kernel extracts of Archidendron bubalinum seeds.

Extract

Gram-

ositive bacteria Gram-negative bacteria

Bacillus

cereus

ATCC11778

Staphylococ

cus aureus

ATCC6538

Acinetobacte

r baumannii

ATCC19606

Escherichia

coli

ATCC35218

Klebsiella

pneumoniae

ATCC13883

Pseudomonas

aeruginosa

ATCC27853

Minimum inhibitory concentration (mg/mL)

Hexane 1.25 1.25 0.31 0.63 0.63 0.63

Eth

l acetate 1.25 1.25 0.31 0.63 1.25 0.63

Ethanol 2.50 1.25 0.31 1.25 1.25 0.63

Wate

2.50 2.50 0.31 1.25 1.25 0.63

Minimum bactericidal concentration (mg/mL)

Hexane 1.25 2.50 2.50 >2.50 0.63 2.50

Eth

l acetate 1.25 2.50 2.50 >2.50 1.25 2.50

Ethanol 2.50 2.50 2.50 >2.50 1.25 2.50

Wate

2.50 2.50 2.50 >2.50 2.50 2.50

Notes: The concentrations are shown in mean values of triplicate.

ABS 2022 - The International Conference on Agricultural and Biological Sciences

3.4 Antioxidant Activity

Two radical scavenging activity assays (DPPH and

ORAC) and an ion reducing activity assay (FRAP)

were deployed to assess the antioxidant activities of

the kernel extracts. Out of the four extracts, only ethyl

acetate extract was able to reduce >50% of DPPH

radicals when the concentration exceeded 0.5 mg/mL

(Figure 1), and the IC

value for this extract was 0.41

± 0.04 mg/mL, much higher than the positive control,

vitamin C (IC

= 2.51 ± 0.12 µg/mL). Besides that,

the highest ORAC value was exhibited by the ethyl

acetate extract (300.13 ± 13.15 mmol Trolox

equivalent/g of extract), followed by the ethanol

extract. Meanwhile, the ORAC values for the hexane

and water extracts were the lowest and similar

(p>0.05). As shown in the FRAP assay (Table 3), the

kernel extracts of A. bubalinum seeds also possessed

the ability to reduce the ion Fe

to Fe

. The ethyl

acetate extract showed the highest FRAP value

(341.36 ± 20.23 mmol Fe

equivalent/g of extract),

followed by the ethanol, water, and hexane extracts.

3.5 Polyphenol Content

All four kernel extracts of A. bubalinum seeds

contained polyphenols. The TPC of the extracts

ranged from the lowest 3.22 ± 0.35 mg gallic acid

equivalent/g of extract for the hexane extract to the

highest 20.19 ± 0.23 mg gallic acid equivalent/g of

extract for the ethyl acetate extract (Table 3). While

for TFC, the ethyl acetate extract also recorded the

highest amount with a value of 4.42 ± 0.08 mg

quercetin equivalent/g of extract. Although the

ethanol extract had the lowest TFC, it was not

significantly different (p>0.05) from that of the

hexane and water extracts (Table 3).

Figure 1: DPPH radical scavenging activity of the kernel

extracts of Archidendron bubalinum seeds. Each value is

shown in mean ± standard deviation of triplicate.

Table 2: Antifungal activities of the kernel extracts of Archidendron bubalinum seeds.

Extract

Yeasts Filamentous Fungi

Candida

albicans

ATCC90028

Candida

krusei

ATCC6258

Candida

parapsilosis

ATCC22019

Aspergillus

fumigatus

ATCC204305

Trichophyton

rubrum

ATCC28188

Minimum inhibitory concentration (mg/mL)

Hexane 0.63 0.63 0.31 0.63 1.25

Ethyl acetate 1.25 0.63 0.63 1.25 2.50

Ethanol 1.25 1.25 0.63 1.25 2.50

Water 1.25 1.25 0.63 2.50 2.50

Minimum fungicidal concentration (mg/mL)

Hexane 0.63 0.63 0.31 0.63 1.25

Ethyl acetate 1.25 0.63 0.63 1.25 2.50

Ethanol 1.25 1.25 0.63 1.25 2.50

Water 1.25 1.25 0.63 2.50 2.50

Notes: The concentrations are shown in mean values of triplicate.

Antibacterial, Antifungal, and Antioxidant Activities and Polyphenol Content of the Edible Seeds of Archidendron bubalinum

Table 3: Antioxidant activities and polyphenol content of the kernel extracts of Archidendron bubalinum seeds.

Extract

DPPH radical

scavenging

activity, half-

maximum

inhibitory

concentration

(

/mL

)

Oxygen radical

absorbance

capacity (mmol

Trolox

equivalent/g of

extract)

Ferric-reducing

antioxidant power

(mmol Fe

equivalent/g of

extract)

Total phenolic

content (mg gallic

acid equivalent/g

of extract)

Total flavonoid

content (mg

quercetin

equivalent/g of

extract)

Hexane Nil 81.63 ± 2.08

25.93 ± 4.70

3.22 ± 0.35

1.25 ± 0.23

Ethyl

acetate

0.41 ± 0.04 300.13 ± 13.15

341.36 ± 20.23

20.19 ± 0.23

4.42 ± 0.08

Ethanol Nil 118.90 ± 5.41

118.13 ± 10.18

8.73 ± 0.10

0.97 ± 0.11

Water Nil 88.62 ± 0.26

75.22 ± 3.47

6.79 ± 0.11

1.16 ± 0.12

Notes: Each value is shown in mean ± standard deviation of triplicate. Values with different alphabetical superscripts

denote significant differences (p<0.05) among the extracts by one-way ANOVA test.

3.6 Correlations between Antioxidant

Activities and Polyphenol Content

The correlation analyses unveiled significant strong

positive correlations between ORAC value and TPC

(r = 0.979; p<0.001) or TFC (r = 0.971; p<0.001)

(Figure 2). Similarly, strong positive correlations

were noted between FRAP value and TPC (r = 0.995;

p<0.001) or TFC (r = 0.937; p<0.001).

Figure 2: Association analysis between total phenolic

content (TPC) or total flavonoid content (TFC) and (a)

oxygen radical absorbance capacity (ORAC) and (b) ferric-

reducing antioxidant power (FRAP) of the kernel extracts

of Archidendron bubalinum seeds using the Pearson

correlation test.

4 DISCUSSIONS

The results of this study indicated that the edible

seeds of A. bubalinum possessed antibacterial,

antifungal, and antioxidant activities. The ability of

all the kernel extracts to exert antibacterial and

antifungal activities implied that different classes of

phytochemicals were present in the kernels and

contributed to the activities. During the sequential

solvent extraction, phytochemicals in the kernels are

segregated according to the polarity of the solvent

used. Non-polar solvents such as hexane and

chloroform commonly remove alkaloids, fatty acids,

sterols, terpenoids, etc. from plants. Phytochemicals

such as anthraquinones, flavones, polyphenols,

saponins, tannins, and terpenoids are obtained by

ethyl acetate and ethanol, which are more polar

solvents. The most polar solvent water could yield

secondary metabolites like polypeptides and lectins

(Heng et al., 2020).

The polyphenols quantified in all the kernel

extracts may account, at least in part, for the

antibacterial and antifungal activities. Polyphenols

constitute one of the biggest groups of

phytochemicals with approximately 8000 different

structures. They are classified into flavonoids,

stilbenes, phenolic acids, coumarins, anthraquinones,

tannins, and xanthones (Forni et al., 2019).

Polyphenols from plants have been reported to have

inhibitory or killing effects against bacteria and fungi

(Kumar et al., 2021; Manso et al., 2022). Polyphenols

exert antimicrobial activities via various mechanisms,

such as damaging fungal cell wall or bacterial

lipopolysaccharide layer, formation of pores in cell

ABS 2022 - The International Conference on Agricultural and Biological Sciences

membrane leading to leakage of cytoplasmic content,

inhibition of metabolic enzymes, ergosterol

biosynthesis, or efflux pumps, repression of genes,

disruption of ionic imbalance, induction of cell death,

and inhibition of biofilm formation (Seleem et al.,

2017; Simonetti et al., 2020; Kumar et al., 2021). The

kernel extracts exhibited a direct killing effect on the

fungi evaluated, suggesting the antifungal

components of A. bubalinum seeds may mainly

disrupt the integrity of the cell wall and cause

cytolysis of the fungal cells.

Riasari et al. (2019) reported the DPPH radical

scavenging activity of the ethanol extracts of the

seeds of A. bubalinum from Lampung and South

Sumatra (Indonesia) with half-maximum inhibitory

concentrations of 163 and 446 µg/mL, respectively.

In contrast, the ethanol kernel extract of A. bubalinum

seeds used in this study exerted weak DPPH radical

scavenging activity (<50% inhibition at 1 mg/mL).

The differences could be attributed to agro-

geographical reasons and/or extraction techniques

used. Ghasemzadeh et al. (2018) studied the stink

beans collected from different regions of Peninsular

Malaysia and found that the sample collected from

Perak had the highest DPPH radical scavenging and

FRAP activities than the ones collected from Negeri

Sembilan and Johor. Another study found that 18

metabolites from black bean and 11 metabolites from

soybean were different significantly when their

metabolite profiles were compared using two

different extraction techniques (Maria John et al.,

2018).

All four kernel extracts of A. bubalinum seeds

exhibited antioxidant activities via the ORAC and

FRAP assays. These antioxidant activities were likely

contributed by polyphenols including flavonoids in

the kernels due to the significant results from the

correlation analyses. Significant strong positive

correlations (r > 0.80) between ORAC values and

polyphenol content (TPC and TFC) have also been

documented for adlay seeds (Xu et al., 2017), seeds

and fruit of Phoenix dactylifera (Djaoudene et al.,

2021), and fruit of Eleiodoxa conferta (Go et al.,

2021). Similarly, strong positive correlations between

FRAP and TPC as well as between FRAP and TFC

have been reported for stink beans (Ghasemzadeh et

al., 2018), kiwifruit (Wang et al., 2018), and peels and

seeds of pomegranates (Sabraoui et al., 2020). The

existence of at least one phenyl ring in the molecular

structure allows polyphenols to have free radical

scavenging, singlet oxygen quenching, and metal ion

reducing or chelating properties (Ullah et al., 2020).

As the ethyl acetate extract was the most active

among all kernel extracts, further analysis using

gas/liquid chromatography-mass spectrometry could

shed some light on the identity of antioxidants in the

extract.

5 CONCLUSIONS

This study suggests that the edible seeds of A.

bubalinum could be promoted as healthy food due to

its health-promoting activities. All four kernel

extracts (hexane, ethyl acetate, ethanol, and water)

from the seeds had bactericidal activities against

Gram-positive and Gram-negative bacteria and

fungicidal activities against yeasts and filamentous

fungi. The antimicrobial activities of the seeds are

likely to be contributed by different classes of

phytochemicals. All the kernel extracts also

possessed antioxidant activities, as revealed by the

ORAC and FRAP assays. Among the extracts, ethyl

acetate extract showed the strongest antioxidant

activity by having the highest ORAC and FRAP

values. The antioxidant activities were attributable

mainly to the polyphenols present in the kernels, due

to the significant strong positive correlations between

antioxidant activities and polyphenol content. Further

work is essential to identify the bioactive components

and elucidate the mechanisms of action. More

research is needed if A. bubalinum is to be cultivated

sustainably as a crop in Malaysia, as currently the

fruit is mainly collected from forests for sales in wet

markets.

ACKNOWLEDGEMENTS

The authors thank Dr. Sugumaran Manickam

(Faculty of Science, University of Malaya, Malaysia)

for the identification of plant species and Lee Vwen

Tan for sourcing the plant material. The work is

supported by the UTAR Research Publication

Scheme (6251/S02).

REFERENCES

Ahmad, M. and Khan, A. U. (2019). Global economic

impact of antibiotic resistance: A review. Journal of

Global Antimicrobial Resistance, 19:313-316.

Bacchetti, T., Turco, I., Urbano, A., Morresi, C., and

Ferretti, G. (2019). Relationship of fruit and vegetable

intake to dietary antioxidant capacity and markers of

oxidative stress: A sex-related study. Nutrition, 61:164-

172.

Antibacterial, Antifungal, and Antioxidant Activities and Polyphenol Content of the Edible Seeds of Archidendron bubalinum

Benzie, I. F. F. and Strain, J. J. (1996). The ferric reducing

ability of plasma (FRAP) as a measure of “antioxidant

power”: The FRAP assay. Analytical Biochemistry,

239:70-76.

Bongomin, F., Gago, S., Oladele, R. O., and Denning, D.

W. (2017). Global and multi-national prevalence of

fungal diseases–estimate precision. Journal of Fungi,

3(4):57.

Chan, Y. H. (2003). Biostatistics 104: Correlational

analysis. Singapore Medical Journal, 44(12):614-619.

Chan, Y. S., Ong, C. W., Chuah, B. L., Khoo, K. S., Chye,

F. Y., and Sit, N. W. (2018). Antimicrobial, antiviral

and cytotoxic activities of selected marine organisms

collected from the coastal areas of Malaysia. Journal of

Marine Science and Technology, 26(1):128-136.

Chokshi, A., Sifri, Z., Cennimo, D., and Horng, H. (2019).

Global contributors to antibiotic resistance. Journal of

Global Infectious Diseases, 11(1):36-42.

Christaki, E., Marcou, M., and Tofarides, A. (2020).

Antimicrobial resistance in bacteria: Mechanisms,

evolution, and persistence. Journal of Molecular

Evolution, 88(1):26-40.

Clinical and Laboratory Standards Institute (CLSI).

(2008a). Reference Method for Broth Dilution

Antifungal Susceptibility Testing of Yeasts; Approved

Standard, CLSI Document M27-A3, 3rd ed. Clinical

and Laboratory Standards Institute.

Clinical and Laboratory Standards Institute (CLSI).

(2008b). Reference Method for Broth Dilution

Antifungal Susceptibility Testing of Filamentous Fungi;

Approved Standard, CLSI Document M38-A2, 2nd ed.

Clinical and Laboratory Standards Institute.

Djaoudene, O., Mansinhos, I., Gonçalves, S., Jara-Palacios,

M. J., Bachir Bey, M., and Romano, A. (2021).

Phenolic profile, antioxidant activity and enzyme

inhibitory capacities of fruit and seed extracts from

different Algerian cultivars of date (Phoenix dactylifera

L.) were affected by in vitro simulated gastrointestinal

digestion. South African Journal of Botany, 137:133-

148.

Fisher, J. F. and Mobashery, S. (2020). Constructing and

deconstructing the bacterial cell wall. Protein Science,

29(3):629-646.

Forni, C., Facchiano, F., Bartoli, M., Pieretti, S., Facchiano,

A., D'Arcangelo, D., Norelli, S., Valle, G., Nisini, R.,

Beninati, S., Tabolacci, C., and Jadeja, R. N. (2019).

Beneficial role of phytochemicals on oxidative stress

and age-related diseases. BioMed Research

International, 2019:8748253.

GBD 2019 Diseases and Injuries Collaborators. (2020).

Global burden of 369 diseases and injuries in 204

countries and territories, 1990–2019: A systematic

analysis for the Global Burden of Disease Study 2019.

The Lancet, 396(10258):1204-1222.

Ghasemzadeh, A., Jaafar, H. Z. E., Bukhori, M. F. M.,

Rahmat, M. H., and Rahmat, A. (2018). Assessment

and comparison of phytochemical constituents and

biological activities of bitter bean (Parkia speciosa

Hassk.) collected from different locations in Malaysia.

Chemistry Central Journal

, 12(1):12.

Global Action Fund for Fungal Infections (GAFFI). (2022).

Fungal disease frequency. Retrieved from

https://gaffi.org/why/fungal-disease-frequency/.

Go, H. C., Low, J. A., Khoo, K. S., and Sit, N. W. (2021).

Nutritional composition, biological activities, and

cytotoxicity of the underutilized fruit of Eleiodoxa

conferta. Journal of Food Measurement and

Characterization, 15(5):3962-3972.

Hanafi, H., Irawan, C., Rochaeni, H., Sulistiawaty, L.,

Roziafanto, A. N., and Supriyono. (2018).

Phytochemical screening, LC-MS studies and

antidiabetic potential of methanol extracts of seed shells

of Archidendron bubalinum (Jack) I.C. Nielson (Julang

Jaling) from Lampung, Indonesia. Pharmacognosy

Journal, 10(6): S77-S82.

Heng, Y. W., Ban, J. J., Khoo, K. S., and Sit, N. W. (2020).

Biological activities and phytochemical content of the

rhizome hairs of Cibotium barometz (Cibotiaceae).

Industrial Crops and Products, 153:112612.

Herald, T. J., Gadgil, P., and Tilley, M. (2012). High-

throughput micro plate assays for screening flavonoid

content and DPPH-scavenging activity in sorghum bran

and flour. Journal of the Science of Food and

Agriculture, 92(11):2326-2331.

Irawan, C., Foliatini, Hanafi, Sulistiawaty, L., and

Sukiman, M. (2018). Volatile compound analysis using

GC-MS, phytochemical screening and antioxidant

activities of the husk of “Julang-jaling” (Archidendron

bubalinum (Jack) I.C Nielsen) from Lampung,

Indonesia. Pharmacognosy Journal, 10(1):92-98.

Ito, N., Fukushima, S., and Tsuda, H. (1985).

Carcinogenicity and modification of the carcinogenic

response by BHA, BHT, and other antioxidants.

Critical Reviews in Toxicology, 15(2):109-150.

Kumar, H., Bhardwaj, K., Cruz-Martins, N., Nepovimova,

E., Oleksak, P., Dhanjal, D. S., Bhardwaj, S., Singh, R.,

Chopra, C., Verma, R., Chauhan, P. P., Kumar, D., and

Kuča, K. (2021). Applications of fruit polyphenols and

their functionalized nanoparticles against foodborne

bacteria: A mini review. Molecules, 26(11):3447.

Liguori, I., Russo, G., Curcio, F., Bulli, G., Aran, L., Della-

Morte, D., Gargiulo, G., Testa, G., Cacciatore, F.,

Bonaduce, D., and Abete, P. (2018). Oxidative stress,

aging, and diseases. Clinical Interventions in Aging,

13:757-772.

Lim, T. K. (2012). Edible Medicinal and Non-Medicinal

Plants: Volume 2, Fruits. Springer Science+Business

Media B.V.

Maria John, K. M., Harnly, J., and Luthria, D. (2018).

Influence of direct and sequential extraction

methodology on metabolic profiling. Journal of

Chromatography B: Analytical Technologies in the

Biomedical and Life Sciences, 1073:34-42.

Manso, T., Lores, M., and de Miguel, T. (2022).

Antimicrobial activity of polyphenols and natural

polyphenolic extracts on clinical isolates. Antibiotics,

11(1):46.

Milow, P., Malek, S. B., Edo, J., and Ong, H. C. (2014).

Malaysian species of plants with edible fruits or seeds

ABS 2022 - The International Conference on Agricultural and Biological Sciences

and their valuation. International Journal of Fruit

Science, 14(1):1-27.

Ong, H. C. and Azliza, M. A. (2015). Medicinal plants for

diabetes by the Orang Asli in Selangor, Malaysia.

Studies on Ethno-Medicine, 9(1):77-84.

Pavithra, K. and Vadivukkarasi, S. (2015). Evaluation of

free radical scavenging activity of various extracts of

leaves from Kedrostis foetidissima (Jacq.) Cogn. Food

Science and Human Wellness, 4(1):42-46.

Pisoschi, A. M., Pop, A., Iordache, F., Stanca, L., Predoi,

G., and Serban, A. I. (2021). Oxidative stress mitigation

by antioxidants–An overview on their chemistry and

influences on health status. European Journal of

Medicinal Chemistry, 209:112891.

Riasari, H., Fauzi, N. I., Anggadiredja, K., Hartati, R., and

Sukrasno. (2021). In vivo antidiabetic screening of

kabau (Archidendron bubalinum (Jack) I.C Nielsen)

seeds. International Journal of Applied Pharmaceutics,

13(S4):228-234.

Riasari, H., Fitriansyah, S. N., Hartati, R., Anggadiredja,

K., and Sukrasno. (2019). Comparison of extraction

methods, antioxidant activities, total phenol in seeds

and seed shells of Kabau (Archidendron bubalinum

(Jack) I.C. Nielsen) from Lampung and South Sumatra.

Pharmacognosy Journal, 11(6):1278-1284.

Revie, N. M., Iyer, K. R., Robbins, N., and Cowen, L. E.

(2018). Antifungal drug resistance: Evolution,

mechanisms and impact. Current Opinion in

Microbiology, 45:70-76.

Rosado-Pérez, J., Aguiñiga-Sánchez, I., Arista-Ugalde, T.

L., Santiago-Osorio, E., and Mendoza-Núñez, V. M.

(2018). The biological significance of oxidative stress,

effects of fruits as natural edible antioxidants. Current

Pharmaceutical Design, 24(40):4807-4824.

Sabraoui, T., Khider, T., Nasser, B., Eddoha, R., Moujahid,

A., Benbachir, M., and Essamadi, A. (2020).

Determination of punicalagins content, metal chelating,

and antioxidant properties of edible pomegranate

(Punica granatum L) peels and seeds grown in

Morocco. International Journal of Food Science,

2020:8885889.

Seleem, D., Pardi, V., and Murata, R. M. (2017). Review of

flavonoids: A diverse group of natural compounds with

anti-Candida albicans activity in vitro. Archives of

Oral Biology, 76:76-83.

Shrestha, P., Cooper, B. S., Coast, J., Oppong, R., Do Thi

Thuy, N., Phodha, T., Celhay, O., Guerin, P. J.,

Wertheim, H., and Lubell, Y. (2018). Enumerating the

economic cost of antimicrobial resistance per antibiotic

consumed to inform the evaluation of interventions

affecting their use. Antimicrobial Resistance and

Infection Control, 7(1):98.

Simonetti, G., Brasili, E., and Pasqua, G. (2020).

Antifungal activity of phenolic and polyphenolic

compounds from different matrices of Vitis vinifera L.

against human pathogens. Molecules

, 25(16):3748.

Styani, E., Hanafi, Sulistiswaty, L., Irawan, C., and Imalia.

(2018). Liquid chromatograph-mass spectrophotometer

and anti-uric acid potential studies of ethyl acetate

extract of Archidendron bubalinum (Jack) I.C. Nielsen

fruit seed shell. In Proceedings of the international

conference on science and technology, pages 293-297.

Atlantis Press.

Sundararajan, A., Rane, H. S., Ramaraj, T., Sena, J.,

Howell, A. B., Bernardo, S. M., Schilkey, F. D., and

Lee, S. A. (2018). Cranberry-derived

proanthocyanidins induce a differential transcriptomic

response within Candida albicans urinary biofilms.

PLoS One, 13(8): e0201969.

Ullah, A., Munir, S., Badshah, S. L., Khan, N., Ghani, L.,

Poulson, B. G., Emwas, A. H., and Jaremko, M. (2020).

Important flavonoids and their role as a therapeutic

agent. Molecules, 25(22):5243.

Urban, K., Chu, S., Scheufele, C., Giesey, R. L., Mehrmal,

S., Uppal, P., and Delost, G. R. (2021). The global,

regional, and national burden of fungal skin diseases in

195 countries and territories: A cross-sectional analysis

from the Global Burden of Disease Study 2017. JAAD

International, 2:22-27.

Vostalova, J., Vidlar, A., Simanek, V., Galandakova, A.,

Kosina, P., Vacek, J., Vrbkova, J., Zimmermann, B. F.,

Ulrichova, J., and Student, V. (2015). Are high

proanthocyanidins key to cranberry efficacy in the

prevention of recurrent urinary tract infection?

Phytotherapy Research, 29(10):1559-1567.

Wang, X., Ouyang, Y. Y., Liu, J., and Zhao, G. (2014).

Flavonoid intake and risk of CVD: A systematic review

and meta-analysis of prospective cohort studies. British

Journal of Nutrition, 111(1):1-11.

Wang, Y., Zhao, C. L., Li, J. Y., Liang, Y. J., Yang, R. Q.,

Liu, J. Y., Ma, Z., and Wu, L. (2018). Evaluation of

biochemical components and antioxidant capacity of

different kiwifruit (Actinidia spp.) genotypes grown in

China. Biotechnology and Biotechnological

Equipment, 32(3):558-565.

Xu, L., Wang, P., Ali, B., Yang, N., Chen, Y., Wu, F., and

Xu, X. (2017). Changes of the phenolic compounds and

antioxidant activities in germinated adlay seeds.

Journal of the Science of Food and Agriculture,

97(12):4227-4234.

Antibacterial, Antifungal, and Antioxidant Activities and Polyphenol Content of the Edible Seeds of Archidendron bubalinum