Metabolites 13 00281 v2

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Article
Breynia cernua: Chemical Profiling of Volatile Compounds in
the Stem Extract and Its Antioxidant, Antibacterial,
Antiplasmodial and Anticancer Activity In Vitro and In Silico
Hesti Lina Wiraswati 1,2,3,4, * , Nisa Fauziah 1,2,3 , Gita Widya Pradini 1,3 , Dikdik Kurnia 5 , Reza Abdul Kodir 6 ,
Afiat Berbudi 1,2,3 , Annisa Retno Arimdayu 4 , Amila Laelalugina 4 , Supandi 7,8 and Ilma Fauziah Ma’ruf 4,9
1 Department of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran,

Sumedang 45363, Indonesia
2 Advance Biomedical Laboratory, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, Indonesia
3 Infection Working Group, Faculty of Medicine, Universitas Padjadjaran, Sumedang 45363, Indonesia
4 Oncology and Stem Cell Working Group, Faculty of Medicine, Universitas Padjadjaran,
Bandung 40161, Indonesia
5 Departement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,
Sumedang 45363, Indonesia
6 Pharmacy Study Program, Faculty of Mathematics and Natural Sciences, Universitas Islam Bandung,
Bandung 40116, Indonesia
7 PT. Borneo Indobara, Central Jakarta 10350, Indonesia
8 Department of Mining Engineering, Faculty of Technology Mineral, Institut Teknologi Nasional Yogyakarta,
Yogyakarta 55281, Indonesia
9 Biochemistry Research Group, Department of Chemistry, Faculty of Mathematics and Natural Sciences,
Institut Teknologi Bandung, Bandung 40132, Indonesia
* Correspondence: hesti.lina@unpad.ac.id
Citation: Wiraswati, H.L.; Fauziah,
N.; Pradini, G.W.; Kurnia, D.; Kodir, Abstract: Breynia cernua has been used as an alternative medicine for wounds, smallpox, cervical
R.A.; Berbudi, A.; Arimdayu, A.R.; cancer, and breast cancer. This plant is a potential source of new plant-derived drugs to cure numerous
Laelalugina, A.; Supandi; Ma’ruf, I.F.
diseases for its multiple therapeutic functions. An in vitro study revealed that the methanol extract of
Breynia cernua: Chemical Profiling of
B. cernua (stem) exhibits antioxidant activity according to DPPH and SOD methods, with IC50 values
Volatile Compounds in the Stem
of 33 and 8.13 ppm, respectively. The extract also exerts antibacterial activity against Staphylococcus
Extract and Its Antioxidant,
Antibacterial, Antiplasmodial and
aureus with minimum bactericidal concentration of 1875 ppm. Further analysis revealed that the
Anticancer Activity In Vitro and In extract with a concentration of 1–2 ppm protects erythrocytes from the ring formation stage of
Silico. Metabolites 2023, 13, 281. Plasmodium falciparum, while the extract with a concentration of 1600 ppm induced apoptosis in
https://doi.org/10.3390/ the MCF-7 breast cancer cell line. GC–MS analysis showed 45 bioactive compounds consisting
metabo13020281 of cyclic, alkyl halide, organosulfur, and organoarsenic compounds. Virtual screening via a blind
docking approach was conducted to analyze the binding affinity of each metabolite against various
Academic Editors: Sameh S. Elhady,
Enas E. Eltamany and Timothy
target proteins. The results unveiled that two compounds, namely, N-[β-hydroxy-β-[4-[1-adamantyl-
O’Toole 6,8-dichloro]quinolyl]ethyl]piperidine and 1,3-phenylene, bis(3-phenylpropenoate), demonstrated
the best binding score toward four tested proteins with a binding affinity varying from −8.3 to
Received: 22 January 2023
−10.8 kcal/mol. Site-specific docking analysis showed that the two compounds showed similar
Revised: 11 February 2023
binding energy with native ligands. This finding indicated that the two phenolic compounds could
Accepted: 13 February 2023
be novel antioxidant, antibacterial, antiplasmodial, and anticancer drugs. A thorough analysis by
Published: 15 February 2023
monitoring drug likeness and pharmacokinetics revealed that almost all the identified compounds can
be considered as drugs, and they have good solubility, oral bioavailability, and synthetic accessibility.
Altogether, the in vitro and in silico analysis suggested that the extract of B. cernua (stem) contains
Copyright: © 2023 by the authors. various compounds that might be correlated with its bioactivities.
Licensee MDPI, Basel, Switzerland.
This article is an open access article Keywords: B. cernua (stem) methanol extract; metabolite profiling; antioxidant; antibacterial;
distributed under the terms and antiplasmodial; anticancer; molecular docking; drug likeness; pharmacokinetics
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Metabolites 2023, 13, 281. https://doi.org/10.3390/metabo13020281 https://www.mdpi.com/journal/metabolites

Metabolites 2023, 13, 281 2 of 28
1. Introduction
From a historical point of view, medicinal plants have been proven to have therapeutic
properties to cure various diseases, and they still represent an important source of novel
drugs today [1,2]. More than 50% of drugs in modern therapeutics are represented by
plant-derived drugs, either in their natural or in their derivative form [3,4]. For exam-
ple, successful plant-derived drugs are aspirin from Filipendula ulmaria (L.) Maxim and
artemisinin from Artemisia annua L. [5,6]. Indonesia is a country with high plant biodiver-
sity and has very diverse plant resources (approximately 30,000–40,000 plant species), 20%
of which are either wild or cultivated medicinal plants [7].
On the other hand, metabolite profiling and the exploration of medicinal plants in
Indonesia are still rarely conducted. For those reasons, the metabolite profile of uninvesti-
gated plants in Indonesia is interesting to explore since they are a promising source of new
plant-derived drugs. Therefore, this research aimed to unveil the metabolite component of
one herbaceous plant as a reservoir of a particular bioactive compound that can potentially
be used as a drug in the future.
Various Breynia species have been traditionally applied as medicine for numerous
diseases [8]. One such species, Breynia cernua (local name: Katuk Hutan), has been used
in Amuntai, Hulu Sungai Utara, and Kalimantan Selatan as herbal medicine for smallpox
and wounds [9], as well as in Papua as a traditional medicine for cervical and breast
cancer [10]. B. cernua has the following anatomical characteristics: semilunar vascular
bundle, paracyitic and anomocytic stomata, irregular shape of the lower epidermis with
a wavy anticlinal wall, polygonal shape of the upper epidermis with a straight anticlinal
wall, single unicellular trichome on the lower epidermis, one-layer upper epidermis with a
regularly shaped cell, one-layer lower epidermis with a papillated cell, one-layer elongated
palisade tissue, loose arrangement of the spongy tissue, druse calcium oxalate on vascular
bundle parenchyma, and hypodermis in the area near leaf bone [11]. It was found that the
ethanol extract of B. cernua has cytotoxic activity according to the brine shrimp lethality
test and MTT assay against MCF-7 [10]. The ethyl acetate or butanol extract of B. cernua
(stem hardwood) and methanol extract of B. cernua (stem bark) exhibited broad-spectrum
antibacterial activities against Micrococcus luteus, Micrococcus roseus, Bacillus coagulans,
Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Citrobacter freundii, Lactobacillus casei,
Staphylococcus albus, Staphylococcus aureus, Staphylococcus epidermidis, Agrobacterium tume-
faciens, Streptococcus faecalis, Streptococcus pneumoniae, Klebsiella pneumoniae, Eschericia coli,
Proteus vulgaris, Proteus mirabili, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Enterobacter
aerogenes, Salmonella Typhi, Salmonella Typhimurium, and Serratia marcescens. Furthermore,
the butanol or methanol extract of B. cernua (root bark) demonstrated antifungal activities
against Aspergillus niger, Aspergillus versicolour, Aspergillus vitis, Candida albican, Candida trop-
icalis, Cladosporium cladosporides, Penicillium notatum, Trichophyton mentagrophyte, Tricophyton
rubrum, and Tricophyton tonsurans [12]. The ethanol extract of B. cernua also was found to
have antiparkinsonism activity in vitro and in vivo [13]. Considering its multiple in vivo
and in vitro bioactivity, B. cernua is a promising source of novel plant-derived drugs. There-
fore, identification of its chemical composition and activity prediction of each metabolite
represent interesting research pathways.
Traditional medicinal plant research has gradually risen worldwide in recent years,
owing to the natural sources and variety of such plants, which allow them to complement
current pharmaceutical treatments [14,15]. The bioactivities of medicinal plants are corre-
lated with the phytochemicals contained in them such as alkaloids, flavonoids, saponins,
terpenoids, tannins, and quinonens [16]. Metabolite profiling through GC–MS using the
silyl derivatization method has been widely applied to identify the composition of plant
extracts [17,18]. In addition, bioinformatics tools aid drug discovery by using all primary
data gathered from in vivo and in vitro studies in a short time and at a lower cost. In silico
molecular docking is one of the most effective ways to find novel ligands for proteins with
known structures, thus playing an essential role in structure-based drug development [19].
A preliminary study (GC–MS) showed that the stem extract of B. cernua contained more
Metabolites 2023, 13, 281 3 of 28
compounds than the leaf extract. Moreover, the stem extract exhibited better antioxidant
and bactericidal activity than the leaf extract (manuscript in preparation). Therefore, in this
report, we performed metabolite profiling, characterized the in vitro antioxidant, antibacte-
rial, antiplasmodial, and anticancer activity, and conducted virtual drug screening through
molecular docking, drug likeness, and pharmacokinetic analyses of the methanol extract of
B. cernua (stem).
2. Materials and Methods

2.1. Plant Material and Preparation of the Extracts
Breynia cernua was collected from a coal mining reclamation area at Site Kusan-
Girimulya of PT Borneo Indobara. The plant material’s taxonomic identification was
validated by reference books and research articles [20]. To obtain the extract, 200 g of
B. cernua stems were cut and oven-dried (50 ◦ C, 48 h) before being macerated in 100 mL of
methanol (24 h, room temperature). The solvent was removed by oven-drying (70 ◦ C, 4 h),
and the extract was solubilized using DMSO (0.5% v/v).
2.2. Gas Chromatography/Mass Spectrometry (GC–MS) Method

GC–MS was used to identify compounds in the methanol extract of B. cernua (stem).
Sample derivatization was conducted using the trimethyl silyl derivatization (TMS) method.
Briefly, 20 µL of extract was dried using an oven (80 ◦ C, 20 min). Immediately after, the
sample was solubilized using 80 µL of methoxyamine hydrochloride solution in pyridine
(2 mg/100 mL), mixed by vortexing for 1 min, and incubated for 90 min at 30 ◦ C. Next,
the solubilized sample was combined with 80 µL of MSTFA and incubated for 30 min at
37 ◦ C prior to GC–MS analysis [21]. GC–MS analyses of B. cernua (stem) extracts were
carried out using an Agilent 5977B GC/MS (made in USA). Pure helium gas was employed
as the carrier gas (1 mL/min), and the injector temperature was set at 270 ◦ C. The oven
temperature program was set as follows: gradual increase from 70 ◦ C to 200 ◦ C (10 ◦ C/min),
then to 310 ◦ C (10 ◦ C/min), before holding at 310 ◦ C for 5 min. The mass spectrometer
was set to electron ionization mode at 70 eV with an electron multiplier voltage of 1859 V.
The retention index of compounds was recorded using Mass Hunter GC/MS Acquisition
10.0.368 software. The phytochemical content determination was conducted by comparing
the results to authentic compound spectral databases stored in the National Institute of
Standards and Technology (NIST) library.
2.3. Antioxidant Activity Test

The antioxidant activity test was conducted using DPPH and SOD methods. The assay
was repeated with solvent (DMSO 0.5% v/v) as a negative control.
2.3.1. DPPH Method

First, 4 × 10−4 M DPPH solution was prepared and placed into a vial, closed tightly
and wrapped using aluminium foil. From the stock solution (1000 ppm), the sample
concentration was varied from 0.5 to 42 ppm and placed in test tubes. After that, 1 mL
of DPPH solution was added to each test tube and allowed to sit for 30 min, followed by
absorbance measurements (λ517 nm). The following equation was used to calculate the
ability to scavenge DPPH free radicals (inhibition): %h = (Ab – As)/Ab × 100%, where %h
is the percentage inhibition (free-radical inhibition), Ab is the absorbance blank, and As is
the sample absorbance. The value of 50% free-radical inhibition concentration (IC50 ) was
calculated using the regression equation y = ax + b.
2.3.2. Method for Superoxide Dismutase (SOD) Activity Assay

From the stock solution (1000 ppm), the sample concentration was varied from 5
to 20 ppm and placed in test tubes. The SOD activity was measured using a xanthine–
xanthine oxidase system capable of generating superoxide radicals. At λ550 nm, the
degree of suppression of nitro blue tetrazolium reduction by O2 was measured. The
Metabolites 2023, 13, 281 4 of 28
amount of enzyme required to inhibit nitro blue tetrazolium degradation by 50% in 1 min
was measured as U/mg protein [22]. The following equation was used to calculate the
percentage inhibition: %h = (Ab – As)/Ab × 100%, where %h is the percentage inhibition
(free-radical inhibition), Ab is the absorbance blank, and As is the sample absorbance. The
value of 50% free-radical inhibition concentration (IC50 ) was calculated using the regression
equation y = ax + b.
2.4. Minimum Inhibitory Concentration against Staphylococcus aureus

The broth microdilution method was used to determine the minimum inhibitory
concentration (MIC) according to CLSI [23]. The extracts were serially diluted twice in
a microtiter plate with Mueller–Hinton broth. Each plate received a bacterial inoculum
with a final concentration of 5 × 105 CFU/mL and was incubated at 37 ◦ C for 24 h. The
MIC was defined as the lowest extract’s concentration that inhibited bacterial growth.
The minimum bactericidal concentration (MBC) was considered as the lowest extract
concentration resulting in no bacterial growth. Treatment with solvent (DMSO 0.5% v/v)
was also used as a negative control.
2.5. Antiplasmodial Assay

P. falciparum strain FCR-3/Gambia was purchased from ATCC (30932TM ). The cells
were grown in Roswell Park Memorial Institute’s medium (RPMI 1640 R8578) supple-
mented with inactivated blood type O serum in a 9:1 ratio. Serum was extracted from
whole blood and centrifuged for 10 min at 1600 rpm. The serum was then inactivated by
heating it at 56 ◦ C for 30 min. P. falciparum cells were cultivated in a complete medium in
red blood cells (RBCs) at 37 ◦ C and 5% CO2 . The medium was regularly replaced twice a
week. After 1 week of maintenance (all RBCs were lysed by parasites), the cells were ready
to be used for the next experiment. The red blood cells (RBCs) were obtained from blood
type O, which was centrifuged at 1600 rpm for 10 min. The pellet of RBC was washed
in phosphate-buffered saline solution and resuspended with complete medium in a ratio
of 1:1. For treatment, infected RBCs were prepared by adding 1 × 106 RBCs to complete
medium and 10 µL of a P. falciparum cell suspension in a 50 mL Falcon tube. Extracts of
B. cernua at various concentrations (600 ppm, 300 ppm, 150 ppm, 100 ppm, 50 ppm, 20 ppm,
10 ppm, 5 ppm, 2 ppm, or 1 ppm) were administered to the same Falcon tube. Treatment
with solvent (DMSO 0.5% v/v) was also used as a negative control. Suspended cells were
grown for 2 × 24 h on six-well plates at 37 ◦ C and 5% CO2 . The positive control consisted
of RBCs and P. falciparum, while the negative control consisted of RBCs. Then, cells were
harvested to calculate the number of intact RBCs using a hemocytometer, and the infected
RBCs were observed using Giemsa staining. Giemsa staining was applied by placing 10 µL
of cell suspension on a slide, fixing it with methanol, and incubating it with Giemsa solution
for 10 min. The presence of P. falciparum in RBCs was determined using a light microscope.
2.6. Cytotoxic Activity

The MCF-7 human breast cancer cell line was purchased from the ATCC (HTB-22TM )
(transcriptome sequence data were deposited in the ArrayExpress database https://www.
ebi.ac.uk/biostudies/arrayexpress/ (accessed on 21 May 2022) by Chiang et al. with
accession number E-MTAB-609) [24]. The cells were grown in Eagle’s minimum essential
medium (EMEM) supplemented with 1% penicillin/streptomycin and 10% heat-inactivated
fetal bovine serum in a 96-well plate (37 ◦ C, 5% CO2 , 20 h). Then, the cells were treated
with various concentrations of B. cernua stem extract (200, 400, 800, and 1600 ppm) for 3, 6,
8, and 24 h. Treatment with solvent (DMSO 0.5% v/v) was also used as a negative control.
The cell viability was estimated by measuring absorbance at λ570 nm using a Quant ELISA
plate reader (Agilent, CA, USA). Trypan blue staining was used to measure the level of cell
death. The morphology of cells was observed using inverted microscopy (Agilent).
Metabolites 2023, 13, 281 5 of 28
2.7. Molecular Modeling (Receptors and Ligand Preparation)

The crystal structure of the selected holoenzymes was downloaded from the RCSB
Protein Databank (PDB): [25] 3EUB [26], 3VSL [27], 3QS1 [28], and 3GD4 [29]. Water and
ligand molecules present in the pdb files were removed using UCSF Chimera [30]. Ligands
were built and energy-minimized (Dreiding force field) using MarvinSketch software
https://chemaxon.com/marvin (accessed on 8 May 2022).
2.8. Molecular Docking

Virtual screening was conducted using PyrX software [31] and Vina software version
1.2.3. [32,33] using a blind docking approach. Specific docking was conducted using UCSF
Chimera software [30] and Vina software version 1.2.3. [32,33]. For the specific docking process,
the grid box of each protein was set as follows: cefotaxime-binding site on 3VSL (center_x = 19.00,
center_y = −50.00, center_z = 24.00, size_x = 17.00, size_y = 17.00, size_z = 17.00); FAD-binding
site on 3EUB (center_x = −17.00, center_y = 13.00, center_z = −84.00, size_x = 19.00,
size_y = 19.00, size_z = 19.00); MTE-binding site on 3EUB (center_x = −42.00, center_y = 18.00,
center_z = −55.00, size_x = 15.00, size_y = 15.00, size_z = 15.00); KNI-10006-binding site on 3QS1
(center_x = 28.00, center_y = −9.00, center_z = 5.00, size_x = 19.00, size_y = 17.00, size_z = 17.00);
FAD-binding site on 3GD4 (center_x = −39.00, center_y = 31.00, center_z = −66.00, size_x = 20.00,
size_y = 22.00, size_z = 20.00); NAD-binding site on 3GD4 (center_x = −50.00, center_y = 35.00,
center_z = −62.00, size_x = 16.00, size_y = 15.00, size_z = 16.00). The best docking results
were chosen and visualized using Biovia Discovery Studio software (https://3ds.com/products-
services/biovia/products) (accessed on 8 May 2022).
2.9. Drug Likeness and Pharmakokinetic Analysis

Drug likeness and pharmacokinetic analyses of molecules were analyzed using the
SwissADME server (http://www.swissadme.ch/)(accessed on 8 May 2022). The drug
likeness analysis was conducted according to the criteria of Lipinski, Ghose, Veber, Egan,
and Muegge.
3. Results and Discussion

3.1. Metabolite Profilling
Phytochemical analysis of leaf extract of several Breynia species such as B. coronata,
B. fruticosa, B. retusa, B. androgyna, B. glauca B. officinalis, B. rostrata, and B. vitis-idaea
was conducted. The identified compounds from several Breynia species were grouped
into alkaloids, aromatic ketones, catechins, flavonoids, glycosides, lignans, neolignans,
steroids, terpenoids, and tannins [8]. Metabolite profiling through GC–MS analysis demon-
strated the presence of 45 compounds in the methanol extract of B. cernua (stem) (Table 1)
which could contribute to the therapeutic effects of this plant. Seven of them were pheno-
lic compounds: N-[β-hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine;
propanoic acid, 2,2-dimethyl-, 2-(1,1-dimethylethyl)phenyl ester; benzenepropanoic acid,
α-hydroxy-, methyl ester; 1,3-phenylene, bis(3-phenylpropenoate); benzenepropanoic
acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, methyl ester; 1,4-bis(trimethylsilyl)benzene;
4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene, 2TMS derivative. In addition, there were
10 cyclic compounds: oxalic acid, cyclohexyl nonyl ester; cyclohexene, 2-ethenyl-1,3,3-
trimethyl-; 1,2:4,5:9,10-triepoxydecane; 1,3-cyclohexadiene, 5-(1,5-dimethyl-4-hexenyl)-2-
methyl-, [S-(R*,S*)]-; 2-piperidinone, N-[4-bromo-n-butyl]-; ethylamine, 2-(adamantan-1-
yl)-1-methyl-; methyl 3-bromo-1-adamantaneacetate; 1-cyclohexylnonene; cyclohexene,
1-nonyl-; cyclotrisiloxane, hexamethyl-. Furthermore, there were alkyl halides (methane,
dichloronitro-; 1-chloroundecane; dodecane, 1-chloro-; decane 1-chloro-; tetradecane, 1-
chloro-; 1-octadecanesulphonyl chloride; 2-bromotetradecane; 2-bromo dodecane; dode-
cane, 1-iodo-; eicosane, 1-iodo-), three organosulfur compounds (sulfurous acid, hexyl
nonyl ester; sulfurous acid, decyl hexyl ester; sulfurous acid, 2-ethylhexyl isohexyl ester),
and one organoarsen compound (arsenous acid, tris(trimethylsilyl) ester). Unfortunately,
compounds with many hydroxyl groups such as flavonoids and phenolic acids were
Metabolites 2023, 13, 281 6 of 28
not detected in this extract. Therefore, this study focused on the volatile compounds of
B. cernua (stem).
Table 1. Metabolite profiling of B. cernua methanol extract using gas chromatography/mass spec-
trometry (GC–MS) method.
Retention Time
No Compound Name Molecular Formula m/z
(minute)
1 Methane, dichloronitro- 3322–3386 CHCl2 NO2 43, 54, 68, 82, 90, 134
2 Ether, hexyl pentyl 4135–4187 C11 H24 O 42, 84, 127
3 1,5-Heptadien-4-ol, 3,3,6-trimethyl- 7410–7453 C10 H18 O 42, 56, 70, 82, 97
Sulfurous acid, 2-ethylhexyl
4 7574–7612 C14 H30 O3 S 42, 56, 82, 90, 112
isohexyl ester
5 Sulfurous acid, hexyl nonyl ester 8008–8046 C15 H32 O3 S 42, 56, 76, 82, 94
6 Decane, 2,4-dimethyl- 8119–8156 C12 H26 43, 84, 112, 126
7 Octane, 3,4,5,6-tetramethyl- 8213–8269 C12 H26 42, 56, 70, 82, 90, 112, 126
8 Oxalic acid, cyclohexyl nonyl ester 8558–8593 C17 H30 O4 42, 56, 68, 82, 94, 110
9 Octane, 2,4,6-trimethyl- 8857–8903 C11 H24 42, 56, 70, 84, 90, 112, 126
N-[β-Hydroxy-β-[4-[1-adamantyl-6,8-
10 9383–9.400 C27 H33 Cl2 NO 43, 54, 69, 97, 118, 134, 149
dichloro]quinolyl]ethyl]piperidine
11 1-Chloroundecane 10,582–10,614 C11 H23 Cl 42, 56, 68, 84, 104, 118, 134, 149
12 Dodecane, 1-chloro- 10,628–10,660 C12 H25 Cl 42, 56, 70, 84, 104, 134
13 Decane, 1-chloro- 10,760–10,803 C10 H21 Cl 42, 56, 68, 84, 104, 134, 149
Propanoic acid, 2,2-dimethyl-,
14 10,897–10,948 C15 H22 O2 43, 56, 78, 90, 106, 118, 134, 149
2-(1,1-dimethylethyl) phenyl ester
15 Sulfurous acid, decyl hexyl ester 11,347–11,377 C16 H34 O3 S 42, 84, 98, 112, 134, 154
16 Tetradecane, 1-chloro- 11,436–11,479 C14 H29 Cl 42, 56, 70, 84, 104, 118, 134, 149
17 Dodecane, 4,6-dimethyl- 11,522–11,557 C14 H30 43, 71, 98, 126, 154
18 Cyclohexene, 2-ethenyl-1,3,3-trimethyl- 1894–11,975 C14 H30 42, 56, 70, 90, 104, 118, 134, 149
42, 56, 70, 84, 104, 118, 134,
19 Pentane, 2,3,3-trimethyl- 12,112–12,134 C8 H18
149, 168
20 Hexadecane 12,182–12,220 C16 H34 42, 56, 84, 98, 112, 126, 140, 154
Benzenepropanoic acid, α-hydroxy-,
21 12,953–13,021 C10 H12 O3 43, 64, 76, 90, 102, 120, 161
methyl ester
22 1,3-Phenylene, bis(3-phenylpropenoate) 13,177–13,231 C24 H18 O4 43, 56, 76, 90, 102, 130, 161
23 1,2:4,5:9,10-Triepoxydecane 13,705–13,754 C10 H16 O3 43, 56, 68, 80, 92, 118, 206
1,3-Cyclohexadiene,
40, 56, 70, 84, 92, 104, 118,
24 5-(1,5-dimethyl-4-hexenyl)-2-methyl-, 14,150–14,20 C15 H24
131, 203
[S-(R*,S*)]-
25 1-Octadecanesulphonyl chloride 14,341–14,379 C18 H37 ClO2 S 42, 70, 76, 92, 118, 140, 154, 201
26 Oxalic acid, allyl pentadecyl ester 15,079–15,125 C20 H36 O4 42, 56, 70, 84, 110
27 2-Piperidinone, N-[4-bromo-n-butyl]- 15,472–15,510 C9 H16 BrNO 43, 56, 70, 90, 104, 122, 206
28 2-Bromotetradecane 16,892–16,959 C14 H29 Br 42, 71, 98, 126, 154, 206
29 Oxalic acid, 6-ethyloct-3-yl heptyl ester 17,021–17,059 C19 H36 O4 42, 56, 84, 98, 110, 126, 206
42, 56, 70, 76, 84, 98, 112, 141,
30 2-Bromo dodecane 17,409–17,504 C12 H25 Br
168, 206
Metabolites 2023, 13, 281 7 of 28
Table 1. Cont.
Retention Time
No Compound Name Molecular Formula m/z
(minute)
Ethylamine,
31 17,991–18,037 C13 H23 N 43, 54, 68, 82, 106, 134, 206
2-(adamantan-1-yl)-1-methyl-
32 Pentadecanal- 18,452–18,511 C15 H30 O 43, 56, 68, 81, 108, 206
33 Di-n-decylsulfone 18,662–18,735 C20 H42 O2 S 43, 56, 70, 84, 98, 206
42, 70, 98, 112, 126, 154,
34 Dodecane, 1-iodo- 19,675–19,756 C12 H25 I
183, 206
42, 54, 73, 86, 96, 110,142, 170,
35 Hexadecanoic acid, methyl ester 20,033–20,093 C17 H34 O2
206, 227, 270
Benzenepropanoic acid, 43, 56, 68, 90, 104, 116, 134,
36 20,136–20,200 C18 H28 O3
3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 146, 206
43, 54, 66, 78, 90, 104, 116, 132,
37 Methyl 3-bromo-1-adamantaneacetate 21,087–21,184 C13 H19 BrO2
156, 190, 206, 256, 280
43, 56, 68, 81, 95, 109, 132, 190,
38 1-Cyclohexylnonene 21,375–21,456 C15 H28
206, 280
4-Methyl-2,4-bis(p-
43, 54, 81, 104, 132, 158,
39 hydroxyphenyl)pent-1-ene, 21,765–21,811 C24 H36 O2 Si2
206, 280
2 TMS derivative
40, 54, 66, 81, 95, 109, 123, 146,
40 Cyclohexene, 1-nonyl- 21,930–21,978 C15 H28
190, 206, 280
41 Cyclotrisiloxane, hexamethyl- 22,121–22,194 C6 H18 O3 Si3 43, 70, 84, 95, 114, 132, 190, 280
43, 68, 84, 95, 118, 132, 146, 190,
42 1,4-Bis(trimethylsilyl)benzene 22,245–22,309 C12 H22 Si2
206, 280
9,12,15-Octadecatrienoic acid, methyl 43, 54, 66, 78, 94, 107, 120, 134,
43 22,323–22,366 C19 H32 O2
ester, (Z,Z,Z)- 148, 190, 280
42, 70, 85, 112, 126, 155, 190,
44 Eicosane, 1-iodo- 22,501–22,608 C20 H41 I
206, 238, 280
43, 54, 74, 104, 132, 162, 190,
45 Arsenous acid, tris(trimethylsilyl) ester 24,536–24,585 C9 H27 AsO3 Si3
206, 236, 280, 314
* Note: TMS = trimethylsilyl.
Medicinal plants are a promising reservoir of bioactive metabolites that can support
the discovery of novel drug molecules. GC–MS analysis was conducted on the B. cernua
(stem) methanol extract and revealed 45 bioactive compounds. Studies in the literature
have revealed the isolation of several metabolites, regardless of whether their specific func-
tion was demonstrated or not. Meanwhile, the origin or bioactivity of a few metabolites
has not been documented. Dichloronitromethane, as the dominant compound in ethyl
acetate fraction of endophytic fungus Alternaria alternata AE1, exhibited antimicrobial prop-
erties [34]. 3,3,6-Trimethyl-1,5-heptadien-4-ol identified, as one of the main compounds
of Artemisia austro-yunnanensis essential oil, possessed antioxidant activity [35]. Sulfurous
acid, 2-ethylhexyl isohexyl ester, contained in the methanol extract of Gracilaria corticata and
Gracilaria edulis, showed antioxidant and antibacterial activity [36]. Sulfurous acid, hexyl
nonyl ester was identified in the ethanol extract of Eclipta prostrata and showed antimicro-
bial activity [37]. Decane, 2,4-dimethyl- was identified in the endophytic bacterium Pantoea
sp. strain Dez632 and possessed antibacterial activity [38]. Octane, 3,4,5,6-tetramethyl-,
contained in the endophytic bacterium Pseudomonas sp. strain Bt851, exhibited antibacterial
activity [38]. Oxalic acid, cyclohexyl nonyl ester, isolated from the methanol extract of
Rheum ribes, showed an inhibitory effect on Escherichia coli [39]. Octane, 2,4,6-trimethyl- was
identified in the essential oil of Rumex hastatus, which exhibited inhibitory and antioxidant
Metabolites 2023, 13, 281 8 of 28
activity [40]. 1-Chloroundecane, contained in the extract of Streptomyces sp., exerted antibac-
terial activity [41]. Dodecane, 1-chloro- was identified in the extract of Spongia officinalis
var ceylonensis and possessed antiproliferative activity against three cancer cell lines [42].
Decane, 1-chloro-, identified in the ethyl acetate fraction of Artocarpus anisophyllus Miq stem
bark, exerted antioxidant activity [43]. Sulfurous acid, decyl hexyl ester was identified in
rambutan seed fat [44] and as a volatile organic compound in olive tree [45]. Tetradecane, 1-
chloro- was identified in the crude extract of medicinal herbs used to treat kidney stones [46].
Dodecane, 4,6-dimethyl-, contained in the essential oil of Ephedra pachyclada, possessed an-
tioxidant and antimicrobial activity [47]. Cyclohexene, 2-ethenyl-1,3,3-trimethyl-, extracted
from the ethyl acetate fraction of Streptomyces sp. Al-Dhabi-90 metabolites, demonstrated
broad-spectrum activity against pathogenic bacteria [48]. Hexadecane was identified as a
major component of Monochaetia kansensis chloroform extract, and this compound is known
to have antioxidant and antibacterial activity [49]. Benzenepropanoic acid, α-hydroxy-,
methyl ester (synonym: lactic acid, 3-phenyl-, methyl ester), contained in Lactobacillus plan-
tarum culture supernatants, possessed potential antipathogenic and chronic skin wound-
healing properties [50]. 1,3-Phenylene, bis(3-phenylpropenoate), identified in the acetone
extract of Jasminum annamense subsp. annamense leaves, exerted antioxidant and antibacte-
rial activity [51]. 1,2:4,5:9,10-Triepoxydecane was extracted from the ethyl acetate fraction
of Alysicarpus glumaceus, which had therapeutic properties such as stimulatory, diuretic, an-
tipsychotic, anti-inflammatory, antidiarrheal, abortifacient, antitussive, and anti-asthmatic
activity [52]. 1,3-Cyclohexadiene, 5-(1,5-dimethyl-4-hexenyl)-2-methyl-, [S-(R*,S*)]- was
identified in the methanol extract of Zingiber officinale, and this compound had pharmaco-
logical properties such as antioxidant, anti-inflammatory, and antinociceptive activities [53].
1-Octadecanesulphonyl chloride was previously isolated from the volatile floral extract
of Acacia auriculiformis, which exhibited antioxidant and antifungal activity [54]. Oxalic
acid, allyl pentadecyl ester was identified in the methanol extract of red alga Laurencia bran-
denii, which showed anti-pest, antimicrobial, termiticidal, and maggoticidal activity [55].
2-Piperidinone, N-[4-bromo-n-butyl]- was contained in various organic solvent extracts
of pomegranate peel and demonstrated antimicrobial activities against P. aeruginosa, P.
mirabilis, and C. albicans [56]. 2-Bromotetradecane, as one of the major constituents of
ethyl acetate fraction of Haematocarpus validus Bakh. F. Ex Forman, exhibited antimicrobial
and antioxidant activity [57]. Oxalic acid, 6-ethyloct-3-yl heptyl ester, as one of the major
constituents of Ricinus communis seed oil, exhibited anti-inflammatory, antibacterial, antiox-
idant, anthelmintic, antidiabetic, anticancer, mosquitocidal, and insecticidal activity [58].
2-Bromododecane was identified in the n-hexane extract of Moringa oleifera root bark and
exhibited antibacterial activity [59]. Ethylamine, 2-(adamantan-1-yl)-1-methyl- was con-
tained in the essential oil from Cinnamomum tamala dried leaves, and this plant has been
reported to have therapeutic activities to treat anorexia, shore throat, skin diseases, colds,
cough, colic, and diarrhea [60]. Pentadecanal, isolated from Pseudoalteromonas haloplanktis
TAC125 culture supernatant, exhibited inhibitory activity toward the biofilm formation of
S. epidermidis [61]. Di-n-decylsulfone was identified in the acetone extract of Spatholobus
purpureus root, and this plant has been used to cure animal hemorrhagic septicemia [62].
Dodecane-1-iodo- was present in Rhazya stricta chloroform extract, which exhibited antidi-
abetic activity [63]. Hexadecanoic acid, methyl ester, contained in the FAME extract of
Sesuvium portulacastrum, possessed antibacterial, anticandidal, and antifungal activity [64].
Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, methyl ester, present in the
hexane, ethyl acetate, and methanol extracts of Ficus bhotanica, had bioactivities such as
antifungal and antioxidant properties [65]. Methyl 3-bromo-1-adamantaneacetate, as the
major component of Caulerpa racemosa, possessed antibacterial and larvicidal activity [66].
1-Cyclohexylnonene was identified in the n-butanol extract of Ehretia serrata and exhibited
broad-spectrum antimicrobial activity [67]. 4-Methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene,
2TMS derivative was identified in the aqueous extract of Luffa acutangula peel [68], and this
compound has been known to have anticancer activity by inducing apoptosis of pancreatic
β-cells [69]. Cyclohexene, 1-nonyl- was identified as a volatile compound in Piper guineense
Metabolites 2023, 13, 281 9 of 28
leaves and seeds, and this spice exerts medicinal functions such as for the treatment of
rheumatism, bronchitis, cough, and intestinal disease. Moreover, the leaf extract exhib-
ited antimicrobial properties [70]. Cyclotrisiloxane, hexamethyl-, mainly contained in the
acetone extract of Turbinaria decurrens, exhibited antioxidant and antidiabetic activity [71].
1,4-Bis(trimethylsilyl)benzene was found in the volatile oil of Glycosmis pentaphylla, and this
plant has in vivo bioactivities such as antidiabetic, antipyretic, antioxidant, antibacterial, an-
thelmintic, antinociceptive, and hepatoprotective properties [72]. 9,12,15-Octadecatrienoic
acid, methyl ester, (Z,Z,Z)- was found in the n-hexane extract of Archidium ohioense (Schimp.
ex Mull), and this compound has been known to have cardioprotective, antioxidant, anti-
cancer, antipyretic, antibacterial, antiarthritic, and antiandrogenic activities [73]. Eicosane,
1-iodo-, contained in the ethyl acetate and petroleum ether extracts of Alnus cremastogyne
pods, exhibited antioxidant activity [74]. Arsenous acid, tris(trimethylsilyl) ester is one of
the major compounds of Momordica cymbalaria methanol extract, and this plant is commonly
used to cure rheumatism, skin disease, diabetes mellitus, diarrhea, and ulcers [75]. From
the above evidence, we can conclude that the B. cernua extract (stem) contains numerous
compounds with various potential bioactivities.
3.2. Antioxidant and Antibacterial Test Results

The extract exhibited remarkable antioxidant activity as measured using the DPPH
(2,2-diphenyl-1-pikrilhidrazil) and SOD (superoxide dismutase) methods, with IC50 values
of 33 and 8.13 ppm, respectively. Numerous phenolic compounds can result in antioxidant
activity since the phenolic and flavonoid groups commonly act as antioxidants, negating
the oxidative damage generated by free radicals at the molecular level [76]. In addition,
the extract also possessed bactericidal activity against pathogenic bacterium S. aureus
at a concentration of 1875 ppm. This finding is in line with a prior study on B. cernua
extract, which exhibited broad-spectrum antimicrobial activity against bacteria, including
S. aureus [12].
3.3. Antiplasmodial Test Result

An antimalarial test was conducted on Plasmodium falciparum FBR3. The test parame-
ters were the number of intact erythrocytes and the parasitemia level of the treatment using
both extracts at various concentrations with an incubation period of 2 × 24 h, compared
to the negative control. The results demonstrated that the methanol extract was active
against P. falciparum. Both methanol extract treatments (1 and 2 ppm) show good activity.
The number of erythrocytes decreased on P. falciparum-infected erythrocytes; however, the
1–2 ppm extract treatment protected cells from lysis (Figures 1 and 2). At 10 ppm, the
extract seemingly did not have a protective effect since the number of intact erythrocytes
was similar to the control (+). Further analysis showed that the methanol extract might
play a role in protecting erythrocytes from ring formation stage of P. falciparum infection
(Figure 3).
3.4. Anticancer Test Result

Previous research stated that n-hexane fraction of B. cernua had the highest cytotoxic
toward MCF-7 breast cancer cells compared to ethyl acetate and water fractions, with
an IC50 value of 165.65 ppm. On the basis of these data, a test was conducted on the
methanol extract of B. cernua using the MCF-7 cell line. The test was conducted at various
concentrations to determine the extract’s activity in killing cancer cells. The result showed
that the extract started killing cancer cells within 3 h of treatment at concentrations of
1600 ppm and 800 ppm. Concentrations under 800 ppm did not show any effects on the
cells. (Figure 4). Further morphology analysis showed that B. cernua (1600 ppm, 3 h)
induced cell shrinkage and apoptotic body formation, which are hallmarks of apoptosis
(Figure 5).
Metabolites 2023, 13, x FOR PEER REVIEW
Metabolites 2023, 13, 281 10 of 28 10 of 27

Metabolites 2023, 13, x FOR PEER REVIEW 10 of 27

120
100
120
Cell population (%)
80 100
60 80
Cell population (%)
40 60
40
20
20
0
0

Figure 1. Quantitative analysis of the impact of B. cernua extract addition to malarial parasite‐in‐
Figure 1. Quantitative analysis of the impact of B. cernua extract addition to malarial parasite-
Figure 1. Quantitative analysis of the impact of B. cernua extract addition to malarial parasite‐in‐
fected erythrocytes.
fected
infectederythrocytes. Control
erythrocytes.Control
Control(−):
(−): ):erythrocytes;
(−erythrocytes; control
control
erythrocytes; (+):
control (+): erythrocytes
erythrocytes
(+): + P.
erythrocytes + P. falciparum;
falciparum;
+ P. falciparum; treatment:
treatment:
treatment:
erythrocytes + P. falciparum + methanol extracts at various concentrations (600 ppm, 300 ppm, 150
erythrocytes + P. falciparum + methanol extracts at various concentrations (600 ppm, 300 ppm, 150
erythrocytes + P. falciparum + methanol extracts at various concentrations (600 ppm, 300 ppm, 150 ppm,
ppm, 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, 2 ppm, or 1 ppm). All of the data except for 1 and
ppm, 100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, 2 ppm, or 1 ppm). All of the data except for 1 and
100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, 2 ppm, or 1 ppm). All of the data except for 1 and 2 ppm
2 ppm extract were considered significantly differ compared to the control (−) (p <0.05).
2 ppm extract were considered significantly differ compared to the control (−) (p <0.05).
extract were considered significantly differ compared to the control (−) (p <0.05).

Figure 2. Qualitative analysis of the impact of B. cernua extract addition to malarial parasite‐infected
Figure 2. Qualitative analysis of the impact of B. cernua extract addition to malarial parasite-infected
erythrocytes. Control (−): erythrocytes; control (+): erythrocytes + P. falciparum; treatment: erythro‐
erythrocytes. Control (−): erythrocytes; control (+): erythrocytes + P. falciparum; treatment: erythro-
cytes + P. falciparum + methanol extracts at various concentrations (600 ppm, 300 ppm, 150 ppm, 100
cytes + P. falciparum + methanol extracts at various concentrations (600 ppm, 300 ppm, 150 ppm,
ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, 2 ppm, or 1 ppm).
Figure 2. Qualitative analysis of the impact of B. cernua extract addition to malarial parasite‐infected
100 ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, 2 ppm, or 1 ppm).
erythrocytes. Control (−): erythrocytes; control (+): erythrocytes + P. falciparum; treatment: erythro‐
cytes + P. falciparum + methanol extracts at various concentrations (600 ppm, 300 ppm, 150 ppm, 100
ppm, 50 ppm, 20 ppm, 10 ppm, 5 ppm, 2 ppm, or 1 ppm).
Metabolites 2023, 13, x FOR PEER REVIEW
Metabolites 2023, 13, 281 11 11
of of27
28

Figure 3. The impact of B. cernua extract addition to the malarial parasite‐infected erythrocytes
(Giemsa staining). Control (−): erythrocytes; control (+): erythrocytes + P. falciparum; treatment:
erythrocytes + P. falciparum + methanol extract at 5 ppm. Red arrow: infected erythrocytes at the ring
formation stage; blue arrow: intact cells following extract treatment.
Previous research stated that n‐hexane fraction of B. cernua had the highest cytotoxic
toward MCF‐7 breast cancer cells compared to ethyl acetate and water fractions, with an
IC50 value of 165.65 ppm. On the basis of these data, a test was conducted on the methanol
extract of B. cernua using the MCF‐7 cell line. The test was conducted at various concen‐
trations to determine the extract’s activity in killing cancer cells. The result showed that
the extract started killing cancer cells within 3 h of treatment at concentrations of 1600
Figure 3.3. The
Figure The impact
impact of of B.
B. cernua
cernua extract
extract addition to the
addition to the malarial
malarial parasite‐infected
parasite-infected erythrocytes
erythrocytes
ppm and 800 ppm. Concentrations under 800 ppm did not show any effects on the cells.
(Giemsa −): erythrocytes; P.falciparum;
falciparum;treatment:
(Giemsa staining).
staining). Control
Control ((−): erythrocytes;control
control(+):
(+):erythrocytes
erythrocytes+ +P. treatment:
(Figure 4). Further morphology analysis showed that B. cernua (1600 ppm, 3 h) induced
stage; blue arrow: intact cells following extract treatment.
cell shrinkage and apoptotic body formation, which are hallmarks of apoptosis (Figure 5).
formation stage; blue arrow: intact cells following extract treatment.
formation
45% Previous research stated that n‐hexane fraction of B. cernua had the highest cytotoxic
40% toward MCF‐7 breast cancer cells compared to ethyl acetate and water fractions, with an
3h
35% IC50 value of 165.65 ppm. On the basis of these data, a test was conducted on the methanol
30% 6h
extract of B. cernua using the MCF‐7 cell line. The test was conducted at various concen‐
Cell death
trations to determine the extract’s activity in killing cancer cells. The result showed that
25% 8h
the extract started killing cancer cells within 3 h of treatment at concentrations of 1600
20% ppm and 800 ppm. Concentrations under 800 ppm did not show any effects on the cells.
24h
(Figure 4). Further morphology analysis showed that B. cernua (1600 ppm, 3 h) induced
15%
cell shrinkage and apoptotic body formation, which are hallmarks of apoptosis (Figure 5).
10%
5% 45%
0% 40% 3h
35%Control 1600 ppm 800 ppm 400 ppm 200 ppm
30% 6h
Cell death
25% Concentration 8h
20%
24h
15% Figure 4. Brenia cernua induced the death of MCF‐7 cancer cells (dose and time dependency). The
Figure 4. Brenia cernua induced the death of MCF-7 cancer cells (dose and time dependency). The cell
10% cell viability was estimated by measuring the absorbance at λ570 nm using a Quant ELISA plate
viability was estimated by measuring the absorbance at λ570 nm using a Quant ELISA plate reader
5% reader (Agilent BioTek Instruments). Control: cancer cells treated with solvent (DMSO 0.5% v/v).
(Agilent BioTek Instruments). Control: cancer cells treated with solvent (DMSO 0.5% v/v).
0%
3.5. Virtual Screening with Blind Docking Approach
Control 1600 ppm 800 ppm 400 ppm 200 ppm
Virtual screening was used to estimate each compound’s affinity to selected target
Concentration
proteins: 3VSL as an antibacterial protein model, 3EUB as an antioxidant protein model,
3QS1 as an antiplasmodial protein model, and 3GD4 as an anticancer protein model. Blind
docking performed using Pyrx software revealed that 44 of 45 compounds were successfully
Figure 4. Brenia cernua induced the death of MCF‐7 cancer cells (dose and time dependency). The
docked into the target proteins. However, the docking analysis could not be performed
cell viability was estimated by measuring the absorbance at λ570 nm using a Quant ELISA plate
reader (Agilent BioTek Instruments). Control: cancer cells treated with solvent (DMSO 0.5% v/v).
using arsenous acid, tris(trimethylsilyl) because the arsenic atom could not be recognized
by AutoDock Vina software (Table 2).

Metabolites 2023, 13, 281
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of of
28 27

Figure 5. The application of Brenia cernua to MCF‐7 cell line. Trypan blue staining was used to meas‐
Figure 5. The application of Brenia cernua to MCF-7 cell line. Trypan blue staining was used to
ure the level of cell death. Control: cancer cells treated with solvent (DMSO 0.5% v/v). The cell mor‐
measure the level of cell death. Control: cancer cells treated with solvent (DMSO 0.5% v/v). The cell
phology was observed using inverted microscopy (Agilent). Apoptotic bodies were characterized
morphology was observed using inverted microscopy (Agilent). Apoptotic bodies were characterized
by cell shrinkage.
by cell shrinkage.
3.5. Virtual Screening with Blind Docking Approach
Table 2. Binding affinity of identified compounds to selected target proteins. Nd: not detected.
Virtual screening was used to estimate each compound’s affinity to selected target
No Name of Compounds Binding Affinity between Ligands and Target Proteins (kcal/mol)
proteins: 3VSL as an antibacterial protein model, 3EUB as an antioxidant protein model,
Antibacterial Antioxidant Antiplasmodial Anticancer
3QS1 as an antiplasmodial protein model, and 3GD4 as an anticancer protein model. Blind
(3VSL) (3EUB) (3QS1) (3GD4)
docking performed using Pyrx software revealed that 44 of 45 compounds were success‐
1 Methane, dichloronitro- −3.9
fully docked into the target proteins. −4.4 the docking
However, −analysis
3.7 could −not
4.2 be per‐
2 Ether, hexyl pentyl: formed using arsenous acid, tris(trimethylsilyl) because the arsenic atom could not be rec‐
−4.2 −4.8 −4.5 −4.9
3 ognized by AutoDock Vina software (Table 2).
1,5-Heptadien-4-ol, 3,3,6-trimethyl- −5.4 −5.3 −5.0 −5.0
4 Sulfurous acid, 2-ethylhexyl isohexyl ester −4.3 −4.9 −4.7 −4.5
Table 2. Binding affinity of identified compounds to selected target proteins. Nd: not detected.
5 Sulfurous acid, hexyl nonyl ester −4.6 −5.3 −4.4 −4.7
6 Decane, 2,4-dimethyl-
Binding Affinity between Ligands and Target Proteins
−4.5 −4.4 −4.6 −4.7
No Name of Compounds
(kcal/mol)
7 Octane, 3,4,5,6-tetramethyl- −5.2 −5.3 −4.7 −4.7
8 Oxalic acid, cyclohexyl nonyl ester −5.4 −5.1 −4.0 −52
(3VSL) (3EUB) (3QS1) (3GD4)
9 Octane, 2,4,6-trimethyl- −5.3 −5.1 −4.7 −5.3
1 Methane, dichloronitro‐ −3.9 −4.4 −3.7 −4.2
2 N-[β-Hydroxy-β-[4-[1-adamantyl-6,8-
Ether, hexyl pentyl:
10 −−4.2
9.2 −4.8
−10.8 −9.0−4.5 −9.3−4.9
3 1,5‐Heptadien‐4‐ol, 3,3,6‐trimethyl‐ −5.4 −5.3 −5.0 −5.0
11 1-Chloroundecane −4.9 −5.2 −4.7 −5.1
4 Sulfurous acid, 2‐ethylhexyl isohexyl ester −4.3 −4.9 −4.7 −4.5
12 Dodecane, 1-chloro- −3.8 −5.0 −4.7 −5.4
5 Sulfurous acid, hexyl nonyl ester −4.6 −5.3 −4.4 −4.7
6 13 Decane, 1-chloro-
Decane, 2,4‐dimethyl‐ −−4.5
4.3 −−4.4
4.8 −3.1−4.6 −4.4−4.7
7 Propanoic acid, 2,2-dimethyl-,
Octane, 3,4,5,6‐tetramethyl‐
14 −−5.2
6.3 −−5.3
6.6 −6.0−4.7 −7.9−4.7
2-(1,1-dimethylethyl)phenyl ester
8 Oxalic acid, cyclohexyl nonyl ester −5.4 −5.1 −4.0 −52
15 Sulfurous acid, decyl hexyl ester −4.9 −4.7 −3.9 −4.6
9 Octane, 2,4,6‐trimethyl‐ −5.3 −5.1 −4.7 −5.3
N‐[β‐Hydroxy‐β‐[4‐[1‐adamantyl‐6,8‐
10 −9.2 −10.8 −9.0 −9.3
11 1‐Chloroundecane −4.9 −5.2 −4.7 −5.1
12 Dodecane, 1‐chloro‐ −3.8 −5.0 −4.7 −5.4
Metabolites 2023, 13, 281 13 of 28
Table 2. Cont.
No Name of Compounds Binding Affinity between Ligands and Target Proteins (kcal/mol)
(3VSL) (3EUB) (3QS1) (3GD4)
16 Tetradecane, 1-chloro- −4.2 −4.8 −3.7 −4.1
17 Dodecane, 4,6-dimethyl- −4.3 −5.7 −4.6 −5.6
18 Cyclohexene, 2-ethenyl-1,3,3-trimethyl- −5.3 −5.7 −5.2 −5.5
19 Pentane, 2,3,3-trimethyl- −4.9 −4.7 −4.3 −4.8
20 Hexadecane −4.0 −5.3 −3.8 −4.5
21 Benzenepropanoic acid,α-hydroxy-, methyl ester −6.2 −6.6 −5.6 −5.9
22 1,3-Phenylene, bis(3-phenylpropenoate) −9.4 −8.4 −8.7 −8.3
23 1,2:4,5:9,10-Triepoxydecane −4.5 −5.7 −4.3 −5.5
1,3-Cyclohexadiene,
24 −5.7 −6.4 −7.4 −5.0
5-(1,5-dimethyl-4-hexenyl)-2-methyl-, [S-(R*,S*)]-
25 1-Octadecanesulphonyl chloride −5.4 −4.5 −4.3 −6.6
26 Oxalic acid, allyl pentadecyl ester −5.4 −4.8 −4.2 −5.2
27 2-Piperidinone, N-[4-bromo-n-butyl]- −4.8 −4.9 −3.7 −5.7
28 2-Bromotetradecane −4.0 −5.2 −4.2 −4.2
29 Oxalic acid, 6-ethyloct-3-yl heptyl ester −5.3 −5.9 −5.4 −7.0
30 2-Bromo dodecane −3.8 −5.3 −3.8 −4.4
31 Ethylamine, 2-(adamantan-1-yl)-1-methyl- −6.0 −7.0 −6.6 −7.3
32 Pentadecanal- −3.8 −5.3 −4.5 −5.0
33 Di-n-decylsulfone −4.6 −4.6 −4.5 −5.0
34 Dodecane, 1-iodo- −3.9 −4.8 −3.2 −5.0
35 Hexadecanoic acid, methyl ester −5.0 −5.4 −3.7 −6.2
Benzenepropanoic acid,
36 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, −6.6 −6.5 −7.0 −6.6
methyl ester
37 Methyl 3-bromo-1-adamantaneacetate −6.3 −6.1 −6.8 −7.4
38 1-Cyclohexylnonene −5.5 −6.2 −3.8 −6.6
4-Methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene,
39 −6.9 −6.5 −7.1 −6.7
2TMS derivative
40 Cyclohexene, 1-nonyl- −5.0 −5.1 −4.7 −5.4
41 Cyclotrisiloxane, hexamethyl- −4.8 −4.9 −4.4 −5.6
42 1,4-Bis(trimethylsilyl)benzene −5.1 −5.7 −4.8 −4.9
9,12,15-Octadecatrienoic acid, methyl
43 −5.3 −6.7 −4.7 −7.6
ester, (Z,Z,Z)-
44 Eicosane, 1-iodo- −4.8 −5.0 −2.9 −4.4
45 Arsenous acid, tris(trimethylsilyl) ester ND ND ND ND
B. cernua is known as an alternative medicine with antiviral properties toward small-

pox [9] and anticancer properties toward breast and cervical cancer [10]. In addition, its
methanol extract also exerts broad-spectrum antibacterial activity [12], as well as antiox-
idant, antiplasmodial, and anticancer properties (this study). Computational docking
tools are used by researchers all around the world to uncover and evaluate the binding
affinity of compounds that fit a protein’s binding site [19]. From the above in vitro and
Metabolites 2023, 13, 281 14 of 28
in vivo evidence, four protein models were chosen as molecular docking targets, as they
were previously crystallized and/or used for in silico studies to investigate the binding
affinity of particular compounds: 3EUB as an antioxidant target protein [77], 3VSL as an
antibacterial target protein [78], 3QS1 as an antiplasmodial target protein [79], and 3GD4 as
an anticancer target protein [80]. According to the virtual screening
Metabolites 2023, 13, x FOR PEER REVIEW results
14 of 27 (Table 2), N-

[β-hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine and 1,3-phenylene,
bis(3-phenylpropenoate) exhibited the best binding affinity toward all protein models.
Hence, the detailed interactions between the two phenolic compounds and the target
anticancer target protein [80]. According to the virtual screening results (Table 2), N‐[β‐
hydroxy‐β‐[4‐[1‐adamantyl‐6,8‐dichloro]quinolyl]ethyl]piperidine
proteins were further investigated. and 1,3‐phenylene,
bis(3‐phenylpropenoate) exhibited the best binding affinity toward all protein models.
Xanthine oxidase (pdb:3EUB) was selected as an antioxidant protein target since
Hence, the detailed interactions between the two phenolic compounds and the target pro‐
the methanol extract of B. cernua stem possessed inhibitory activity against the enzyme
teins were further investigated.
according to the SOD assay.The binding interaction between 3EUB and N-[β-hydroxy-β-[4-
Xanthine oxidase (pdb:3EUB) was selected as an antioxidant protein target since the
[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine revealed one conventional hydrogen
methanol extract of B. cernua stem possessed inhibitory activity against the enzyme ac‐
cording to the SOD assay.The binding interaction between 3EUB and N‐[β‐hydroxy‐β‐[4‐
bond formed between Leu257 and the oxygen atom of the ligand. One electrostatic in-
[1‐adamantyl‐6,8‐dichloro]quinolyl]ethyl]piperidine
teraction was formed between the ligand and revealed
Lys256 one on
conventional hydro‐
3EUB (π–cation). Furthermore,
gen bond formed between Leu257 and the oxygen atom of the ligand. One electrostatic
there were 16 hydrophobic interactions involving Ile264 (alkyl),
interaction was formed between the ligand and Lys256 on 3EUB (π–cation). Furthermore,
Leu257, Ile353, Lys395,
Leu398 (π–alkyl), and 11 other amino acids including Arg394 (van
there were 16 hydrophobic interactions involving Ile264 (alkyl), Leu257, Ile353, Lys395, der Waals) (Figure 6).
The binding interaction between 3EUB and 1,3-phenylene, bis(3-phenylpropenoate) re-
Leu398 (π–alkyl), and 11 other amino acids including Arg394 (van der Waals) (Figure 6).
The binding
vealed twointeraction between
conventional 3EUB and bonds
hydrogen 1,3‐phenylene,
formed bis(3‐phenylpropenoate)
between Leu257 and re‐ Val259 and the
vealed
carboxyltwo group
conventional
of thehydrogen bonds formed abetween
ligand. Meanwhile, van derLeu257
Waalsand Val259 and
interaction wasthe formed between
carboxyl group of the ligand. Meanwhile, a van der Waals interaction was formed be‐
Lys256 and 1,3-phenylene, bis(3-phenylpropenoate). Moreover, there were 16 hydrophobic
tween Lys256 and 1,3‐phenylene, bis(3‐phenylpropenoate). Moreover, there were 16 hy‐
interactions involving Ala301 (π–alkyl), Leu257 (π–sigma), and 14 other amino acids (van
drophobic interactions involving Ala301 (π–alkyl), Leu257 (π–sigma), and 14 other amino
der Waals)
acids (van der (Figure S1). Arg880
Waals) (Figure andand
S1). Arg880 Glu1261 are
Glu1261 active
are active residues
residues of of xanthine oxidase [26].
xanthine
oxidase [26]. Both plant‐derived molecules could be antioxidant drugs by binding to xan‐
Both plant-derived molecules could be antioxidant drugs by binding to xanthine oxidase in
thine oxidase in a competitive manner, leading to the reduction in reactive oxygen species
a competitive manner, leading to the reduction in reactive oxygen species in the cell.
in the cell.

(a)

(b) (c)
Figure 6. Binding interaction of N-[β-hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl] piperidine

compound with antioxidant target protein (3EUB) based on the binding energy generated by PyRx program:

Metabolites 2023, 13, 281 15 of 28
Metabolites 2023, 13, x FOR PEER REVIEW 15 of 27

Figure 6. Binding interaction of N‐[β‐hydroxy‐β‐[4‐[1‐adamantyl‐6,8‐dichloro]quinolyl]ethyl] pi‐

(a) a close-up view of ligand binding on 3EUB (the binding sites of the native ligand and docked ligand
peridine compound with antioxidant target protein (3EUB) based on the binding energy generated
are indicated by red and black circles, respectively); (b) 3D diagram of ligand–protein binding pose;
by PyRx program: (a) a close‐up view of ligand binding on 3EUB (the binding sites of the native
ligand and docked ligand are indicated by red and black circles, respectively); (b) 3D diagram of
(c) 2D illustration of ligand–protein interactions.
ligand–protein binding pose; (c) 2D illustration of ligand–protein interactions.
Penicillin-binding protein 3 (PBP3) from methicilin-resistant S. aureus (pdb:3VSL) was
Penicillin‐binding
selected protein
as an antibacterial 3 (PBP3)
target from
since the methicilin‐resistant
methanol extract of S. B.
aureus (pdb:3VSL)
cernua stem exhibited bac-
was selected as an antibacterial target since the methanol extract of B. cernua stem exhib‐
tericidal activity against pathogenic bacteria. The binding interaction between 3VSL and
ited bactericidal activity against pathogenic bacteria. The binding interaction between
N-[β-hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine revealed two conven-
3VSL and N‐[β‐hydroxy‐β‐[4‐[1‐adamantyl‐6,8‐dichloro]quinolyl]ethyl]piperidine re‐
tional hydrogen bonds formed between Asn487 and Gly492 and the oxygen atom of ligand.
vealed two conventional hydrogen bonds formed between Asn487 and Gly492 and the
Furthermore, there were 17 hydrophobic interactions involving Pro253, Leu256, Lys273 (alkyl),
oxygen atom of ligand. Furthermore, there were 17 hydrophobic interactions involving
Tyr278, Leu256,
Pro253, Tyr275, Lys273
Ile381, Ile507
(alkyl), (π–alkyl), and 10Ile381,
Tyr278, Tyr275, other amino acids (vanand
Ile507 (π–alkyl), der10
Waals)
other (Figure S2).
The binding interaction between 3VSL and 1,3-phenylene, bis(3-phenylpropenoate) revealed
amino acids (van der Waals) (Figure S2). The binding interaction between 3VSL and 1,3‐
phenylene, bis(3‐phenylpropenoate) revealed three conventional hydrogen bonds formed
three conventional hydrogen bonds formed between Tyr275, Arg483, and Arg504 and the
between Tyr275, Arg483, and Arg504 and the carboxyl groups of the ligand. One electro‐
carboxyl groups of the ligand. One electrostatic interaction was formed between the ligand
static interaction was formed between the ligand and Arg483 on 3VSL (π–cation). Moreo‐
and Arg483 on 3VSL (π–cation). Moreover, there were 11 hydrophobic interactions involving
ver, there were 11 hydrophobic interactions involving Ile381 (π–alkyl), Arg483 (π–sigma),
Ile381 (π–alkyl), Arg483 (π–sigma), and nine other amino acids (van der Waals) (Figure 7).
and nine other amino acids (van der Waals) (Figure 7). Ser392, Ser393, Val394, Lys395,
Ser392, Ser393, Val394, Lys395, Ser448, Ser449, Asn450, Lys608, Thre609, and Gly610 are
Ser448, Ser449, Asn450, Lys608, Thre609, and Gly610 are active residues of methicillin‐
active residues
resistant ofpenicillin‐binding
S. aureus methicillin-resistant S. aureus
protein 3/PBP3 penicillin-binding protein
[27]. Both plant‐derived 3/PBP3 [27]. Both
molecules
plant-derived molecules could be promising antibacterial drugs by binding to PBP3 in a
could be promising antibacterial drugs by binding to PBP3 in a noncompetitive manner,
noncompetitive manner, leading to the inhibition of bacterial cell-wall formation.
leading to the inhibition of bacterial cell‐wall formation.

(a)

(b) (c)
Figure 7. Binding interaction of 1,3-phenylene, bis(3-phenylpropenoate) compound with antibacterial

target protein (3VSL) based on the binding energy generated by PyRx program: (a) a close-up view
of ligand binding on 3VSL (binding sites of the native ligand and docked ligand are indicated by red
and black circles, respectively); (b) 3D diagram of ligand–protein binding pose; (c) 2D illustration of
ligand–protein interactions.
Metabolites 2023, 13, 281 16 of 28
Plasmepsin I (PMI) from P. falciparum (pdb:3QS1) was selected as an antiplasmodial

target since the methanol extract of B. cernua (stem) was proven to inhibit parasite growth,
as indicated by healthy erythrocytes (Figure 3). The binding interaction between 3QS1
and N-[β-hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine only revealed
hydrophobic interactions such as Ile287 (alkyl), Val12, Ile30, Ala111, Phe117, Ile287, Pro343
(π–alkyl), and 14 other amino acids including Ser77, Phe109, Ile120, Thr218, Ser219, Thr222,
and Phe242 (Van der Waals) (Figure S3). Ile30, Ile287, Ser77, Phe109, Ile120, Thr218, Ser219,
Thr222, and Phe242 are part of the Plasmepsin 1-binding site [28]. The binding interaction
between 3QS1 and 1,3-phenylene, bis(3-phenylpropenoate) revealed only hydrophobic
interactions: Ile30, Pro110, Ala111, Leu243, (π–alkyl), Phe117 (π–sigma), and eight other
amino acids including Phe109, Ile120, and Phe242 (van der Waals) (Figure S4). Ile30, Phe109,
Ile120, and Phe242 are part of the Plasmepsin 1-binding site [28]. Both molecules could be
promising antiplasmodial drug candidates by inhibiting P. falciparum Plasmepsin I, leading
to inhibition of haemoglobin degradation.
Apoptosis induction factor (AIF) was selected as an anticancer target protein since
the methanol extract of B. cernua (stem) induced apoptotic cell death of the MCF-7 cell line
(Figures 4 and 5). The binding interaction between AIF (3GD4) and N-[β-hydroxy-β-[4-
[1-adamantyl-6,8-dichloro]quinolyl]ethyl] piperidine revealed the following hydrophobic
interactions: Phe583 (alkyl) Pro172, Phe192, Trp482 (π–alkyl), Trp482 (π–π stacked), and
11 other amino acids including Lys176 (van der Waals) (Figure S5). Interestingly, Trp482,
Pro172, and Lys 176 are part of the quinone (menadione)-binding site [80], and this molecule
has been proven to induce apoptosis in cancer cells via thiodione formation mediated by
AIF [81]. N-[β-Hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine is a
quinone derivative that could be a new anticancer drug candidate with a mechanism of
action by inducing apoptosis in cancer cells. The binding interaction between 3GD4 and
1,3-phenylene, bis(3-phenylpropenoate) revealed one conventional hydrogen bond formed
between Arg568 and the carboxyl group of the ligand. In addition, there was one π–donor
hydrogen bond (Arg448) and one electrostatic (π–cation) interaction between the ligand
and Arg449. Moreover, there were 15 hydrophobic interactions involving Val451, Leu501
(π–alkyl), Leu296 (π–sigma), and 12 other amino acids (van der Waals) (Figure S6).
3.6. Site-Specific Docking on Binding Site of Native Ligands

Site-specific docking was performed to compare the binding energy of N-[β-hydroxy-
β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine and 1,3-phenylene, bis(3-phenyl-
propenoate) with the native ligands of each protein. Table 3 reveals that the two compounds
exhibited almost similar binding energy to the native ligands of each protein, except for the
binding energy of N-[β-hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl]piperidine
on MTE binding site of 3EUB, which was much lower than that of the native ligand.
Figures S7–S12 visualize the binding interactions between the native ligands and the iso-
lated compounds and each target protein. The result demonstrates that the two compounds
have potential application as novel drugs for the inhibition of antibacterial, antioxidant,
antiplasmodial, and anticancer protein targets in a competitive manner. Nevertheless, an
experimental investigation is needed to validate these findings. Therefore, the next step
will be to obtain these compounds naturally or synthetically.
3.7. Drug Likeness and Pharmacokinetic Analyses

The qualification of each identified compound as an oral drug was conducted accord-
ing to the criteria of Lipinski, Ghose, Veber, Egan, and Muegge (Table 4). Lipinski’s rule of
five is as follows: molecular weight < 500 Da, <5 hydrogen bond donors, <10 hydrogen
bond acceptors, and log P < 5. Ghose utilizes four parameters to qualify a molecule as drug:
160 Da ≤ molecular weight ≤ 480 Da, −0.4 ≤ log P ≤ 5.6, 40 ≤ omlar refractivity ≤ 130, and
20 ≤ total atom number ≤ 70. Veber bases drug likeness on two parameters: topological
polar surface area (TPSA) < 140 A2 and number of rotatable bonds <10. Egan considers
three criteria: TPSA <132 A2 and −1 ≤ log P ≤6. Muegge establishes drug likeness as fol-
Metabolites 2023, 13, 281 17 of 28
lows: 200 Da ≤ molecular weight ≤ 400 Da, −2 ≤ log P ≤ 5, TPSA ≤ 150 A2 , <5 hydrogen
bond donors, <10 hydrogen bond acceptors, <15 rotatable bonds, <7 rings, and at least four
carbon atoms and one heteroatom. Solubility was measured using the ESOL model, where
a log S value ≤−6 indicates a poorly soluble compound, while a log S value ≤−10 indicates
an insoluble compound. Additionally, a compound with 0.55 ≤ bioavailability score ≤ 0.85
and 0.25 ≤ Csp3 (degree of flexibility) score ≤1 is qualified as orally bioavailable [82].
Compounds with no more than one violation are promising drug candidates. According to
Lipinski’s criteria, all of the identified molecules in the methanol extract of B. cernua (stem)
can be considered oral drug candidates. According to Ghose’s criteria, almost all molecules
can be considered drug candidates except for compounds 1 and 10. The filter process using
Veber and Egan criteria showed that all molecules qualified as drug candidates. On the
basis of Muegge’s criteria, only compounds 4, 5, 8, 10, 14, 21, 22, 23, 27, 35, 36, 37, 39,
and 41 qualified as drug candidates. The bioavailability and Csp3 score revealed that all
compounds were orally bioavailable except compound 22.
Table 3. Binding affinity of identified compounds against selected target proteins.
Binding Energy
with N-[β- Binding Energy
Binding Energy Hydroxy-β-[4-[1- with 1,3-Phenylene,
No Native Ligand with Native adamantyl-6,8- bis(3-
Ligand (kcal/mol) dichloro]quinolyl] phenylpropenoate)
ethyl]piperidine (kcal/mol)
(kcal/mol)
Antibacterial protein
1 Cefotaxime −7.6 −7.8 −8.3
target: 3VSL
Flavin adenine
−13.0 −10.1 −10.6
dinucleotide (FAD)
Phosphonic acidmono-(2-
Antioxidant protein
2 amino-5,6-dimercapto-4-oxo-
target: 3EUB
3,7,8A,9,10,10A-hexahydro-4H-
−9.9 −3.6 −7.8
8-oxa-1,3,9,10-tetraaza-
anthracen-7-
ylmethyl)ester (MTE)
(4R)-3-[(2S,3S)-3-{[(2,6-
Dimethylphenoxy)acetyl]amino}-
2-hydroxy-4-phenylbutanoyl]-
Antiplasmodial N-[(1S,2R)-2-hydroxy-2,3-
3 −10.5 −10.6 −9.2
protein target: 3QS1 dihydro-1H-inden-1-yl]-5,5-
dimethyl-1,3-thiazolidine-4-
carboxamide
(KNI-10006)
Flavin adenine
−14.6 −9.1 −10.1
dinucleotide (FAD)
Anticancer protein
4 target: 3GD4 Nicotinamide adenine
−10.5 −11.1 −10.5
dinucleotide (NAD)
The pharmacokinetic parameters of each compound (Table 5) were predicted in terms

of gastrointestinal absorption (GI), blood–brain barrier (BBB) permeability, and skin perme-
ability (log Kp). A more negative log Kp indicates lower skin permeability of the molecule.
Another factor underlying pharmacokinetics is the interaction between the molecule and
particular proteins such as permeability glycoprotein (P-gp) and cytochromes P450 (CYPs).
P-gp is a key factor in active efflux toward the biological membrane, while CYPs are proteins
that play a role in drug biotransformation. The synthetic accessibility score ranges from 1
(very easy) to 10 (very difficult) [83]. Table 5 shows that half of the compounds had high GI
absorption, whereas the other half had low GI absorption. To solve this challenge, scientists
Metabolites 2023, 13, 281 18 of 28
have been using particular strategies such as encapsulating drugs in lipid formulations [84].
The pharmacokinetic analysis also revealed that half of the compounds exhibited low pene-
tration into the BBB and half of the compounds exhibited high penetration into the BBB.
Generally, a low penetration of drugs into the BBB is required to minimize the side-effects
on the central nervous system (CNS). On the other hand, drugs with high permeability
across the BBB are needed to cure cancer patients with brain metastases [85]. The prediction
of the interactions with pharmacokinetic proteins revealed that almost all compounds
had a low interaction with CYP enzymes, thus indicating minimal drug–drug interactions.
Moreover, all compounds had easy to moderate synthetic accessibility. In view of this
thorough analysis, these compounds can be good drug leads of natural origin. Further
studies on the purification or synthesis of these can be conducted to reveal the mechanisms
underlying their antioxidant, antibacterial, antiplasmodial, and anticancer activities.
Metabolites 2023, 13, 281 19 of 28
Table 4. Drug likeness results of the identified compounds from B. cernua. Nd: not detected.
H-Bond Fraction Rotatable Consensus ESOL ESOL Violation

Molecule MW MR TPSA
Acceptors Donors Csp3 Bonds Log P Log S Class Lipinski Ghose Veber Egan Muegge
Very
1 129.93 2 0 1.00 1 24.62 45.82 0.84 −1.49 0 3 0 0 2
soluble
2 172.31 1 0 1.00 9 56.08 9.23 3.66 −2.97 Soluble 0 0 0 0 2
3 154.25 1 1 0.60 3 50.14 20.23 2.59 −2.53 Soluble 0 1 0 0 2
Moderately
4 294.49 3 0 1.00 12 86.78 54.74 3.66 −4.67 0 1 1 0 1
soluble
Moderately
5 292.48 3 0 1.00 15 84.67 54.74 4.71 −4.75 0 1 1 0 1
soluble
Moderately
6 170.33 0 0 1.00 7 59.80 0 4.78 −4.30 1 0 0 0 3
soluble
Moderately
7 170.33 0 0 1.00 5 59.80 0 4.48 −4.07 1 0 0 0 3
soluble
Moderately
8 298.42 4 0 0.88 12 84.29 52.60 4.31 −4.79 0 0 1 0 1
soluble
9 156.31 0 0 1.00 5 54.99 0 4.26 −3.82 Soluble 1 1 0 0 3
Poorly
10 457.46 2 1 0.67 4 130.69 33.12 5.98 −8.06 1 2 0 1 1
soluble
Moderately
11 190.75 0 0 1.00 9 59.79 0 4.75 −4.41 1 0 0 0 3
soluble
Moderately
12 204.78 0 0 1.00 10 64.59 0 5.12 −4.77 1 0 0 0 2
soluble
Moderately
13 176.73 0 0 1.00 8 54.98 0 4.37 −4.04 1 0 0 0 3
soluble
Moderately
14 232.36 1 0 0.56 4 74.65 17.07 4.07 −4.19 0 0 0 0 1
soluble
Moderately
15 306.50 3 0 1.00 16 89.47 54.74 5.18 −5.11 0 1 1 1 2
soluble
Metabolites 2023, 13, 281 20 of 28
Table 4. Cont.

Molecule MW MR TPSA
Moderately
16 232.83 0 0 1.00 12 74.21 0 5.86 −5.49 1 1 1 1 2
soluble
Moderately
17 198.39 0 0 1.00 9 69.41 0 5.50 −5.02 1 0 0 0 3
soluble
18 150.26 0 0 0.64 1 51.61 0 3.44 −2.9 Soluble 0 1 0 0 2
19 114.23 0 0 1.00 2 40.31 0 3.17 −2.82 Soluble 1 1 0 0 2
Moderately
20 226.44 0 0 1.00 13 79.03 0 6.42 −5.60 1 1 1 1 2
soluble
Very
21 180.2 3 1 0.30 4 48.28 46.53 1.43 −1.95 0 0 0 0 1
soluble
Moderately
22 374.43 4 0 0.17 8 109.67 52.6 4.68 −5.06 1 0 0 0 1
soluble
Very
23 184.23 3 0 1.00 6 47.10 37.59 1.68 −1.09 0 0 0 0 1
soluble
Moderately
24 204.35 0 0 0.60 4 70.68 0 4.47 −4.10 1 0 0 0 2
soluble
Poorly
25 353.00 2 0 1.00 17 102.40 42.52 6.68 −6.72 1 1 1 1 2
soluble
Moderately
26 340.50 4 0 0.80 19 100.35 52.60 5.78 −5.89 0 0 1 0 2
soluble
27 234.13 1 0 0.89 4 58.14 20.31 2.17 −2.35 Soluble 0 0 0 0 0
Moderately
28 277.28 0 0 1.00 11 77.28 0 5.83 −5.61 1 1 1 1 2
soluble
Moderately
29 328.49 4 0 0.89 16 96.02 52.60 5.33 −5.46 0 0 1 0 2
soluble
Moderately
30 249.23 0 0 1.00 9 67.67 0 5.10 −4.89 1 0 0 0 2
soluble
Metabolites 2023, 13, 281 21 of 28
Table 4. Cont.

Molecule MW MR TPSA
31 193.33 1 1 1.00 1 60.71 26.02 3.05 −3.13 Soluble 0 0 0 0 2
Moderately
32 226.40 1 0 0.93 13 74.42 17.07 5.04 −4.49 0 0 1 0 2
soluble
Poorly
33 346.61 2 0 1.00 18 107.22 42.52 6.68 −6.22 1 1 1 1 2
soluble
Moderately
34 296.23 0 0 1.00 10 72.76 0 5.41 −5.68 1 0 0 0 2
soluble
Moderately
35 270.45 2 0 0.94 15 85.12 26.30 5.54 −5.18 1 1 1 0 1
soluble
Moderately
36 292.41 3 1 0.61 6 87.68 46.53 4.22 −4.5 0 0 0 0 0
soluble
37 287.19 2 0 0.92 3 67.20 26.30 3.28 −3.53 Soluble 0 0 0 0 0
Moderately
38 208.38 0 0 0.87 7 71.63 0 5.43 −5.07 1 0 0 0 2
soluble
Poorly
39 412.71 2 0 0.42 8 128.08 18.46 6.22 −7.76 1 1 0 1 1
soluble
Moderately
40 208.38 0 0 0.87 8 71.63 0 5.43 −4.76 1 1 0 0 2
soluble
41 222.46 3 0 1.00 0 55.70 27.69 1.16 −3.12 Soluble 0 0 0 0 0
Moderately
42 222.47 0 0 0.50 2 72.40 0 3.44 −4.89 0 0 0 0 2
soluble
43 292.42 2 0 0.63 14 93.31 26.30 nd nd nd nd nd nd nd nd
Poorly
44 408.44 0 0 1.00 18 111.22 0 8.36 −8.58 1 1 1 1 3
soluble
Moderately
45 326.49 2 0 1.00 4 77.56 26.30 1.43 −4.64 0 0 0 0 0
soluble
Metabolites 2023, 13, 281 22 of 28
Table 5. Pharmacokinetic results of the identified compounds from B. cernua.
Bioavailability GI BBB P-gp Cyp Inhibitor Log Kp Alerts Likeness Synthetic

Molecule Absorption Accessibility
Score Permeant Substrate 1A2 2C19 2C9 2D6 3A4 (cm/s) PAINS BRENK Violation
1 0.55 High Yes No No No No No No −6.07 0 3 1 2.46
2 0.55 High Yes No Yes No No No No −4.36 0 0 3 2.22
4 0.55 High No Yes No No No No Yes −3.82 0 0 2 4.31
5 0.55 High No No No No Yes No Yes −3.48 0 0 2 3.97
6 0.55 Low No No No No Yes No No −2.98 0 0 2 2.28
7 0.55 Low No Yes No No No No No −3.39 0 0 2 2.88
9 0.55 Low Yes No No No No No No −3.48 0 0 2 2.44
10 0.55 Low No Yes No No Yes No No −3 0 0 2 5.58
11 0.55 Low No No Yes No No No No −2.98 0 1 3 2.58
14 0.55 High Yes No No No No Yes No −4.44 0 0 2 2.12
15 0.55 High No Yes No No Yes No Yes −3.18 0 0 2 4.08
18 0.55 Low Yes No No No No No No −4.72 0 0 2 3.35
19 0.55 Low Yes No No No No No No −4.28 0 0 2 1
22 0.55 High Yes No No Yes Yes No Yes −4.9 0 3 3 3.77
24 0.55 Low No No No Yes Yes No No −3.88 0 1 2 4.81
Metabolites 2023, 13, 281 23 of 28
Table 5. Cont.
Bioavailability GI BBB P-gp Cyp Inhibitor Log Kp Alerts Likeness Synthetic

Molecule Absorption Accessibility
Score Permeant Substrate 1A2 2C19 2C9 2D6 3A4 (cm/s) PAINS BRENK Violation
25 0.55 Low No No Yes No Yes No No −1.91 0 0 3 4.36
26 0.55 High Yes No No No Yes No No −2.52 0 3 2 3.43
29 0.55 High Yes Yes Yes Yes Yes No No −3.07 0 2 2 3.73
36 0.55 High Yes No No No No Yes No −4.66 0 0 1 2.13
37 0.55 High Yes No No No Yes No No −5.68 0 1 0 4.94
39 0.55 Low No Yes No No No Yes No −2.54 0 1 3 3.75
42 0.55 Low Yes No No No No Yes No −3.73 0 1 2 3.66
43 nd nd nd nd nd nd nd nd nd nd nd nd nd nd
44 0.55 Low No Yes Yes No No No No −0.46 0 2 3 4.28
45 0.55 High Yes Yes No No No No No −4.86 0 1 1 5.21
Metabolites 2023, 13, 281 24 of 28
4. Conclusions
As a country with huge biodiversity, Indonesia has abundant plant resources to be
used as herbal medicine or as metabolite reservoirs for drug discovery. Amongst them,
B. cernua is a potential medicinal plant to be explored since it has been traditionally used to
treat various diseases. We provided in vitro evidence that the methanol extract of B. cernua
(stem) exhibits antioxidant, antibacterial, antiplasmodial, and anticancer activities. In
addition, the plant has been traditionally demonstrated to have antiviral activity. Further
analysis revealed that the extract induced apoptosis in the MCF-7 breast cancer cell line
and protected erythrocytes from the ring formation stage of P. falciparum. Then, to iden-
tify important phytoconstituents, the extract’s metabolites were profiled using GC–MS
analysis, which revealed 45 compounds that could contribute to the plant’s therapeutic
functions. Bioinformatics was used for drug discovery to save time and chemical reagents.
Therefore, blind docking was performed to identify the binding affinity of the metabo-
lites toward target proteins, which revealed that N-[β-hydroxy-β-[4-[1-adamantyl-6,8-
dichloro]quinolyl]ethyl]piperidine and 1,3-phenylene, bis(3-phenylpropenoate) exhibited
the best results. Further analysis showed that the two molecules were predicted to have an
inhibition effect toward antioxidant, antiplasmodial, and anticancer protein models in a
competitive manner. Meanwhile, the two molecules were predicted to have an inhibitory
effect toward the antibacterial protein model in a noncompetitive manner. Site-specific
docking analysis showed that the two compounds exhibited similar binding energy to
the native ligands. Interestingly, their bioactivities have not yet been documented. The
drug likeness and pharmacokinetic analyses revealed that almost all identified compounds
can be considered promising drugs, showing good oral bioavailability, high absorption,
and good synthetic availability. Our results clearly indicate that the in silico studies were
supported by in vitro studies.
Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/metabo13020281/s1, Figure S1. Binding interaction of1,3-Phenylene,
bis(3-phenylpropenoate) compound with antioxidant target protein (3EUB) based on the binding
energy generated by PyRx program.A; Figure S2: Binding interaction of N-[.beta.-Hydroxy-.beta.-
[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl] piperidine compound with antibacterial target protein
(3VSL) based on the binding energy generated by PyRx program; Figure S3: Binding interaction of
N-[.beta.-Hydroxy-.beta.-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl] piperidine compound with an-
tiplasmodial target protein (3QS1) based on the binding energy generated by PyRx pro-gram; Figure
S4: Binding interaction of1,3-Phenylene, bis(3-phenylpropenoate) compound with an-tiplasmodial
target protein (3QS1) based on the binding energy generated by PyRx program; Figure S5: Bind-
ing interaction of N-[.beta.-Hydroxy-.beta.-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl] piperidine
compound with anticancer target protein (3GD4) based on the binding energy gener-ated by PyRx
program; Figure S6: Binding interaction of 1,3-Phenylene, bis(3-phenylpropenoate) compound with
an-ti-cancer target protein (3GD4) based on the binding energy generated by PyRx program; Figure
S7: 2D interaction of Antioxidant protein target (3EUB) and ligands; Figure S8: 2D interaction of
Antibacterial protein target (3VSL) and ligands; Figure S9: 2D interaction of Antioxidant protein
target (3EUB) and ligands; Figure S10: 2D interaction of Antiplasmodial protein target (3QS1) and
ligands; Figure S11: 2D interaction of Anticancer protein target (3GD4) and ligands; Figure S12: 2D
interaction of Anticancer protein target (3GD4) and ligands.
Author Contributions: Conceptualization, H.L.W. and S.; methodology, H.L.W. and D.K.; software,
H.L.W. and I.F.M.; validation, G.W.P. and A.R.A.; formal analysis, A.L.; investigation, A.R.A. and A.L.;
resources, S; data curation, H.L.W.; writing—original draft preparation, H.L.W.; writing—review and
editing, H.L.W., N.F., G.W.P., D.K., R.A.K., A.B. and I.F.M.; visualization, A.L. and I.F.M.; supervision,
H.L.W., N.F., G.W.P., D.K., R.A.K. and A.B.; project administration, A.L.; funding acquisition, H.L.W.
All authors have read and agreed to the published version of the manuscript.
Funding: The APC was funded by Directorate of Research and Community Engagement Universi-
tas Padjadjaran.
Institutional Review Board Statement: Not applicable.
Metabolites 2023, 13, 281 25 of 28
Informed Consent Statement: Not applicable.

Data Availability Statement: The main article and supplementary materials have already provided
all the data.
Acknowledgments: The authors acknowledge PT. Borneo Indobara-Indonesia and the Directorate of
Research and Community Engagement Universitas Padjadjaran.
Conflicts of Interest: The authors declare no conflict of interest.
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1 Department of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran,

Metabolites 2023, 13, 281. https://doi.org/10.3390/metabo13020281 https://www.mdpi.com/journal/metabolites

2. Materials and Methods

2.2. Gas Chromatography/Mass Spectrometry (GC–MS) Method

2.3. Antioxidant Activity Test

2.3.1. DPPH Method

2.3.2. Method for Superoxide Dismutase (SOD) Activity Assay

2.4. Minimum Inhibitory Concentration against Staphylococcus aureus

2.5. Antiplasmodial Assay

2.6. Cytotoxic Activity

2.7. Molecular Modeling (Receptors and Ligand Preparation)

2.8. Molecular Docking

2.9. Drug Likeness and Pharmakokinetic Analysis

3. Results and Discussion

3.2. Antioxidant and Antibacterial Test Results

3.3. Antiplasmodial Test Result

3.4. Anticancer Test Result

Metabolites 2023, 13, x FOR PEER REVIEW 10 of 27

B. cernua is known as an alternative medicine with antiviral properties toward small-

Figure 6. Binding interaction of N-[β-hydroxy-β-[4-[1-adamantyl-6,8-dichloro]quinolyl]ethyl] piperidine

Figure 6. Binding interaction of N‐[β‐hydroxy‐β‐[4‐[1‐adamantyl‐6,8‐dichloro]quinolyl]ethyl] pi‐

Figure 7. Binding interaction of 1,3-phenylene, bis(3-phenylpropenoate) compound with antibacterial

Plasmepsin I (PMI) from P. falciparum (pdb:3QS1) was selected as an antiplasmodial

3.6. Site-Specific Docking on Binding Site of Native Ligands

3.7. Drug Likeness and Pharmacokinetic Analyses

Table 3. Binding affinity of identified compounds against selected target proteins.

The pharmacokinetic parameters of each compound (Table 5) were predicted in terms

H-Bond Fraction Rotatable Consensus ESOL ESOL Violation

H-Bond Fraction Rotatable Consensus ESOL ESOL Violation

H-Bond Fraction Rotatable Consensus ESOL ESOL Violation

Table 5. Pharmacokinetic results of the identified compounds from B. cernua.

Bioavailability GI BBB P-gp Cyp Inhibitor Log Kp Alerts Likeness Synthetic

Bioavailability GI BBB P-gp Cyp Inhibitor Log Kp Alerts Likeness Synthetic

Informed Consent Statement: Not applicable.

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