In vitro antibacterial, antioxidant activities, molecular docking, and ADMET analysis of phytochemicals from roots of Hydnora johannis

Abstract

Hydnora johannis is a medicinal plant traditionally used to treat various ailments. Chemical investigation of the dichloromethane (DCM)/methanol (MeOH) (1:1) roots extract of Hydnora johannis afforded four compounds (1–3), reported herein for the first time from the species. The structures of the isolated compounds 1–3 were elucidated using 1D and 2D NMR spectroscopic analysis and comparison with literature data. The highest zone of inhibition value was measured for DCM/MeOH extract (10.75±0.25 mm) against Staphylococcus aureus at concentration of 0.25 mg/mL, promising in comparison to the standard amoxicillin (16.0±0.0 mm, 0.25 mg/mL). At concentration of 0.25 mg/mL,
the largest mean inhibition zone of 12.0±0.0 mm was measured for compound 2 against Pseudomonas aeruginosa, comparable to the standard drug amoxicillin (16.0±0.0 mm, 0.25 mg/mL). Compound 2 displayed better binding affinity with minimum binding energy of − 8.7 kcal/mol (PqsA), − 7.6 kcal/mol (DNA gyrase), and −7.4 kcal/mol ( S aureus PK) than amoxicillin (− 7.3, − 6.1, and −7.0 kcal/mol, respectively). This suggests that compound 2 may act as potential inhibitor of the tested bacterial proteins. Compound 1 satisfies the Lipinski’s rule of five with zero violations. Compound 2 obey the MW 452.4 g/mol) and iLogP (< 5) rules, and compound 3 obey the NHD (4) and NHA (6)
rules. Compounds 2 recorded iLogP value less enough than five (1.55), implying its optimal lipophilicity. Compounds 1 and 3 satisfy the veber’s rule (NRB < 12, and TPSA < 140 unit). Compound 2 and 3 exhibited negligible acute toxicity (LD50 > 5000, Toxicity class > 5. Compound 2 demonstrated maximum scavenging activity (67.87%) with IC50 value of 0.190 µg/mL, compared to ascorbic acid (78.21%) with IC50 value of 0.014 µg/mL at concentration of 12.5 µg/mL. Overall, the in vitro antibacterial activity of the extracts and compounds, molecular docking analysis and radical scavenging activity results of the isolated compounds suggest DCM/MeOH crude extract and compound 2 are promising antibacterial agents whereas compound 2 and 3 are promising antioxidants which corroborates with the traditional uses of the roots of H. johannis.

Keywords: Antibacterial, Molecular docking, Antioxidant, Hydnora johannis

Full text

Degeetal. Applied Biological Chemistry (2022) 65:76
https://doi.org/10.1186/s13765-022-00740-8
ARTICLE
In vitro antibacterial, antioxidant activities,
molecular docking, andADMET analysis
ofphytochemicals fromroots ofHydnora
johannis
Teshome Degfie1, Milkyas Endale1*, Tarekegn Tafese1, Aman Dekebo1,2* and Kebede Shenkute1
Abstract
Hydnora johannis is a medicinal plant traditionally used to treat various ailments. Chemical investigation of the
dichloromethane (DCM)/methanol (MeOH) (1:1) roots extract of Hydnora johannis afforded three compounds (1–3),
reported herein for the first time from the species. The structures of the isolated compounds 1–3 were elucidated
using 1D and 2D NMR spectroscopic analysis and comparison with literature data. The highest zone of inhibition
value was measured for DCM/MeOH extract (10.75 ± 0.25 mm) against Staphylococcus aureus at concentration of
0.25 mg/mL, promising in comparison to the standard amoxicillin (16.0 ± 0.0 mm, 0.25 mg/mL). At concentration of
0.25 mg/mL, the largest mean inhibition zone of 12.0 ± 0.0 mm was measured for compound 2 against Pseudomonas
aeruginosa, comparable to the standard drug amoxicillin (16.0 ± 0.0 mm, 0.25 mg/mL). Compound 2 displayed better
binding affinity with minimum binding energy of − 8.7 kcal/mol (PqsA), − 7.6 kcal/mol (DNA gyrase), and − 7.4 kcal/
mol ( S aureus PK) than amoxicillin (− 7.3, − 6.1, and − 7.0 kcal/mol, respectively). This suggests that compound 2
may act as potential inhibitor of the tested bacterial proteins. Compound 1 satisfies the Lipinski’s rule of five with
zero violations. Compound 2 obey the MW (452.4 g/mol) and iLogP (< 5) rules, and compound 3 obey the NHD (4)
and NHA (6) rules. Compounds 2 recorded iLogP value less enough than five (1.55), implying its optimal lipophilicity.
Compounds 1 and 3 satisfy the veber’s rule (NRB < 12, and TPSA < 140 unit). Compound 2 and 3 exhibited negligible
acute toxicity (LD50 > 5000, Toxicity class > 5. Compound 2 demonstrated maximum scavenging activity (67.87%) with
IC50 value of 0.190 µg/mL, compared to ascorbic acid (78.21%) with IC50 value of 0.014 µg/mL at concentration of
12.5 µg/mL. Overall, the in vitro antibacterial activity of the extracts and compounds, molecular docking analysis and
radical scavenging activity results of the isolated compounds suggest DCM/MeOH crude extract and compound 2 are
promising antibacterial agents whereas compound 2 and 3 are promising antioxidants which corroborates with the
traditional uses of the roots of H. johannis.
Keywords: Antibacterial, Molecular docking, Antioxidant, Hydnora johannis
© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
Introduction
e genus Hydnora (Hydnoraceae) includes approxi-
mately eight taxonomically identified species [1], the only
known angiosperm with no leaves or scales of any sort [2,
3], all are chiefly hypogenous, root-parasitic herbs, with
an extremely reduced vegetative body (Additional file1:
Fig. S13), which has no leaves or bracts and consists of
a massive, fleshy horizontal rhizome like root covered
Open Access
*Correspondence: milkyas.endale@astu.edu.et; amandekeb@gmail.com
1 Department of Applied Chemistry, Adama Science and Technology
University, P.O. Box 1888, Adama, Ethiopia
2 Institute of Pharmaceutical Sciences, Adama Science and Technology
University, P.O. Box 1888, Adama, Ethiopia

Page 2 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
with haustoria roots as wart like outgrowth [1, 2]. Mem-
bers of this genus have long been used in folk medicine
to treat various ailments, such as diarrhea, cholera, hem-
orrhoid, swollen abdomen, lung cancer, breast cancer,
and diabetic [4, 5]. Phytochemical constituents of genus
Hydnora include flavonoids, tannins, terpenoids, sapo-
nins, alkaloids, and essential oils [6, 7]. ese compounds
exhibit diverse bioactivities, including antioxidant, anti-
bacterial, antifungal, hepatoprotective, and anticancer
[8]. Hydnora johannis Becc is a subterranean holopara-
sitic (non-photosynthetic) herb with often massive root
systems spreading laterally from the host. It is an obli-
gate root parasite found on Acacia and Albiziz species
as well as Kigelia africana; growing between 1000 and
1500 m altitudes [1]. It is widely distributed in Ethiopia,
Eritrea, Somalia, Kenya, Tanzania, Rwanda and Demo-
cratic republic of Congo [2, 9]. In Ethiopia, it is identified
by vernacular name Licce, Like, Likke, Lipti (Som), Dech-
Merech (Oro), and available in different part of the coun-
try including Shewa, Sidamo, Bale, Arsi and Hararge,
[10, 11]. Various traditional uses of the plant have been
reported in Ethiopia, including treating breast cancer,
lung cancer, diarrhea, hemorrhage, wound and painful
body swelling, locally called GOFLA (Oromo language)
around Erer Valley of Babile Wereda, Eastern Ethiopia
[10–12]. Despite the widespread ethnomedicinal uses of
this plant for ailments of various diseases, there exists
no detailed study reports on the phytochemical analysis
and pharmacological properties of H. johannis. Hence,
reported herein are the molecular docking analysis (for
the first time), antibacterial, antioxidant, and isolation
of threecompounds from the root of dichloromethane/
methanol (1:1) extract of H. johannis.
Experimental part
General experimental procedure
e NMR spectra were recorded on Bruker Avance
400 MHz spectrometer with deuterated chloroform
and methanol using TMS as internal standard. Analyti-
cal TLC plates with silica gel 60 F254 TLC (Merck, Ger-
many) were used to determine TLC profiles. e spots
on TLC plates were visualized using a UV lamp (254 and
365 nm). Silica gel column chromatography was per-
formed at silica gel (60–120 mesh). All chemicals, sol-
vents and reagents were used to analytical grade level.
Plant material collection andidentication
e roots of H. johannis were collected from the district
of Huruta town, Lode Hetosa Woreda, Arsi Zone, Oro-
mia, Ethiopia, in September 2019. e plant material
was authenticated by Melaku Wendafrash (Chief Tech-
nician, Botanist), and voucher specimen (TCY010) was
deposited at the National Herbarium, Department of
Biology, Addis Ababa University, Ethiopia.
Extraction and isolation of compounds from the roots of
H. johannis
e powdered roots of H. johanns (500 g) were extracted
with n-hexane for 72 h, three times at room tempera-
ture to obtain 18.3 g crude extract. e marc was fur-
ther extracted with dichloromethane (DCM)/methanol
(MeOH) (1:1), and MeOH (100%). e filtrates were
concentrated in vacuum a rotary evaporator at 40oC to
obtain 30.6 g, and 24 g of crude extracts, respectively.
e DCM/MeOH (1:1) root extract (10 g) was sub-
jected to silica gel column chromatography (silica gel
180 g) and eluted with increasing gradient of n-hexane/
EtOAc followed by dichloromethane/methanol mix-
tures. A total of 119 fractions were collected (each
50 mL). Compound 1 (58 mg) was obtained from fraction
27-31using n-hexane/EtOAc (7:3) as eluent. Fractions
100–105 obtained with DCM/MeOH (7:3) were com-
bined and purified using isocratic elution with 5% MeOH
in DCM as eluent to afforded compound 2, (57 mg) and
compound 3, (20 mg).
Antibacterial activity
Isolated compounds 2 and 3, and n-hexane, DCM/
MeOH (1:1), and MeOH extracts were studied for in-
vitro antibacterial activity against four standard human
bacterial pathogens; namely, Staphylococcus aureus (S.
aureus, ATCC 25,923), Escherichia coli (E. coli, ATCC
25,922), Pseudomonas aeruginosa (P. aeruginosa, ATCC
27,853) and Streptococcus pyogen (S. pyogen ATCC19615)
collected from Ethiopian Public Health Institute (EPHI).
Experiments were done at the microbiology laboratory
of the AppliedBiology Department, Adama Science and
Technology University, in collaboration with microbiolo-
gists. e degree of susceptibility of each bacterial strain
to the isolated compounds was evaluated by using the
agar medium disc-diffusion technique as per the stand-
ard protocols of Clinical and Laboratory Standards Insti-
tute (CLSI) [13]. at is, about 2–4 fresh colonies with
similar morphology of each bacterial species were asep-
tically transferred into a saline solution using a sterile
inoculating loop, and bacterial turbidity was adjusted in
reference to 0.5 McFarland standard solution (108 CFU/
mL). e Mueller Hinton Agar (HiMedia) medium was
prepared as per the manufacturer’s instruction. e bac-
terial suspension was then streaked by swapping with
cotton swap onto Petri plates containing the medium.
A sterilized Whatman No. 1 filter paper discs (6 mm in
diameter) were prepared using a puncher to hold sam-
ples. A stock solution of each isolated compound (5 mg
in 5 mL) was prepared in 4% DMSO. ereafter, different

Page 3 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
solutions, 1.00 mg/mL, 0.5 mg/mL and 0.25 mg/mL of
each extract, and 0.5 mg/mL and 0.25 mg/mL of each
compound (2 and 3) were prepared from their corre-
sponding stock solutions. Standard antibiotic disc of
Amoxicillin (0.25 mg/mL) and DMSO were served as
positive and negative controls, respectively. Each solution
of 100 µL was loaded onto separate paper discs (6 mm
in diameter), placed discs onto the Petri plates with the
bacterial culture inoculated MHA, and incubated at 37
ºC for 18–24 h. Inhibition zones were monitored by the
paper disc’s clear area and measured by a caliper (in mm)
[13, 14]. e experiment was done in duplicate asepti-
cally, and the result was expressed as mean ± standard
deviation using statistical analysis software of SPSS (ver-
sion 20).
Molecular docking studies oftheisolated compounds
In this study, the isolated compounds 1–3 were docked
with proteins E. coli DNA gyrase B (PDB ID: 6F86), PqsA
(PDB ID: 3T07) and S. aureus Pyruvate Kinase (PK) (PDB
ID: 5OE3) as target proteins. AutoDock Vina 4.2 (MGL
tools 1.5.7) was used to perform the molecular dock-
ing analysis to predict the potential binding modes of
the test compounds with the target bacterial proteins
[15]. e docking calculation results include binding
energy (kcal/mol), hydrogen bond distances, and picto-
rial representation of viable docked poses. Chem Office
tools (ChemDraw 16.0) was used to draw the structures
of the compounds (1–3) with appropriate 2D orienta-
tions. Minimized energy of the compounds/ligands
were obtained using ChemDraw3D ultra. e energy
minimized ligand molecules were then used as input for
AutoDock Vina to carry out the docking simulation.
Crystal structures of the receptor protein molecules
were obtained from the protein data bank (PDB). e
coordinates of the structures were complexed with
water molecules and other atoms which are responsi-
ble for increased resolution. e proteins were prepared
using protein preparation protocol [16], applying default
parameters. e target protein file was generated by leav-
ing the associated residue (remove water molecules and
cofactors) with protein by using Auto Preparation of
target protein file AutoDock 4.2 (MGL tools1.5.7). e
grid box for docking simulation with size of 50 × 50 × 54
Å points with a grid spacing of 0.375 Å was adjusted
using the graphic user interface program in such a way
that it surrounds the region of interest in the macro-
molecule. e potential binding energy between ligand
and protein was obtained using the docking algorism
provided by AutoDock Vina. e conformations with
the most favorable (least) binding energy between the
target proteins and compounds were selected for ana-
lyzing the interactions between the target receptor and
ligands by Discovery studio visualizer and PyMOL. e
ligands are represented in different colors, H-bonds and
the interacting residues are represented in stick model
representation.
In Silico pharmacokinetic analysis
Pharmacokinetic parameters were evaluated for the isolated
compounds to investigate their drug candidate chances. e
ADMET properties were evaluated with the aid of Swis-
sADME, an online ADME prediction tool [17]. e drug-
likeness of the compounds were predicted by adopting the
Lipinski’s Rule of five [18]. Structure of the isolated com-
pounds (1–3) were converted to their canonical simplified
molecular-input line-entry system (SMILE) and submitted
to SwissADME and PreADMET tool to estimate in-silico
pharmacokinetic parameters [17]. e toxicity profile of the
studied compound (1–3) was predicted using ProTox-II Web
tool [19]. e selection of the compounds as drug candidates
was determined by a parameter called drug score. e higher
the drug score value, the higher the compound’s chance is
considered a drug candidate [20].
Radical scavenging activity
e antioxidant activity of each of the isolated compounds
from the root of H. johannis was evaluated based on the
scavenging activity of the stable2,2-diphenyl-1-picrylhydra-
zyl (DPPH) free radical [21]. Each sample was separately dis-
solved in methanol and serially diluted to give 200, 100, 50,
and 25 µg/mL concentrations. Freshly prepared 0.04% DPPH
solution in methanol was added to each concentration. e
mixture was shaken and incubated in an oven at 37oC for
30 min. e absorbance of the resultant solution was meas-
ured at 517 nm using UV-Vis spectrophotometry. Ascorbic
acid with a similar concentration of test sample was used as
the positive control. e DPPH radical scavenging activity of
each of the tested compounds was reported by percentage
inhibition using the following formula:
where
Acontrol
is the absorbance of DPPH solution and
Asample
is the absorbance of the test sample (DPPH solu-
tion plus compound) [21]. e DPPH radical scaveng-
ing activity of the compounds was also expressed as
IC50, the concentration of the test compound to give a
50% decrease of the absorbance from that of the control
solution.
Results anddiscussion
e DCM/MeOH (1:1) root extracts of H. johannis was
subjected to gravity column chromatography over silica
gel to afford three compounds(1–3) (Fig. 1), reported
%
DPPH inhibition =
(A
control −
A
sample
)
Acontrol
×
100%

Page 4 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
from this plant for the first time. e structures of each
of the isolated compounds were elucidated by different
spectroscopic techniques and comparison of their spec-
troscopic data to those previously reported in the litera-
ture. Compound 1 was isolated as a yellowish solid. e
melting point of the compound was found to be in the
range of 134 to 136oC [22]. e TLC profile showed Rf
value of 0.52 with n-hexane/EtOAc (7/3) mobile phase.
e 1H NMR (400 MHz, CDCl3) spectrum displayed sig-
nals of olefinic protons at δ 5.33 (m, H6), methine pro-
ton signal at δ 3.51 (H3) assignable to an sp3 oxygenated
methine proton (Table1, Additional file1: Fig. S1).e 1H
NMR also showed proton signals at δ 2.00 (m, 2H, H7),
2.33 (m, 2H, H4) attributed to methylene and methine
protons, respectively, and six methyl protons at 0.99
(H19), δ 0.87 (H21), 0.85 (H26), 0.81 (H27), 0.76 (H29),
and 0.67 (H18). e remaining proton signals overlapped
in the range of δ 1.26 to 2.00. e 13C NMR (100 MHz,
CDCl3) spectrum displayed 29 carbon signals includ-
ing two olefinic carbon signals at δ 140.8 (C5), 121.8
(C6), of which the former is sp2 quaternary carbon, one
sp3methine at δ 71.9 (C3), two sp3 quaternary carbons at
δ 36.6 (C10) and 42.4 (C13), seven methine signals at δ
32.0 (C8), 50.2 (C9), 56.8 (C14), 56.1 (C17), 36.2 (C20),
45.9 (C24) and 29.2 (C25), six methyl carbons at δ 19.9
(C26), 19.5 (C27), 19.1 (C19), 18.8 (C21), 12.1 (C29) and
11.9 (C18), and eleven methylene carbon signals [δ 21.2
(C11), 23.1 (C28), 24.4 (C15), 26.1 (C23), 28.3 (C16),
31.6 (C2), 32.0 (C7), 34.1 (C22), 37.3 (C1), 39.8 (C12),
42.2 (C4)] (Table 1, Additional file 1: Figs. S2 and S3).
e obtained 1H/13C NMR spectral data are found to be
consistent with reported literature data of β-sitosterol,
Figs.1, [23, 24] reported herein for the first time from
roots of H. johannis.
Compound 2, was obtained as a brown solid. e TLC
profile showed Rf value of 0.47 with DCM/MeOH (9/1)
mobile phase. e 1H NMR spectrum revealed the pres-
ence aromatic protons at δ 6.86 (d, J = 1.9 Hz, 1H, H2’),
δ 6.78 (d, J = 8.1 Hz, 1H, H5’) and δ 6.73 (dd, J = 8.1,
1.9 Hz, 1H, H6’) with AMX spin system (ring B), and
at δ 5.95 (d, J = 2.3 Hz, 1H, H6) and δ 5.87 (d, J = 2.3 Hz
1H, H8) assignable to AX type system (ring A) (Addi-
tional file1: Table S-1; Fig. S4). e protons signals at δ
4.58 (d, J = 7.5 Hz, 1H, H2)/3.94 (m, 1H, H3), and at 2.87
(dd, J = 16.1, 5.5 Hz, 1H, H4a)/2.52 (dd, J = 16.1, 5.5 Hz,
1H, H4b) were assignable to methine and methylene
protons of ring C, respectively. e 1H NMR also dis-
played proton signals assignable to sugar moiety at δ 4.31
(d, J = 7.7 Hz, 1H, H1’’), and ranging δ 3.21 to 3.71 (6H,
H2’’ to H6’’) (Table2). e 13C NMR (100 MHz, CD3OD)
spectra displayed 21 well resolved carbon peaks. e
peaks at δ 157.6 (C5), 157.4 (C7), 156.7 (C9), 146.1 (C3’),
146.0 (C4’), 132.1 (C1’) and 100.7 (C10), are attributed to
aromatic quaternary carbons of ring A and B of the flavan
moiety (Additional file1: Table S-1; Fig. S5 and S6). e
carbon signals at δ 96.2 (C6), 94.5 (C8), 115.2 (C2’), 116.1
(C5’) and 120.0 (C6’) are in agreement with aromatic
methine signals of ring A and B. e DEPT-135 spectrum
(Additional file1: Fig. S6) displayed methine and methyl-
ene carbons but no methyl carbon. e carbon methine
carbon signals at δ 82.6 and 68.6, and methylene signals
at δ 28.3 (C4) attributed to the oxy-methine (C2 and C3,
respectively) and methylene (C4) of ring C of the flavan
moiety. Furthermore, the 13Cspectrum revealed charac-
teristic sugar peaks at δ 104 (C1”), 75.0 (C2”), 77.9 (C3”),
72.0 (C4”), 77.7 (C5”), and 61.6 (C1’’). e methylene
absorption at δ 61.6 suggests the sugar to be glucopyra-
nose group. e linkages and substitution patterns were
established from the COSY and HMBC analyses (Addi-
tional file1: Table S-1; Figs. S7–S9). e HMBC spectrum
displayed 3J correlations between proton at δ 4.31 (H1”)
Table 1
1H, 13C NMR spectral data of compound 1
Compound 1 13C [23]
Position 1H 13C
1 37.3 37.2
2 31.6 31.6
3 3.51 (m, 1 H) 71.9 71.8
4 2.33 (m, 1 H) 42.2 42.3
5 140.8 140.7
6 5.33 (m, 1 H) 121.8 121.7
7 2.00 (d, J = 7.0 Hz, 2 H) 32.0 31.9
8 32.0 31.9
9 50.2 50.1
10 36.6 36.5
11 22.8 21.1
12 39.8 39.8
13 42.4 42.3
14 56.8 56.7
15 24.4 24.3
16 28.3 28.2
17 56.1 56.0
18 0.67 (s, 3 H) 11.9 11.8
19 0.99 (s, 3 H) 19.1 19.4
20 36.2 36.1
21 0.87 (d, J = 6.9 Hz, 3 H) 18.8 18.8
22 34.1 33.9
23 26.1 26.1
24 45.9 45.8
25 29.2 29.1
26 0.85 (d, J = 6.9 Hz, 3 H) 19.9 19.8
27 0.81 (d, J = 7.2 Hz, 3 H) 19.5 19.0
28 23.1 23.1
29 0.76 (m, 3 H) 12.1 12.0

Page 5 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
with 96.2 (C6) and proton 5.87 (H8) with 104.1 (C1”) sug-
gests the attachment of the glucopyranose moiety to C7
of ring A. e spectral data of compound 2 obtained in
this study is found to be in good agreement with reported
data of catechine-7-O-glucoside [25]. us, based on the
reported spectral data and the data from literature [25],
compound 2 is identified to be flavan-3-ol-7-O-glucoside
(catechine-7-O-glucoside, Fig.1), reported herein for the
first time from roots of H. johannis.
Compound 3 was obtained as a pale brown solid. e
TLC profile showed Rf value of 0.56 with DCM/MeOH
(9/1) mobile phase. e 1H NMR (400 MHz, CD3OD)
spectrum displayed signals assignable to olefinic pro-
tons at δ 5.25 (H6), oxygenated methine proton at δ 4.29
(H3), methylene protons at δ 2.31 (dd, 2H, H4), and six
methyl protons at δ 1.14 (H19), 0.89 (H29), 0.81 (H21),
0.80 (H18), 0.73 (H26), and 0.71 (H27) (Table2, Addi-
tional file 1: Fig. S10). e proton signals observed in
the range of 3.12 to 4.29 were assignable to sugar moi-
ety. e remaining proton signals overlapped in the range
of δ 1.26 to 2.00. e 13C NMR (100 MHz, CDCl3) spec-
trum displayed 35 carbon signals including olefinic car-
bon at δ 140.7 (C5) and 121.7 (C6), of which the former
suggest sp2 quaternary carbon, sp3oxy-carbon at δ 78.9
(C3), two sp3 quaternary carbons at δ 36.5 (C10) and 42.3
(C13), and methine carbons at δ 31.6 (C8), 49.9(C9), 56.5
(C14), 55.8 (C17), 35.9 (C20), 45.6 (C24) and 28.8 (C25),
six methyl carbons at δ 19.5 (C19), 19.0 (C26), 18.7 (C27),
18.5 (C21), 11.6 (C29) and 11.5 (C18), and six carbons
assignable to sugar moiety at 100.8 (C1’), 76.1 (C5’), 75.5
(C3’), 73.3 (C2’), 69.7 (C4’) and 61.4 (C6’) (Table2, Addi-
tional file1: Fig. S11). e DEPT-135 spectrum displayed
the presence of 12 methylene signals including one
belong to the glucopyranose moiety (Additional file 1:
Fig. S12). e spectral data of compound 3 that obtained
in this study is found to be in good agreement with pre-
viously reported data of β-Sitosterol-3-O-β- D-glucoside
[26], Fig.1, reported herein for the first time from the
roots of H. johannis.
Antibacterial activity
e invitro antibacterial activities of n-hexane, DCM/
MeOH (1:1) and MeOH (100%) extracts from the root
of H. johannis and isolated compounds 2 and 3 from
DCM/MeOH (1:1) were tested against E. coli, S. aureus,
P. aeruginosa, and S. pyogen pathogens at concentrations
of 0.25, 0.50, and 1.00 mg/mL. e average inhibitory
zone diameters (in mm) of the extracts and identified
compounds (2 and 3) against the growth of each bacte-
rial strain are shown in Tables3 and 4, respectively. In
most cases, the DCM/MeOH (1:1) extract was shown to
be more active than the n-hexane and MeOH extracts
in inhibiting the growth of each of the tested bacterial
strains Table3). At the concentration of 0.25 mg/mL, the
Table 2
1H, 13C NMR spectral data of compound 3
position Comppund 3 13C [26] Position Compound 3 13C [26]
1H 13C 1H 13C
1 1.89 (m) 37.0 36.7 19 1.14 (s) 19.5 19.4
2 1.37 (m) 29.4 29.2 20 35.9 35.4
3 3.59 (m, 1 H) 78.9 76.8 21 0.81 (d) 18.5 18.5
4 2.31 (dd) 39.5 39.2 22 1.18 (m) 33.7 33.2
5 140.7 140.4 23 1.16 (m) 25.7 25.3
6 5.25 (bs, 1 H) 121.7 121.2 24 45.6 45.1
7 1.94 (s) 31.7 31.3 25 1.50 (m) 28.8 28.6
8 1.48 (m) 31.6 31.2 26 0.73 (d) 19.0 19.6
90.89 (m) 49.9 49.5 27 0.71 (d) 18.7 18.8
10 36.5 36.1 28 1.37 (m) 22.8 22.1
11 1.48 (m) 20.8 20.5 29 0.89 (m) 11.6 11.7
12 1.15 (m) 38.4 38.2 1’ 4.42 (d) 100.8 100.7
13 42.3 41.8 2’ 3.20 (m) 73.3 73.4
14 1.04 (m) 56.5 56.1 3’ 3.31 (m) 75.5 76.8
15 24.0 23.8 4’ 3.30 (m) 69.8 70.0
16 1.75 (m) 28.0 27.7 5’ 3.46 (m) 76.1 76.7
17 1.13 (m) 55.8 55.3 6’ 3.71 (m) 61.4 62.8
18 0.80 (s) 11.5 11.6

Page 6 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
extracts displayed promising growth inhibitory effect
against all the selected bacterial strains. e highest
inhibition zone was measured for DCM/MeOH extract
(10.75 ± 0.25 mm) against S. aureus, which is promising in
comparison to the standard, amoxicillin (16.0 ± 0.0 mm).
In addition, the DCM/MeOH extract exhibited growth
inhibition effect against P. aeruginosa, E. coli, and S. pyo-
gen, with mean inhibition zones of 10.5 ± 0.5, 9.5 ± 0.5,
and 8.5 ± 0.5 mm, respectively. At the same concentration
of 0.25 mg/mL, the n-hexane extract scored maximum
mean inhibition zone of 9.5 ± 0.5 mm against S. pyogen,
followed by 9.25 ± 0.25, 8.5 ± 0.5, and 8.25 ± 0.25 mm
against S. aureus, P. aeruginosa, and E. coli, respectively.
e MeOH extract displayed highest inhibitory effect
against S. aureus, with inhibition zone of 8.5 ± 0.0 mm,
and then against S. pyogen (8.25 ± 0.25 mm), P. aerugi-
nosa (8.00 ± 1.0 mm), and E. coli (7.75 ± 0.75 mm). In
comparison to the n-hexane and MeOH extract, at higher
concentration of 1.0 mg/mL) also, DCM/MeOH extract
showed the highest mean inhibition zone (14.25 ± 0.25)
against P. aeruginosa, than against the other studied bac-
terial strains. Likewise, the n-hexane and MeOH extract
showed good antibacterial activity against S. pyogen
(13.75 ± 0.75, and 12.25 ± 0.75 mm respectively) than the
other tested bacterial strains. e result also showed that,
at 0.25 mg/mL, all the three extracts (n-hexane, DCM/
MeOH, and MeOH) showed significant growth inhibi-
tion against the gram-positive bacterial strain S. aureus
(9.25 ± 0.25, 10.75 ± 0.25, and 8.5 ± 0.0 mm), which is
considerable compared to the standard drug, amoxicillin
(16.0 ± 0.0 mm). e in vitro antibacterial activity result
shows that the tested bacterial strains are susceptible
to each extract. In a previous study, the MeOH extract
of roots of H. johannis showed mean inhibitory effects
against E. coli, P. aeruginosa, and S. aureus at mean inhi-
bition zone of in 9 mm, 8 mm, and 8 mm, respectively [7],
which is in agreement with the result reported in current
study.
e antibacterial activity of compound 1 (β-sitosterol)
has been studied by many researchers. According to
Sileshi and his co-workers, report, β-sitosterol found
to have low/moderate antibacterial activity against sev-
eral bacterial species that include E. coli, S. aureus and
P. aeruginosa [24]. Other reports also indicate that the
compound demonstrated significant growth inhibition
effect against both gram-positive and gram-negative
bacterial strains [22, 23]. In this work the invitro anti-
bacterial of compounds 2 and 3 were reported. Table4
Table 3 Inhibition zone (mean ± SD, in mm) of n-hexane, DCM/MeOH (1:1) and MeOH extracts
Samples Concentrations (mg/mL) Bacterial strains
E. coli P. aeruginosa S. aureus S. pyogen
n-Hexane 0.25 8.25 ± 0.25 8.5 ± 0.5 9.25 ± 0.25 9.5 ± 0.5
0.5 10.25 ± 0.25 9.5 ± 0.5 10.5 ± 0.5 11.25 ± 0.25
1 11.75 ± 0.25 11.5 ± 0.5 12.5 ± 0.5 13.75 ± 0.75
DCM/MeOH 0.25 9.5 ± 0.5 10.5 ± 0.5 10.75 ± 0.25 8.5 ± 0.5
0.5 11 ± 0.0 12.25 ± 0.25 11.5 ± 0.5 9.5 ± 0.5
1.0 13.75 ± 0.25 14.25 ± 0.25 13 ± 0.0 11.5 ± 0.5
MeOH 0.25 7.75 ± 0.75 8.00 ± 1.0 8.5 ± 0.0 8.25 ± 0.25
0.5 9 ± 1.0 9.25 ± 1.25 10 ± 1.0 9.75 ± 0.75
1.0 10.25 ± 0.5 11.5 ± 1.0 12.25 ± 0.75 11.5 ± 0.5
Amoxicillin (0.25 mg/mL) 15.25 ± 0.25 15.25 ± 0.25 16.0 ± 0.0 16.25 ± 0.25
Table 4 Inhibition zone (mean ± SD, in mm) of isolated compounds 2 and 3
Conc. (mg/mL) Compounds Bacterial strains
E. coli P. aeruginosa S. aureus S. pyogen
0.25 2 11.5 ± 0.5 12.0 ± 0.0 10.75 ± 0.25 11.0 ± 0.0
3 9.0 ± 0.0 10.75 ± 0.25 10.0 ± 0.5 9.5 ± 1.5
0.50 2 12.75 ± 0.75 13.5 ± 0.5 11.75 ± 0.25 12.0 ± 0.0
3 11.25 ± 0.25 11.75 ± 0.75 11.5 ± 0.5 11.0 ± 0.5
Amoxicillin (0.25 mg/mL) 15.25 ± 0.25 16.0 ± 0.0 15.25 ± 0.25 16.25 ± 0.25

Page 7 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
displayed the mean inhibition zone of isolated com-
pounds 2 and 3 against E. coli, P. aeruginosa, S. aureus,
and S. pyogen. e obtained reported shows that the
tested compounds 2 and 3 exhibited growth inhibi-
tory effect against all the tested bacterial strains in a
dose dependent manner. At concentration of 0.25 mg/
mL, the larger average growth inhibition zone of
12.0 ± 0.0 mm was measured for compound 2 against P.
aeruginosa, which is good in comparison to the stand-
ard drug, amoxicillin (16.0 ± 0.0 mm). Compound 2
also showed the highest antibacterial activity against
E.coli, S. pyogen, and S. aureus with average inhibition
zone of 11.5 ± 0.5, 11.0 ± 0.0, and 10.75 ± 0.25 mm,
respectively. Compound 3 recorded the next largest
inhibitory effect against P. aeruginosa with inhibition
zone of 10.75 ± 0.25 mm, and then against S. aureus
(10.0 ± 0.5), 9.5 ± 1.5 (S. pyogen), and 9.0 ± 0.0 mm (E.
coli). Similarly, at higher concentration, 0.50 mg/mL,
the larger mean inhibition zone of 13.5 ± 0.5 was meas-
ured for compound 2 against P aeruginosa. Results
from previous antibacterial activity study result, using
agar diffusion method, showed that compound 3 exhib-
ited mean inhibition zone of 0.9 mm against each P. aer-
uginosa and S. aureus at concentration of 0.25 mg/mL
[27]. e mean inhibition zone reported for compound
3 in our study is good agreement with the previous one.
e overall antibacterial activity result shows, irrespec-
tive of their lower antibacterial activities relative to the
standard compound (amoxicillin), indicated that com-
pounds 2 and 3 exhibited promising growth inhibitory
effect against the tested bacterial strains, which support
the use of H. johannis in tradition medication system.
Molecular docking studies oftheisolated compounds
e molecular docking study was carried out to assess
the binding affinity and binding interactions of isolated
compounds 1–3 toward target proteins E.coli DNA
gyrase B (PDB ID 6F86), S. aureus PK (PDB ID: 3T07),
and PqsA (PDB ID: 5OE3). e binding affinity, H-bond,
and residual interaction of all the isolated compounds are
summarized in Tables5, 6 and 7.e docked compounds
1–3 displayed minimum binding energies ranged from
− 7.6 to −6.2 kcal/mol, −8.7 to −8.0 kcal/mol, and − 7.4
to −7.0 kcal/mol, with DNA gyrase, PqsA, and S. aureus
PK, respectively (Tables5, 6 and 7). Such low docking
score values signify a good interaction of the compounds
with protein binding pocket. In the study, each of the
isolated compounds (1–3) demonstrated better binding
affinity (binding energy of −7.6 to −6.2 kcal/mol) against
protein DNA gyrase than the standard drug, amoxicillin
(−6.1 kcal/mol). e highest binding affinity (−7.6 kcal/
mol) was recorded by compound 2 (− 7.6 kcal/mol) fol-
lowed by compound 3 (−6.5 kcal/mol). When docked to
the protein PqsA, each compound (1–3) displayed higher
binding affinities (−8.7 to −8.0 kcal/mol) compared to
the standard, amoxicillin (− 7.3 kcal/mol). Compound 2
showed the highest binding affinity (− 8.7 kcal/mol), fol-
lowed by compound 3 (−8.6 kcal/mol) and compound
1 (−8.0 kcal/mol). e binding affinities of the ligands
(1–3) to the protein S. aureus PK (−7.4 to − 7.0 kcal/
mol) were found to be equal to or better than amoxicillin
(−7.0 kcal/mol). Of these, the strongest binding affinity
was displayed by compound 2 (− 7.4 kcal/mol). In gen-
eral, at each ligand-protein pose, compound 2 scored the
highest binding affinity [− 8.7 kcal/mol (against DNA
gyrase), − 7.6 kcal/mol (PqsA), and − 7.4 kcal/mol (S
aureus PK)]. Compounds 1 and 3 also scored significant
binding affinity [DNA gyrase (−6.2 and − 6.5 kcal/mol),
PqsA (−8.0 and − 8.6 kcal/mol, S. aureus PK (−7.0 kcal/
mol each)].
Based on the molecular docking analysis results, the
isolated compounds (1–3) displayed H-bond, hydro-
phobic van der Waals interactions with binding pockets
of DNA gyrase, PqsA, and S. aureus PK (Tables6, 7 and
8). e amino acid residues that are involved in hydro-
gen bonds, hydrophobic, and van der Waals interactions
at each ligand- protein complexes were also mapped
using AutoDock Vina (Figs. 2, 3 and 4, and Tables 6, 7
and 8).e binding affinity of compound 2 with gyraseB
(-7.6 kcal/mol) was stabilized by hydrogen bonds at
amino acid residues Glu-50, Gly-77 and Ser-121, hydro-
phobic interaction at Ile-94 and Ile-78, and van der Waals
interaction at Arg-76, Pro-79, Gly-75, Asp-73, r-165,
Table 5 Docking scores of isolated compounds 1–3 against E. coli DNA (PDB ID: 6F86)
Cmpds Anity (kcal/
mol) H-bond Residual interactions
Hydrophobic Van Dar Waals
1− 6.2 Val-97 Ile-78, Ile-94, Pro-79 Arg-76, Glu-50, Asn-46, Asp-49, Ale-98, Ser-121
2− 7.6 Glu-50, Gly-77, Ser-121 Ile-94, Ile-78 Arg-76, Pro-79, Gly-75, Asp-73, Thr-165, Gly-164, Asn-46,
Gly-117, Val-118, Leu-98, Val-97, Gly-119, Val-120
3− 6.5 Asn-46, Gly-77, Arg-76 Leu-52, Ala-53, Pro-79 Sp-49, Ile-78, Ile-94, Ala-47, Gly-75
Amox − 6.1 Arg-76, Asp-49 Asp-73, Ala-47, Ile-78, Asn-46, Ile-94, Pro-79, Gly-75, Gly-164

Page 8 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
Gly-164, Asn-46, Gly-117, Val-118, Leu-98, Val-97, Gly-
119 and Val-120. e docking of compound 1 and 3 with
DNA gyrase stabilized by H-bond, hydrophobic van der
Waals interactions across various amino acid residues
(Table5; Fig.2). At its heavy negative binding energy of
−8.7 kcal/mol with protein PqsA, compound 2 displayed
hydrogen bonds with residual amino acids Pro-85, Lys-
86, and Ala-108, hydrophobic interaction with Ala-124,
Leu-60, Arg-128, and Ala-108, and large number (ten)
of Van Dar Waals interaction with Arg-106, Pro-111,
Ala-110, Asp-109, Asn-61, Phe-134, Leu-66, Ser-65, Ala-
125, A: Gly-127, and Ala-126. Similarly, the low binding
energy interactions of compound 1 (− 8.0 kcal/mol) and
3 (−8.6 kcal/mol) with protein PqsA involved H-bonds,
hydrophobic and van der Waals interactions across vari-
ous residual amino acids at its binding pocket (Table6;
Fig.3). In its interaction with S. auruesPK, compound 2
(binding energy of −7.4 kcal/mol) displayed seven hydro-
gen bonds at Glu-234, Leu-233, Ser-236, Asp-237, Lys-
271, Asn-357, and Leu-269, which is assumed to boost
its binding affinity (Table7; Fig. 4). Likewise, the dock-
ing result revealed that the recorded binding affinities of
compound 1 and 3 with the protein S. aureus (−7.0 kcal/
mol each) were mediated by hydrogen bond, hydro-
phobic, and van der Waals interactions that are formed
across various amino acids. Overall, the results obtained
from the molecular docking study agreed with the
invitro antibacterial activity against E.coli, P. aeruginosa,
and S. aureus. e highest binding affinities of compound
2 to DNA gyrase (−7.6 kcal/mol), PqsA (−8.7 kcal/mol),
and S. aureus (−7.4 kcal/mol) are in agreement with its
highest invitro antibacterial activity result. In addition,
at each docking pose, all the tested compounds (1–3)
scored binding affinities better than the standard amoxi-
cillin. e binding affinity, H-bond and residual amino
acid interactions of the three compounds and amoxicillin
are summarized in Tables5, 6 and 7. e mapped binding
interactions of compounds 1–3 against targeted proteins
are displayed in Figs.2, 3 and 4. Ribbon model shows the
binding pocket structure of target proteins with the com-
pounds. Hydrogen bond between compounds and amino
acids are shown as green dash lines, hydrophobic interac-
tion are shown as pink lines.
In silico pharmacokinetic analysis
e in silico drug-likeness of the isolated compounds
1–3 was determined based on the concept of Lipinski’s
rule of five [18] and Veber’s rule [28]. Results are given
in Table8. Lipinski’s rule of five states that a compound
with molecular weight (MW) < 500 Daltons, H-bond
donors (NHD) < 5, H-bond, acceptors (NHA) < 10, and a
log P of < 5 could be a good drug candidate. Veber’s rule
says that a compound with rotatable bonds (RTB) < 10
and a polar surface area (TPSA) < 140 ´Å2 should pre-
sent good oral bioavailability. Compound with a fewer or
preferably no violations are likely to be considered as a
Table 7 Docking score of compounds 1–3 against S. aureus Pyruvate Kinase
Cmpds Anity
(kcal/mol) H-bonds Residual interactions
Hydrophobic Van dar Waals
1− 7.0 -- Lys-260 Arg-264, Asp-346, Asp-303, Asp-261, Met-260, Tyr-302, Gly-338
2− 7.4 Glu-234, Leu-233, Ser-236, Asp-
237, Lys-271, Asn-357, Leu-269 Val-356 Arg-204, Val-235, Glu-352, Lys-390, Gly-270,Arg-386
3− 7.0 Ser-411 Val-419 Glu-440, Val-420, Asp-58, Ile-60, Val-416, B: Lys-5, Gln-417 Thr-441
Amox − 7.0 Lys-390, Arg-386 Asp-237, Arg-386,Val-356 Asn-357, Thr-387, Ser-383, Lys-271, Ser-381, Ser-215, Pro-272
Table 6 Docking score of isolated compounds 1–3 against Pseudomonas aeruginosa PqsA
Cmpds Anity
(kcal/
mol)
H-bond Residual interactions
Hydrophobic/Pi-sigma/alkly/Pi-alkalyl Van dar Waals
1− 8.0 Gly-302 Phe-209, Val-254, Pro-234, Ile-204 Ile-257, pro-205, Lys-206, Trp-233, Gly-279, Tyr-211,
Phe-208, Ala-303, Phe-209
2− 8.7 Pro-85, Lys-86, Ala-108 Ala-124, Leu-60, Arg-128, Ala-108 Arg-106, A: Pro-111, Ala-110, Asp-109, Asn-61, Phe-134,
Leu-66, Ser-65, Ala-125, A: Gly-127, Ala-126
3− 8.6 Gly-279, Asp-282 Val-254, A: Pro-205 A: Ile-204, A; Ile-257, Phe-209 Pro-234, Tyr-211, Phe-208, Trp-233, Gly-210, Ala-303,
Gly-302, Arg-397, His-394, Ile-301, Thr-304
Amox − 7.3 Lys-86, Pro-129, Ala-125 Arg-128, Ala-124, Pro-129 Glu-107, Asp-231, Tyr-25, Ser-63, Pro-64, Leu-130, Ser-
65, Asp-132, Ala-126, Gly-127, Thr-232

Page 9 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
potential drug candidate. e chemical structures of the
compounds (1–3) were converted to their corresponding
canonical simplified molecular-input line-entry system
(SMILE) and submitted to the SwissADME tool to gener-
ate the physicochemical and pharmacokinetic properties
of the compounds. e SwissADME prediction outcome
showed that compound 1 satisfies the Lipinski’s rule of
five with zero violations. As shown in Table8, the com-
puted molecular weights for compounds 1 and 2 are
found to be asper the rule of five (< 500 g/mol). e NHD
and NHA for compounds 1 and 3 were predicted to be
according to the Lipinski’s rule of five. According to the
obtained result, compound 2 obey the MW (< 500 g/mol)
and iLogP (1.55) rules, and compound 3 obey the NHD
and NHA rules. e LogP value is a significant descriptor
for the affinity of molecules to a lipophilic environment
[29]. Compounds 2 recorded iLogP value less enough
than five (1.55), implying its optimal lipophilicity. e
prediction result showed that compound 1 and 3 obeyed
the Veber’s rule (NRB < 12, and TPSA < 140 unit). Veber’s
rule says that a compound with 10 or fewer rotatable
bonds (NRB) and a polar surface area (TPSA) no greater
than 140 ´Å2 should present good oral bioavailability.
e total polar surface area (TPSA) value is sufficiently
below the cut-off value (140 2 Å2) for compounds 1 and
3 (20.23 and 99.38, respectively), indicating very good
absorption by the gut. Our prediction results regard-
ing the drug-like features indicated that Lipinski’s rule is
obeyed by compound 1 with zero violation. Compound
2 and 3 obeyed the rule of MW and LogP, and NHD and
NHA, respectively. Veber’s rule is valid for compound 1
and 3. e ADME properties of compounds 1–3 were
performed using the SwissADME tool [17].
e main ADME parameters concerning pharma-
cokinetic behavior of the tested compounds are pre-
sented in Table9. e logKp (with Kp in cm/s) values of
compounds 1–3 were predicted to be -2.69, -10.04 and
− 4.32 cm/s, respectively, inferring low skin permeabil-
ity of each. e more negative the logKp value, the lesser
skin permeant the molecule [17]. Compound 2 scored
the most negative LogKp value of -10.04 cm/s, indicating
its lowest skin permeability compared to other isolated
compounds (1 & 3). e ADME prediction outcome
displayed that, all tested compounds (1–3) were found
to be non-substrate of permeability glycoprotein (P-gp),
and hence non-inhibitors of all the selected cytochromes
(CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4).
e toxicity profiles of the studied compound were car-
ried out using ProTox II. e predicted toxicity results of
Table 8 In silico pharmacokinetic predictions (drug likeness and
ADTE) of isolated compounds 1–3 computed by SwissADME and
PreADMET tool
NHD Number of Hydrogen donor, NHA Number of Hydrogen acceptor, NRB
Number of rotatable bonds, TPSA total polar surface area, MW Molecular weight,
LogKp Skin permeation value, GI Gastro-Intestinal, BBB Blood Brain Barrier, P-gp P
glycoprotein, CYP Cytochrome-P.
Predicted
Parameter Compounds
123
Drug likeness
Formula C29H50O C21H24O11 C35H60O6
MW (g/mol) 414.71 452.41 576.85
NHD 1 8 4
NHA 1 11 6
LogP (iLogP) 4.89 1.55 5.29
Lipinski’s RO5 Yes No, 2 violation No, 1 violation
NRB 6 4 9
TPSA (A°2) 20.23 189.53 99.38
Veber’s rule Yes No Yes
ADME predic-
tions
log Kp cm/s − 2.69 − 10.08 − 4.32
GIA Low Low Low
BBB No No No
Inhibitory
interaction
P-gp sub-
strate No No No
CYP1A2
inhibitor No No No
CYP2C19
inhibitor No No No
CYP2C9
inhibitor No No No
CYP2D6
inhibitor No No No
CYP3A4
inhibitor No No No
Table 9 Prediction of Toxicity of isolated compounds 1–3 computed by ProTox-II.
Compounds LD50 (mg/Kg) Toxicity class Organ toxicity
Hepato Carcino Immuno Mutagen Cyto
1 890 4 Inactive Inactive Active Inactive Inactive
2 10,000 6 Inactive Inactive Inactive Inactive Inactive
3 8000 6 Inactive Inactive Active Inactive Inactive

Page 10 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
the studied compounds (1–3) were presented in Table9.
Compound 2 and 3 exhibited negligible acute toxicity
(LD50 > 5000, Toxicity class > 5). e ProTox-II prediction
also gives toxicological endpoints such as hepatotoxicity,
carcinogenicity, immunotoxicity, mutagenicity and cyto-
toxicity results. Hepatotoxicity predictions indicate that
all studied compounds (1–3) are unlikely to disturb the
liver normal function. e studied compounds 1–3 also
exhibited no carcinogenicity, mutagenicity, and cytotox-
icity. Compound 2 and 3 have shown immunotoxicity.
Based on the reported ADMET prediction analysis, the
studied compounds can be good drug candidates, with
partial or complete structural modifications.
Radical scavenging activity
e radical scavenging activity of compound 1–3
isolated from the root extract of H. johannis were
evaluated using the DPPH assay, a simple method to
evaluate antioxidant activities by measuring absorbance
at 517 nm due to the formation of stable DPPH radical
[21]. At all the tested concentrations of each compound,
the purple color of the DPPH solution was changed
to yellow color indicating potential radical scaven-
ger activity of the compounds. e absorbance of the
DPPH radical at 517 nm was also reduced. e DPPH
assay result indicated that the compounds showed
radical scavenging activity in dose-dependent manner
(Table10). e scavenging activity result evident that of
the studied compounds (1–3) showed promising radi-
cal scavenging activities at all tested concentrations.
At the lowest tested concentration of 12.5 µg/mL, the
strongest DPPH scavenging activity was recorded by
compound 2 (67.87%), with IC50 value of 0.190 µg/mL,
which is in good comparison to the standard, ascorbic
acid (78.21%), with IC50 value of 0.014 µg/mL. Com-
pound 1 and 3 also showed considerable percentage
Table 10 DPPH radical inhibition of isolated compounds 1–3
Samples were reported as Mean ± SD; Ascorbic acid was used as positive control
200 µg/mL 100 µg/mL 50 µg/mL 25 µg/mL 12.5 µg/mL
1 61.44 ± 0.3 57.47 ± 0.1 56.43 ± 0.01 51.16 ± 0.25 49.76 ± 0.66 14.668
2 79.74 ± 0.04 77.36 ± 0.02 75.03 ± 0.01 70.99 ± 0.1 67.87 ± 0.02 0.190
3 67.07 ± 0.01 65.48 ± 0.01 61.44 ± 0.03 57.77 ± 0.02 52.75 ± 0.01 6.250
Asc acid 90.51 ± 0.03 82.19 ± 0.03 80.17 ± 0.02 79.50 ± 0.02 78.21 ± 0.03 0.014
Fig. 1 Structures of compounds (1–3) isolated from the root of H. johannis

Page 11 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
scavenging activity (52.75%, and 49.76%), with IC50
value of 6.250 and 14.668 µg/mL, respectively, which
is lower in comparison to the standard. e strongest
DPPH scavenging activity of compound 2 compared
to other tested compounds, which is good compared
ascorbic acid, in this report can be justified due to its
proton-donating ability (Fig.1), which make it to have
higher NHD and NHA reported in the drag likeness
in this study. Overall, the radical scavenging activity
results of the studied compounds are consistent with
the drug likeness predictions outcomes.
Fig. 2 The 2D (top) and 3D (bottom) binding interactions of isolated compounds 1–3 and Amoxicillin against protein DNA gyrase B
Fig. 3 2D (top) and 3D (bottom) binding interaction of isolated compounds 1–3 and Amoxicillin against protein P. aeruginosa PqsA

Page 12 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13765- 022- 00740-8.
Additional le1: Table S-1. 1H, 13C, HMBC spectral data of compound
2. Fig. S1. 1H NMR spectra of compound 1. Fig. S2. 13C NMR spectra of
compound 1. Fig. S3. DEPT-135 spectra of compound 1. Fig. S4. 1H NMR
spectra of compound 2. Fig. S5. 13C NMR spectra of compound 2. Fig.
S6. DEPT-135 spectra of compound 2. Fig. S7. COSY spectra of compound
2. Fig. S8. HSQC spectra of compound 2. Fig. S9. HMBC spectra of com-
pound 2. Fig. S10. 1H NMR spectra of compound 3. Fig. S11. 13C spectra
of compound 3. Fig. S12. DEPT-135 spectra of compound 3. Fig. S13.
Photo of Hydnora johannis. (Source: Photo taken by Teshoem Degfie).
Acknowledgements
We thank Adama Science and Technology University for the research fund
with grant number: ASTU/AS-R/003/2020. We are also grateful to the World
Academy of Sciences (TWAS) and the United Nations Educational, Scientific
and Cultural Organization (UNESCO) for financing this research with funds
allocated to the AD research team under the TWAS Research Grant RGA No.
20–274 RG/CHE/AF/AC-G - FR3240314163.
Author contributions
AD and ME designed the experiments. TD conducted the isolation, biological
activity assay and elucidation the structures. TT conducted molecular docking
studies. KS assisted the laboratory work for isolation. TD, AD and ME revised
interpreted the data and prepared the paper. AD, ME and TD revised the
manuscript. All authors read and approved the final manuscript.
Funding
Adama Science and Technology University for the research fund with grant
number: ASTU/AS-R/003/2020. The World Academy of Sciences (T WAS)
Research Grant RGA No. 20–274 RG/CHE/AF/AC-G-FR3240314163.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article.
Declarations
Ethics approval and consent to participate
Not applicable.
Competing interests
There is no conflict of interests.
Received: 13 July 2022 Accepted: 24 October 2022
References
1. Thorogood C (2019) Hydnora: the strangest plant in the world ? Plants.
People Planet 1(1):5–7
2. Musselman LJ, Visser JH (1989) Taxonomy and natural history of Hydnora
(Hydnoraceae). Aliso: J Syst Flor Bot 12(2):317–326
3. Yagi S, Chrétien F, Duval RE, Fontanay S, Maldini M, Piacente S, Henry M,
Chapleur Y, Laurain-Mattar D (2012) Antibacterial activity, cytotoxicity and
chemical constituents of Hydnora johannis roots. S Afr J Bot 78:228–234
4. Musa MS, Abdelrasool FE, Elsheikh EA, Ahmed LAMN, Mahmoud ALE, Yagi
SM (2011) Ethnobotanical study of medicinal plants in the Blue Nile State,
South-eastern Sudan. J Med Plants Res 5:4287–4297
5. Yagi S, Drouart N, Bourgaud F, Henry M, Chapleur Y, Laurain-mattar D
(2013) Antoxidant and antiglycation properties of Hydnora johannis roots.
S Afr J Bot 84:124–127
6. Al-Fatimi M, Ali NA, Kilian N, Franke K, Arnold N, Kuhnt C, Schmidt J,
Lindequist U (2016) Ethnobotany, chemical constituents and biological
activities of the flowers of Hydnora abyssinica A. Br. (Hydnoraceae). Die
Pharm Int J Pharm Sci 71(4):222–226
7. Saadabi AMA, Ayoub SMH (2009) Comparative bioactivity of Hydnora
abyssinica A. Braun against different groups of fungi and bacteria. J Med
Plants Res 3:262–265
8. Nethathe BB, Ndip RN (2011) Bioactivity of Hydnora africana on selected
bacterial pathogens: preliminary phytochemical screening. Afr J Micro-
biol Res 5:2820–2826
9. Dagne E (2016) Species details http://www. alnap netwo rk. com/ Speci
esDet ail. aspx SEARCH. http://www.alnapnetwork.com/SpeciesDetail.
aspx. Accessed 17 Sep 2022
Fig. 4 2D (top) and 3D (bottom) binding interaction of isolated compounds 1–3 and Amoxicillin against protein S. aureus PK.

Page 13 of 13
Degeetal. Applied Biological Chemistry (2022) 65:76
10. Bussa NF, Belayneh A (2019) Traditional medicinal plants used to treat
cancer, tumors and inflammatory ailments in Harari Region, Eastern
Ethiopia. S Afr J Bot 122:360–368
11. Girma B, Mulisa E, Amelo W (2018) Ethnomedicine claim activity directed
in silico prediction of anticancer. Ethiop J Heal Sci 28:83–92
12. Belayneh A, Asfaw Z, Demissew S, Bussa NF (2012) Medicinal plants
potential and use by pastoral and agro-pastoral communities in Erer Val-
ley of Babile Wereda, Eastern Ethiopia. J Ethnobiol Ethnomed 8:1–11
13. Balouiri M, Sadiki M, Ibnsouda SK (2016) Methods for in vitro evaluating
antimicrobial activity: a review. J Pharm Anal 6:71–79
14. Zaidan MR, Noor Rain A, Badrul AR, Adlin A, Norazah A, Zakiah I (2005)
In vitro screening of five local medicinal plants for antibacterial activity
using disc diffusion method. Trop Biomed 22:165–170
15. Trott O, Olson AJ (2009) AutoDock Vina: Improving the speed and accu-
racy of docking with a new scoring function, efficient optimization, and
multithreading. J Comput Chem 31:455–461
16. Narramore S, Stevenson CEM, Maxwell A, Lawson DM, Fishwick CWG
(2019) New insights into the binding mode of pyridine-3-carboxamide
inhibitors of E. coli DNA gyrase. Bioorg Med Chem 27:3546–3550
17. Daina A, Michielin O, Zoete V (2017) SwissADME: a free web tool to evalu-
ate pharmacokinetics, drug-likeness and medicinal chemistry friendliness
of small molecules. Sci Rep 7(1):1–13
18. Lipinski’s CA, Dominy BW, Feeney PJ, (2012) Experimental and com-
putational approaches to estimate solubility and permeability in drug
discovery and development settings. Acta Petrol Sin 28:1765–1784
19. Banerjee P, Eckert AO, Schrey AK, Preissner R (2018) ProTox-II: A web-
server for the prediction of toxicity of chemicals. Nucleic Acids Res
46(W1):W257–W263
20. Behrouz S, Soltani Rad MN, Taghavi Shahraki B, Fathalipour M, Behrouz
M, Mirkhani H (2019) Design, synthesis, and in silico studies of novel
eugenyloxy propanol azole derivatives having potent antinociceptive
activity and evaluation of their β-adrenoceptor blocking property. Mol
Divers 23:147–164
21. Gülçin I (2010) Antioxidant properties of resveratrol: a structure-activity
insight. Innov Food Sci Emerg Technol 11:210–218
22. Pierre Luhata L, Usuki T (2021) Antibacterial activity of β-sitosterol isolated
from the leaves of Odontonema strictum (Acanthaceae). Bioorg Med
Chem Lett 48:128248
23. Ododo MM, Choudhury MK, Dekebo AH (2016) Structure elucidation
of β-sitosterol with antibacterial activity from the root bark of Malva parvi-
flora. Springerplus 5:1–11
24. Sileshi W, Adane L, Tariku Y, Muleta D, Begashaw T (2012) Evaluation of
antibacterial activities of compounds isolated from Sida. Nat Prod Chem
Res 1:1–8
25. Friedrich W, Galensa R (2002) Identification of a new flavanol gluco-
side from barley (Hordeum vulgare L.) and malt. Eur Food Res Technol
214:388–393
26. Peshin T, Kar HK (2017) Isolation and characterization of β-sitosterol-3-O-
β-D-glucoside from the extract of the flowers of Viola odorata. Br J Pharm
Res 16:1–8
27. Wong KC, Hag Ali DM, Boey PL (2012) Chemical constituents and antibac-
terial activity of Melastoma malabathricum L. Nat Prod Res 26:609–618
28. Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD (2002)
Molecular properties that influence the oral bioavailability of drug candi-
dates. J Med Chem 45:2615–2623
29. Daina A, Zoete V (2016) A boiled-egg to predict gastrointestinal
absorption and brain penetration of small molecules. Chem Med Chem
11:1117–1121
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.