nanomaterials

Article
Loaded n-Hydroxyapatite/SSG 3D Scaffolds as a Drug Delivery
System of Nigella sativa Fractions for the Management of Local
Antibacterial Infections
Mohammed Dalli 1, * , Abdelqader El Guerraf 2 , Salah-eddine Azizi 1 , Karim Benataya 2 , Ali Azghar 3 ,
Jeong Mi-Kyung 4 , Adil Maleb 3 , Kim Bonglee 5,6, * and Nadia Gseyra 1

1 Laboratory of Bioresources, Biotechnology, Ethnopharmacology and Health, Faculty of Sciences,

University Mohammed the First, P.O. Box 524, Oujda 60000, Morocco; [email protected] (S.-e.A.);
[email protected] (N.G.)
2 Laboratory of Applied Chemistry and Environment, Faculty of Sciences, University Mohammed the First,
P.O. Box 524, Oujda 60000, Morocco; [email protected] (A.E.G.); [email protected] (K.B.)
3 Laboratory of Microbiology, Hospital University Center/Faculty of Medicine and Pharmacy,
P.O. Box 724, Oujda 60000, Morocco; [email protected] (A.A.); [email protected] (A.M.)
4 KM Convergence Research Division, Korea Institute of Oriental Medicine, Yuseong-daero, Yuseong-gu,
Daejeon 34054, Korea; [email protected]
5 Korean Medicine-Based Drug Repositioning Cancer Research Center, College of Korean Medicine,
Kyung Hee University, Seoul 02447, Korea
6 Department of Pathology, College of Korean Medicine, Kyung Hee University, Seoul 02447, Korea
* Correspondence: [email protected] (M.D.); [email protected] (K.B.)




Abstract: As a result of their close similarities to the inorganic mineral components of human

 bone, hydroxyapatite nanoparticles (n-HAp) are widely used in biomedical applications and for
Citation: Dalli, M.; El Guerraf, A.; the elaboration of biocompatible scaffold drug delivery systems for bone tissue engineering. In this
Azizi, S.-e.; Benataya, K.; Azghar, A.; context, a new efficient and economic procedure was used for the consolidation of n-HAp in the
Mi-Kyung, J.; Maleb, A.; Bonglee, K.; presence of various Nigella sativa (NS) fractions at a near-room temperature. The research conducted
Gseyra, N. Loaded n-Hydroxyapatite/ in the present study focuses on the physicochemical properties of loaded n-HAp 3D scaffolds by
SSG 3D Scaffolds as a Drug Delivery
NS fractions and the in vitro antibacterial activity against Gram-negative (Escherichia coli ATCC
System of Nigella sativa Fractions for
25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 27853), and Gram-positive
the Management of Local
(Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 700603) bacteria. In order to better
Antibacterial Infections.
Nanomaterials 2022, 12, 856. https://
understand the effect of the inserted fractions on the HAp molecular structure, the elaborated samples
doi.org/10.3390/nano12050856 were subject to Fourier transform infrared (FTIR) and X-ray diffraction (XRD) spectroscopic analyses.
In addition, the morphological investigation by scanning electron microscope (SEM) of the loaded
Academic Editor: Hangrong Chen
n-HAp 3D scaffolds demonstrated the presence of a porous structure, which is generally required
Received: 17 January 2022 in stimulating bone regeneration. Furthermore, the fabricated 3D composites exhibited significant
Accepted: 28 February 2022 antibacterial activity against all tested bacteria. Indeed, MIC values ranging from 5 mg/mL to
Published: 3 March 2022 20 mg/mL were found for the HAp-Ethanol fraction (HAp-Et) and HAp-Hexane fraction (HAp-Hex),
Publisher’s Note: MDPI stays neutral
while the HAp-Aqueous fraction (HAp-Aq) and HAp-Methanol fraction (HAp-Me) showed values
with regard to jurisdictional claims in between 20 mg/mL and 30 mg/mL on the different strains. These results suggest that the HAp-NS
published maps and institutional affil- scaffolds were effective as a drug delivery system and have very promising applications in bone
iations. tissue engineering.

Keywords: hydroxyapatite; 3D scaffolds; Nigella sativa; organic fractions; antibacterial activity

Copyright: © 2022 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article 1. Introduction
distributed under the terms and
Calcium phosphate biomaterials are widely adopted in the reconstruction of large
conditions of the Creative Commons
Attribution (CC BY) license (https://
bone defects, which improves the health state of patients every day. Nano-crystalline
creativecommons.org/licenses/by/
hydroxyapatite (n-HAp: Ca10 (PO4 )6 (OH)2 ) is one of the main components of human bones
4.0/). and constitutes up to 50% by volume, and 65% by weight, of bone [1,2]. This explains

Nanomaterials 2022, 12, 856. https://doi.org/10.3390/nano12050856 https://www.mdpi.com/journal/nanomaterials

Nanomaterials 2022, 12, 856 2 of 14

their suitable biological characteristics, including osteoinductivity and good bioactivity,

larger surface area (100 m2 g−1 ), and biocompatibility with hard and soft tissues [3]. Addi-
tionally, n-HAp possess the ability to promote bone healing and induce cell viability and
proliferation of mesenchymal stem (MSC) and osteosarcoma cells [4]. Furthermore, in vitro
studies show that n-HAp can stimulate the osteogenic trans-differentiation of smooth
muscle cells (SMCs) and support calcification on the extracellular matrix (ECM) [5]. What
is more, n-HAp is used to inhibit proliferation and induce apoptosis in numerous cancer
cells, including liver [6], breast [7], colon [8], and gastric cancer cells [9]. They are also
commonly being used as carriers of various proteins, genes, drugs, anti-tumor agents, and
other macromolecules [10,11]. Therefore, n-HAp is of interest in various biomedical fields,
especially for bone tissue engineering and dental implant applications [12]. Thus, extensive
research efforts have been conducted for the densification of n-HAp in order to produce
bulk materials. In fact, the most adopted consolidation techniques are mainly carried out at
very high temperatures (e.g., 1250 ◦ C or higher). However, n-HAp is thermodynamically
unstable at an extremely high sintering temperature, which influences, scientifically, the
outstanding physicochemical and biological characteristics of the apatite phase, such as the
low crystallinity and nanometric dimension of its particles [13]. Consequently, using such
a conventional consolidation technique, the obtained materials are crystalline, with larger
grain size and reduced in vivo reactivity. In our previous investigations, a novel approach
at a near-room temperature was adopted to elaborate a highly biodegradable and biocom-
patible composite material based on n-HAp and sodium silicate glass (SSG) [14]. In this new
method, a sodium silicate solution is used as a mineral binder to consolidate n-HAp while
preserving their chemical and structural characteristics with a three-dimensional structure
that possesses the adequate macro- and micro-porosities and mechanical profile that are
required for bone-healing applications. The in vitro biodegradability and bioactivity tests
demonstrated a fast reactivity of the elaborated scaffolds. In addition, the cytocompatibility
tests assessed by cell viability and morphology proved that the n-HAp/SSG has a non-toxic
effect and can show enhanced cell proliferation.
On the other hand, despite their good bioactivity, the n-HAp has no antibacterial
properties, which restrict their clinical applications [15,16]. Additionally, various inves-
tigations found that bone tissue infections at implanted sites are usually associated with
Gram-negative and Gram-positive bacteria, which can lead to serious complications after
surgery [17,18]. Thus, a composite material with a chemical structure similar to the natural
bone, in combination with antibacterial properties, is a highly demanded orthopedic bio-
material. Recent studies in the field have shown the use of hydroxyapatite as a local drug
delivery system. Among them, we cite the study based on the absorption of the basilic and
lavender essential oils on the hydroxyapatite surface that showed a good release of the
tested essential oils and verified an important antibacterial activity [16]. In a similar vein,
Huang et al. [19] described the antibacterial effect of chitosan-coated hydroxyapatite, and
demonstrated the cytotoxic effect of the composite on normal cell lines.
Nigella sativa (NS), commonly known as black cumin, is a well-known plant with
high distribution in Europe, the Middle East, Asia, and the north of Africa [20]. NS
is found to be rich in different secondary metabolites such as polyphenols, flavonoids,
tannins [21], and alkaloids [22,23]. It was also noted that black cumin has a large spectrum
of pharmacological activities. It was found that the plant extracts exert an antibacterial [24],
antifungal [25], antidiabetic [26], immunomodulatory [27], and analgesic effect [28].
The current study focuses on the antibacterial activity of n-HAp 3D scaffolds loaded
with different NS fractions. This work combines the antibacterial activity exerted by
the different fractions and the hydroxyapatite/SSG capacity to encapsulate the bioactive
compounds present in each fraction. The morphology of the loaded n-HAp was observed
using a scanning electron microscope (SEM), and the influence of the inserted fractions on
the molecular structure of HAp was investigated by Fourier transform infrared (FTIR) and
X-ray diffraction (XRD) spectroscopies.
Nanomaterials 2022, 12, 856 3 of 14

2. Materials and Methods

2.1. Chemicals
Diammonium hydrogen phosphate ((NH4 )2 HPO4 ), calcium nitrate tetrahydrate
(Ca(NO3 )2 ·4H2 O), ammonium hydroxide (NH4 OH, 35%), sodium hydroxide (NaOH),
methanol (CH3 OH, ≥99.8%), ethanol (C2 H5 OH, ≥99.8%), dimethyl sulfoxide (C2 H6 OS ,
99.9%) and hexane (C6 H14 , ≥97.0%) were purchased from Sigma-Aldrich (St. Louis, MO,
USA) and used as received. Silica gel (SiO2 , high-purity grades (Davisil Grade 633)) was
also purchased from Sigma-Aldrich and used to prepare the sodium silicate solution. All
solutions were prepared using highly purified water.

2.2. Plant Material

The Nigella sativa seeds were purchased and identified by professional botanists.
A voucher specimen was deposited at the herbarium of the faculty under the number
(HUMPOM471).

2.3. Preparation of Nigella sativa Fractions

The different Nigella sativa seeds were taken and turned into fine powder, then 100 g
of that powder was taken and put in a Soxhlet apparatus formed by an extractor, fitted in
between a round-bottom flask at the bottom and a condenser at the top. Inside the extractor
glass, the NS powder was placed within the thimble. The n-hexane was used for degreasing
the plant. After that, the residual plant was extracted using ethanol. The remaining plant
material was airdried again, and the extraction was assessed by methanol and water by
following the same procedure after each extraction (Figure 1, step 1). The yields obtained
ranged from 2.53% to 21.51%. The different obtained fractions were collected and stored at
4 ◦ C for further use.

Figure 1. Schematic representation of the NS extraction method and 3D n-HAp-based composite

scaffold fabrication. (1) extraction, (2) scaffolds preparation and (3) drying.
Nanomaterials 2022, 12, 856 4 of 14

2.4. Synthesis of Hydroxyapatite Nanoparticles and Sodium Silicate Solution

HAp nanoparticles were prepared according to the method described in our previous
work [14]. At room temperature, an aqueous solution of 0.20 M diammonium hydrogen
phosphate was added drop-wise to a solution of 0.3 M calcium nitrate. The pH was
maintained at 10 by adding 0.5 M ammonium hydroxide solution, and the mixture was
kept in maturation for 30 min. Afterward, the precipitated n-HAp was filtered, washed
with distilled water, and dried at 80 ◦ C for 24 h.
A sodium silicate solution (Na2 SiO3 , H2 O) with a molar ratio of SiO2 /Na2 O = 1 was
prepared at 90 ◦ C by dissolving pure NaOH pellets in distilled water, then an adequate
amount of SiO2 was added to obtain a liquid glass containing, by weight, 25% SiO2 , 25%
Na2 O, and 50% water.

2.5. n-HAp 3D Scaffolds Preparation

After the preparation of n-HAp, it was directly mixed with the sodium silicate solution
and different NS fractions at a fixed liquid-solid ratio until a homogenous paste was
obtained (Figure 1, step 2). The portion of n-HAp, sodium silicate solution and NS fractions
in the prepared scaffolds were 70 wt%, 25 wt% and 5 wt%, respectively.
The malleable pastes were molded and oven-dried at 40 ◦ C for 2 weeks to obtain dried
bulk n-HAp/SSG/fractions composites (Figure 1, step 3).

2.6. Characterization
2.6.1. XRD
The phases of the consolidated composites with different fractions were characterized
through X-ray diffraction (XRD, XRD-6000, SHIMADZU, Kyoto, Japan) in the range of 2θ,
an angle of 10–65◦ using Cu Kα radiation (λ = 0.154 nm), and a scanning speed of 4◦ /s.
The crystallite size of the (002) plane was estimated by the Scherrer formula, as follows:

Kλ
D= (1)
β cos θ

D represents the crystallite size (nm); K is the Scherrer constant (k = 0.89); β is the
half-width height of the diffraction peak; and θ is the diffraction angle of the associated
(hkl) plane.

2.6.2. FTIR
Fourier-transform infrared (FTIR) analysis in attenuated total reflection (ATR) mode is
used for complementary structure identification. The experiment was conducted using a
Jasco 4700-ATR spectrophotometer in the spectral range between 4000 and 500 cm−1 with a
4 cm−1 resolution.

2.6.3. SEM
The microstructure of elaborated loaded composites was observed by scanning elec-
tron microscopy (FE-SEM) using a JEOL-JSM7001F apparatus. The working distance was
maintained at 6 mm (the distance between the sample and the objective lens remains) and
the SEM filament was operated at variable currents and a voltage of 5 kV using differ-
ent magnifications. The elemental composition was determined using an X-ray energy
dispersive spectroscopy (EDS).

2.7. Bacterial Strains

The bacterial strains used in the test were Staphylococcus aureus ATCC 29213 and
Enterococcus faecalis ATCC 700603 (Gram-positive), and Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 27853 (Gram-
negative) were purchased from Thermoscientific.
Nanomaterials 2022, 12, 856 5 of 14

2.8. Agar Diffusion Method

For the determination of the antibacterial activity of the different HAp 3D scaffolds, the
agar diffusion method was adopted. All strains’ turbidity was normalized to 0.5 McFarland
and then inoculated on the surface of the Petri dish. The HAp 3D scaffolds, loaded with
different fractions, were placed on the surface of the Mueller–Hinton Agar (MHA). After
24 h of incubation at 37 ◦ C, the inhibition diameter was measured. All measurements of
inhibition zones were performed in triplicate.

2.9. Minimal Inhibitory and Minimal Bactericidal Concentration

The microdilution technique was used for the determination of the minimal inhibitory
concentration [29]. The different tested fractions were dissolved in 2% DMSO, and then a
serial dilution was prepared by Mueller–Hinton Broth. A total of 180 µL of each concentra-
tion was taken and added to each well. After that, 20 µL of the bacterial suspension was
added to all wells. All plates were then incubated at 37 ◦ C for about 24 h. To facilitate the
determination of the MIC value, the resazurin, an indicator of bacterial growth, was added
to the mixture (10 µL) and the plates were incubated for an additional 2 h. The well with
no color change was considered as the MIC value.
For the determination of the minimal bactericidal concentration, 20 µL was taken from
each well with no color change and inoculated on the surface of the Petri dish, and then
incubated at 37 ◦ C for about 24 h. The dish with no subculture was considered as the MBC.
All tests were performed in triplicate.

3. Results and Discussion

3.1. Physicochemical Characterization of the Composite Scaffold
Consolidated composite materials are characterized by ATR-FTIR and depicted in
Figure 2A. The ATR-FTIR spectra highlight that all consolidated materials present char-
acteristic vibrational bands of phosphate groups (PO4 3− ) of n-HAp structure, specifically
at nearly 570, 603.7 cm−1 , 960 cm−1 , and between 1010 and 1110 cm−1 . The two vibra-
tional bands observed at 630 and 3540 cm−1 are assigned to the hydroxyl group (O–H) of
the n-HAp structure. The small band around 1420 cm−1 in the pure composite material
n-HAp/SSG is attributed to the carbonate ions CO3 2− [30]. The intensity of this band
increased as the fractions were incorporated in the materials. Additionally, two more peaks
appeared at approximately 1640 cm−1 , and between 2700 and 3200 cm−1 , corresponding to
the functional groups present in the organic compounds in the NS fractions. The sharp band
at 1640 cm−1 can be assigned to the C=C stretching vibration [16]. In the 2700–3200 cm−1
spectral region, the bands situated between 2700 and 3200 cm−1 are attributed to the C–H
stretching vibrations [31].
The structure and phase composition of the consolidated materials loaded are investi-
gated as well, using XRD analysis (Figure 2B). As observed in the figure, the XRD patterns
of all elaborated samples exhibited peaks with high-intensity characteristics that are in good
agreement with JCPDS data for HAp (JCPDS file No. 09-0432). Additionally, the broadened
peaks indicate the low crystallinity of the apatitic phase in the elaborated materials. Based
on the XRD diffraction peak (002), the crystallite size of n-HAp is determined to be in the
range of 37 to 40 nm for non-loaded and loaded scaffolds, respectively, which illustrates
the nanocrystalline feature of the consolidated HAp phases. These results suggest that 3D
composites are mainly formed by an apatitic phase with low crystallinity and nanoparticles
similar to the inorganic phase of young bones (newly formed bone tissues) [32].
Nanomaterials 2022, 12, 856 6 of 14

Figure 2. ATR-FTIR (A) and XRD patterns (B) of consolidated samples. (a) HAp-Aq, (b) HAp-Me,
(c) HAp-Et, and (d) HAp-Hex.

On the other hand, SEM investigation was achieved to describe the resulting structure
of the 3D composites. The micrographs of various NS fraction-loaded composites are
presented in Figure 3. For comparison, the analysis was also achieved for non-loaded
n-HAp/SSG and the images are gathered in the same figure. As shown, the morphology
of n-HAp is affected by the introduced molecules. Indeed, each NS fraction interacts
differently with HAp, giving various topographies.
The SEM revealed a compact and homogeneous structure where n-HAp particles are
totally integrated within the SSG material (Figure 3A). Hence, this results in the formation
of a high number of pores spread equally throughout the 3D surface. After the modification
of n-HAp/SSG, each introduced NS fraction presents a specific morphology. It can be
seen that the fraction prepared with methanol solvent (HAp-Me) can block some of the
pores. As shown, a smooth coating layer has formed, which covers the entire outer surface
of n-HAp/SSG (Figure 3C). However, many micropores with a diameter generally lower
than 50 µm are still present. On the other hand, the aqueous-, ethanol-, and hexane-based
fractions demonstrate the presence of randomly distributed and interconnected micro-
porosity (Figure 3B,D,E). To obtain a comprehensive understanding of the pore space of
the samples under investigation, the histogram of the pore size distribution, plotted using
processed SEM micrographs from ImageJ, is presented in Figure 4. The specimens display
a unimodal distribution with a mean pore size of 68.63 µm. In fact, the pore diameters
ranged between 10 and 200 µm for practically all elaborated loaded scaffolds.
Commonly, the presence of abundant irregular open and interconnected pores on the
outer and inner surfaces of a biomaterial is an important key for industrial applications.
This interesting property has a crucial role in stimulating bone regeneration, as it provides
a great ability to induce osteointegration and improve implant adhesion to the host tissue.
Nanomaterials 2022, 12, 856 7 of 14

Figure 3. SEM micrographs and the corresponding EDS patterns of consolidated samples.
(A) HAp/SSG, (B) HAp-Aq, (C) HAp-Me, (D) HAp-Et and (E) HAp-Hex.

The EDS analysis of the NS-based composite scaffolds, presented in the same figure,
clearly indicates the presence of calcium (Ca) and phosphorus (P) from the n-HAp particles,
and silicon (Si) and sodium (Na) resulted from the precipitated sodium silicate glass.
Additionally, carbon (C) and oxygen (O) can be associated with either n-HAp or SSG.
By comparison to the atomic percentage of carbon in n-HAp/SSG before and after the
integration of NS fractions, we note an important increase in the organic matter in our
composites. Indeed, C% of about 9.9% was obtained for the unmodified composite (as
shown in Figure 3 from our previous paper [14]); it passes to 27%, 28%, 25%, and 32% for
HAp-Aq, HAp-Me, HAp-Et, and HAp-Hex, respectively.

3.2. Antibacterial Activity

The antibacterial susceptibility testing of the 3D HAp scaffolds loaded with different
NS fractions was assessed on the following Gram-positive and Gram-negative bacterial
strains: Staphylococcus aureus ATCC 29213, E. coli ATCC 25922, Pseudomonas aeruginosa
ATCC 27853, Klebsiella pneumoniae ATCC 27853, and Enterococcus faecalis ATCC 700603. The
results obtained using the agar diffusion method are given in Table 1.
Nanomaterials 2022, 12, 856 8 of 14

Figure 4. Statistical histogram of the pore size distribution derived from the SEM micrographs.

Table 1. Inhibition zone diameters of NS-loaded fractions.

S. aureus E. faecalis P. aeruginosa K. pneumoniae E. coli

ATCC 29213 ATCC 25922 ATCC 27853 ATCC 27853 ATCC 700603
HAp/SSG 0 0 0 0 0
HAp-Aq 14.67 ± 0.26 9.50 ± 0.19 16.2 ± 0.13 15.67 ± 0.44 17 ± 0.45
HAp-Me 11.5 ± 0.26 11.17 ± 0.32 14.7 ± 0.25 14.83 ± 0.22 16.67 ± 0.26
HAp-Et 13.75 ± 0.19 14.50 ± 0.19 14 ± 0.001 14.17 ± 0.556 15.67 ± 0.26
HAp-Hex 11.17 ± 0.13 11 ± 0.38 16 ± 0.577 15 ± 0.7 15.50 ± 0.38
Inhibition diameter of the different tested strains (mm).

The agar diffusion method was used for testing the antibacterial capacity of the differ-
ent NS fractions. The results obtained for the 3D HAp scaffold-free fractions demonstrate
no activity on the tested strains, while loading the 3D scaffolds with aqueous, methanol,
ethanol, and n-hexane fractions showed that all strains were sensitive toward all n-HAp
scaffolds. Due to the fact that the HAp 3D scaffolds free of any additional fraction have no
effect, the antibacterial activity was attributed to the NS fractions.
Concerning the MIC and MBC values, the results are depicted in Table 2. The different
MIC values were ranging from 5 to 20 mg/mL, while the MBC values were from 5 to
50 mg/mL.
Nanomaterials 2022, 12, 856 9 of 14

Table 2. MIC and MBC in mg/mL of Nigella sativa-loaded fractions in HAp 3D scaffolds against
tested bacteria.

S. aureus E. faecalis P. aeruginosa K. pneumoniae E. coli

ATCC 29213 ATCC 25922 ATCC 27853 ATCC 27853 ATCC 700603
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
HAp-Aq 20 20 30 50 20 30 20 30 20 30
HAp-Me 20 20 20 30 20 30 20 30 20 30
HAp-Et 5 5 5 5 10 20 20 30 10 20
HAp-Hex 5 5 5 5 20 20 20 20 10 20
MIC and MBC are minimal inhibitory concentration and minimal bactericidal concentration, respectively.

The present study aimed to investigate the antibacterial activity of different n-HAp-
loaded scaffolds with different NS fractions obtained from Soxhlet apparatus by using a
new consolidation technique. According to our previous studies, it was mentioned that
the NS seeds are of great richness with phenolic and flavonoid compounds [21]. For exam-
ple, the aqueous fraction was found to be rich in vanillic acid, ferulic acid, salicylic acid,
vanillin, and rutin (Figure 5A(1)). In addition, the MeOH fraction was characterized by
the presence of gallic acid, vanillic acid, rutin, and naringenin (Figure 5A(2)), while the
EtOH fraction was rich with three flavonoids (rutin, catechin, kaempferol) (Figure 5A(3)).
On the other hand, the n-hexane methyl ester was distinguished by the presence of thy-
moquinone, linoleic acid, palmitic acid, carvacrol, and stearic acid, with different amounts
(Figure 5B) [26].
Regarding the antibacterial activity, the results obtained indicate that the different
tested fractions are endowed with a strong antibacterial activity that could be attributed to
the bioactive compounds present in each fraction. The smallest MIC value was registered
in Gram-positive bacteria (S. aureus and E. faecalis) with a value of 5 mg/mL in HAp-
Hex and HAp-Et. The highest MIC value was 30 mg/mL for E. faecalis and 20 mg/mL
for the other bacteria. From the results, it was observed that the Gram-positive bacteria
were very sensitive to the n-HAp-Hex and n-HAp-Et, while the Gram-negative bacteria
were less sensitive to the tested fractions. This difference in response between the Gram-
positive bacteria and the Gram-negative bacteria is principally due to the morphology of
the bacterial wall. The Gram-positive bacteria are characterized by their richness with
peptidoglycans, which facilitate the passage of hydrophobic molecules to the inside of the
bacteria. The Gram-negative bacteria wall is rich with lipopolysaccharides, which allow
the passage of the hydrophilic molecules from the external environment to the internal
environment [33].
In a study performed on the methanolic extract of NS, it was noted that the extract
exerts a strong activity on S. aureus, while it was inactive on K. pneumoniae. In the same
context, Zuridah et al. [34] demonstrated a weak activity on E. coli and P. aeruginosa. These
findings were different when compared with our results, where the MeOH fraction was
able to inhibit the K. pneumoniae, E. coli, and P. aeruginosa at an MIC value of 20 mg/mL.
Regarding the black cumin ethanolic extract tested on bacteria that alter food, it
was shown that this extract inhibits the bacteria S. aureus and E. coli, with no effect on
K. pneumoniae growth [35], which was contradictory to our findings, where the EtOH
fraction showed a great inhibitory potential toward all strains tested with an MIC value
ranging from 5 to 20 mg/mL. The study of Khalid et al. [36] demonstrated that the aqueous
extract has a weak antibacterial activity toward S. aureus, E. coli, P. aeruginosa, and E. faecalis,
while in the present study it was demonstrated that all tested strains manifested a high
sensitivity to the NS aqueous fraction. Concerning the n-hexane fraction, the MIC values
ranged from 5 to 20 mg/mL, which was close to those obtained in the Abraham et al.
study [37].
Nanomaterials 2022, 12, 856 10 of 14

Figure 5. HPLC profiles of the different tested fractions (A) and the total ion chromatogram of the
n-hexane fraction (B). (1) aqueous fraction, (2) methanol fraction and (3) ethanol fraction.

Several studies have reported the antibacterial potential of the different molecules
identified in our fractions. For example, the salicylic acid present in our aqueous fraction
was found to be endowed with an important inhibitory activity on different bacteria such
as S. aureus, E. coli, P. aeruginosa, and E. faecalis with an MIC (250–500 µg/mL). In the
same study, it was registered that the quercetin present in our MeOH fraction, and the
kaempferol present in our EtOH and aqueous fractions, were found to play a moderate
antibacterial role with an MIC value of about 500–1000 µg/mL. Moreover, the rutin was
found to be capable of inhibiting the different strains tested with an MIC ranging between
500 and 1000 µg/mL [38]. The two phenolic acids (gallic and ferulic acids) were reported
to exhibit an important antibacterial activity on different bacteria such as S. aureus, E. coli,
and P. aeruginosa, with an MIC value between 100 and 1750 µg/mL. It was also noted that
exposure to gallic acid and ferulic acid induced membrane damage in the tested bacterial
strains [39]. The catechin present in our MeOH, EtOH, and aqueous fractions was revealed
Nanomaterials 2022, 12, 856 11 of 14

to have an important antibacterial activity characterized by creating damage to the cell

membrane [40]. On the other hand, it was noted that long-chain unsaturated fatty acids are
capable of inhibiting Gram + and Gram—bacteria [41], while TQ present in the n-hexane
fraction was mentioned to be endowed with a great antibacterial activity, especially on
multidrug-resistant bacteria [42]. It was also mentioned that carvacrol causes an alteration
in the fluidity and permeability of the membrane, which is attributed to the hydroxyl group
present on the molecule. In addition, this terpenoid compound could cause a leakage of
K+ ions and an exhaustion of intracellular ATP, which explains the various alterations
induced [43,44]. Likewise, the carvacrol was found to be capable of blocking the flagellins,
which induces a cessation of bacterial motility [45], and it was registered that the linoleic
acid has an antibacterial activity, especially on methicillin-resistant staphylococcus aureus,
which could be used as a therapeutic agent. Finally, in a previous study, the different
fractions were found to be non-toxic, which indicates that these fractions are free of any
hazardous effects [26].
In summary, the synthesized 3D scaffolds based on HAp and NS fractions exhibited
interesting antibacterial activities due to the interaction of various bioactive compounds
present in each fraction with the outer membrane of the bacteria. The amelioration of
n-HAp biological properties by loading with an NS fraction has very promising clinical
applications that can eliminate any bone tissue infections (Figure 6).

Figure 6. Schematic representation of the loaded hydroxyapatite/SSG 3D scaffold as a promising

biomaterial for orthopedic applications.

4. Conclusions and Future Perspectives

In this paper, hydroxyapatite nanoparticles were successfully consolidated in the
presence of various NS fractions. The findings reveal that the NS exerted antibacterial
activity on several Gram-positive and Gram-negative bacteria that were loaded on hy-
droxyapatite 3D scaffolds as a drug delivery system. This effect could be attributed to the
different bioactive compounds identified in the tested fractions, such as gallic acid, salicylic
acid, vanillic acid, rutin, catechin, kaempferol, thymoquinone, linoleic acid, and carvacrol.
These obtained results could be considered a significant contribution to the development
Nanomaterials 2022, 12, 856 12 of 14

of new applications in the medical field. The use of these bioactive compounds loaded
in hydroxyapatite/SSG composite in bone reconstruction could help in the amelioration
of the health state by reducing the number of postoperative infections after the implants.
More studies are in progress in order to investigate, in vivo, the capacity of the loaded
hydroxyapatite 3D scaffolds.

Author Contributions: Conceptualization, M.D. and N.G.; methodology, M.D.; software, A.E.G.;
validation, S.-e.A., A.A. and J.M.-K.; formal analysis, K.B. (Karim Benataya); investigation, M.D.;
resources, K.B. (Karim Benataya); data curation, M.D. and A.E.G.; writing—original draft preparation,
M.D.; writing—review and editing, M.D. and A.E.G.; visualization, A.M.; supervision, N.G.; funding
acquisition, K.B. (Kim Bonglee). All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported by Korea Institute of Oriental Medicine: KSN2021240, Basic
Science Research Program through the National Research Foundation of Korea (NRF) funded by the
Ministry of Education (NRF-2020R1I1A2066868), the National Research Foundation of Korea (NRF)
grant funded by the Korea government (MSIT) (No. 2020R1A5A2019413).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available within the article.
Acknowledgments: The authors would like to thank LEGSSYER Bouchra and LAKRAT Mohammed
for their precious help during this study.
Conflicts of Interest: The authors declare no conflict of interest.

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