crystals

Article
Evaluation of Antioxidant, Cytotoxic, Mutagenic and Other
Inhibitory Potentials of Green Synthesized
Chitosan Nanoparticles
Narayanasamy Duraisamy 1 , Sangeetha Dhayalan 1, *, Mohammed Rafi Shaik 2, * , Althaf Hussain Shaik 3 ,
Jilani P. Shaik 4 and Baji Shaik 5

1 Department of Microbiology, Faculty of Science, Annamalai University, Annamalai Nagar,

Chidambaram 608002, Tamil Nadu, India
2 Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
3 Department of Zoology, College of Science, King Saud University, P.O. Box 2454, Riyadh 11451, Saudi Arabia
4 Department of Biochemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
5 School of Chemical Engineering, Yeungnam University, Gyeongsan 38541, Korea
* Correspondence: ds13134@annamalaiuniversity.ac.in (S.D.); mrshaik@ksu.edu.sa (M.R.S.);
Tel.: +91-9486680815 (S.D.); +966-11-46-70439 (M.R.S.)

Abstract: The current study was performed with aim of evaluating antioxidant, cytotoxicity, α-amylase,
and α-glucosidase inhibitory activities and mutagenicity properties of Martynia annua mediated
Chitosan nanoparticles (MAL-CNPs). The green synthesized MAL-CNPs were characterized and
confirmed through several characterization techniques, including UV-visible spectroscopy (UV-
Vis), high-resolution transmission electron microscopy (HRTEM), scanning electron microscopy
(SEM), Fourier-transform infrared spectroscopy (FT-IR), and dynamic light scattering (DLS). The
HR-TEM analysis exhibited that the as-synthesized chitosan nanoparticles are spherical in shape.
Citation: Duraisamy, N.; Dhayalan, Furthermore, the DLS analysis exhibited that the average size of MAL-CNPs was 53 nm and the
S.; Shaik, M.R.; Shaik, A.H.; Shaik, maximum diameter was 130.7 nm. The antioxidant activity results revealed that the MAL-CNPs
J.P.; Shaik, B. Evaluation of showed DPPH (2,2-diphenyl-1-picrylhydrazyl) (66.78%) and H2 O2 (91.65%) scavenging activities
Antioxidant, Cytotoxic, Mutagenic
at 50 µg/mL concentration. The IC50 values were 2.431 µg/mL and 50 µg/mL for DPPH and
and Other Inhibitory Potentials of
H2 O2 , respectively. MTT (3-4, 5 dimethylthiazol-2yl-2, 5-diphenyltetrazolium bromide) assay results
Green Synthesized Chitosan
exhibited dose-dependent cytotoxicity found from 50 µg/mL concentration of MAL-CNPs. The
Nanoparticles. Crystals 2022, 12, 1540.
https://doi.org/10.3390/
MAL-CNPs showed remarkable α-glucosidase and α-amylase inhibitory activity (IC50 1.981 µg/mL
cryst12111540 and 161.8 µg/mL). No toxic effect of MAL-CNPs was found through the Ames test. Further, the
study concluded that MAL-CNPs are non-toxic and possess adequate antioxidants and cytotoxicity
Academic Editor: Kyeong Kyu Kim
activity against cancer cells, α-glucosidase, and α-amylase inhibitory activity. Hence, the MAL-CNPs
Received: 30 September 2022 were considered for biomedical applications after the assessment of their efficiency and safety.
Accepted: 25 October 2022
Published: 28 October 2022 Keywords: Martynia annua; antioxidant activity; MTT assay; α-glucosidase inhibitory assay

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Nanobiotechnology is an advanced research field that entails configuring, synthesizing,
and applying particle sizes ranging from 1 to 100 nm [1]. In recent decades, the discovery
of nanoparticles’ distinctive attributes has enabled their use in various fields, including
Copyright: © 2022 by the authors. biomedicine, drug and gene delivery, antimicrobials, antioxidants, tumor detection, etc. [2].
Licensee MDPI, Basel, Switzerland. Among different nanoparticles, chitosan is the most popular biodegradable nanoparticle as
This article is an open access article
of yet. Chitosan nanoparticles have many therapeutic applications, including drug delivery,
distributed under the terms and
antidiabetic agents, wound healing, antimicrobial agents with the shipping carrier, and
conditions of the Creative Commons
effective drug delivery control [3–5].
Attribution (CC BY) license (https://
The potential of such polymer-based nanomaterials to combat particles with special-
creativecommons.org/licenses/by/
ized surface receptors and enter cells can help with more efficient and secure regenerative
4.0/).

Crystals 2022, 12, 1540. https://doi.org/10.3390/cryst12111540 https://www.mdpi.com/journal/crystals

Crystals 2022, 12, x FOR PEER REVIEW 2 of 17

Crystals 2022, 12, 1540 The potential of such polymer-based nanomaterials to combat particles with special- 2 of 16
ized surface receptors and enter cells can help with more efficient and secure regenerative
medicine [6]. As small generalized polypeptide adsorption properties, polymer nanopar-
ticles,
medicine particularly some
[6]. As small with a hydrophilic
generalized polypeptide exterior, are widely
adsorption usedpolymer
properties, as carriers. Provi-
nanoparti-
dentially, this chitosan polymeric compound occurs naturally in abundance
cles, particularly some with a hydrophilic exterior, are widely used as carriers. Providen- [7–9]. Gener-
ally,
tially,chitosan has several
this chitosan polymeric applications
compound due to its naturally
occurs excellent in phytochemical
abundance [7–9]. characteristics
Generally,
and distinctive applications, including healthcare, nourishment,
chitosan has several applications due to its excellent phytochemical characteristics chemical, cosmetic prod-
and
ucts, water applications,
distinctive purification, metal
including production andnourishment,
healthcare, recovery, andchemical,
metaboliccosmetic
and bioengineer-
products,
ing
waterindustries [10]. metal production and recovery, and metabolic and bioengineering
purification,
Chitosan
industries [10]. comprises a few functional groups that allow graft alteration, which confers
specialChitosan comprises a the
characteristics on fewcustomized chitosan
functional groups that[11,12]. Thesealteration,
allow graft improvements can be
which confers
used
special to characteristics
attain chemically on altered chitosan chitosan
the customized to increase its solubility
[11,12]. and thus broaden
These improvements can itsbe
biological
used to attain applications.
chemically Such chemical
altered modifications
chitosan to increaseresult in a wide
its solubility andrange of chitosan
thus broaden its
derivatives with long-term
biological applications. release
Such characteristics,
chemical modificationsnontoxicity,
result inexcellent biocompatibility,
a wide range of chitosan
and compostability
derivatives [11,13]. release
with long-term As a positively charged
characteristics, polymer, excellent
nontoxicity, it has bio-adhesion,
biocompatibility,anti-
hypercholesterolemic,
and compostability [11,13]. cell membrane transfection,
As a positively charged andpolymer,
anti-inflammatory properties anti-
it has bio-adhesion, that
hypercholesterolemic,
can be improved by blending cell membrane
this withtransfection,
other substances,and anti-inflammatory
making it an attractive properties
candidatethat
canbiological
for be improved andby blending
medical this with other
applications [14]. substances, making itnanoparticles
Moreover, chitosan an attractive can
candidate
boost
for biological
the immune system,and medical
resultingapplications
in antitumor [14].activity.
Moreover, chitosan nanoparticles
Additionally, can boost
chitosan nanoparticles
the being
are immune usedsystem,
as drug resulting
delivery in carriers
antitumor dueactivity.
to theirAdditionally, chitosan nanoparticles
high biodegradability and biocom-
are being used
patibility as drug
and their delivery carriers
convenience due to their[15,16].
of modification high biodegradability
The presence ofand biocompati-
hydroxyl and
bility and their convenience of modification [15,16]. The presence
amino groups in chitosan makes it the ideal forum for complex formation with some other of hydroxyl and amino
groups in chitosan
compounds, activelymakes it the
helping inideal forum for of
the formation complex formation with
more sustainable some other
complexes withcom-im-
pounds, actively helping in the formation of more sustainable
proved pharmacokinetics and pharmacodynamics [17]. Furthermore, the functional complexes with improved
pharmacokinetics
groups are abundant, andand pharmacodynamics
chitosan could be [17]. alteredFurthermore,
in various waysthe functional
to producegroups
swapped, are
abundant, bonded,
covalently and chitosan could beionic,
carboxylate, alteredandinenclosed
various ways to produce
derivative products swapped,
to meetcovalently
a variety
bonded,
of future carboxylate,
research [18].ionic, and enclosed derivative products to meet a variety of future
research [18].
In this context, the medicinal plant, Martynia annua was used to synthesize chitosan
In this context,
nanoparticles the medicinal
(MAL-CNPs), henceplant, Martyniapharmaceutical-grade
it possessed annua was used to synthesize preciouschitosan
phyto-
nanoparticles (MAL-CNPs), hence it possessed pharmaceutical-grade
chemical constituents and has been used to treat venomous bites, tuberculosis, precious andphyto-
sore
chemical constituents and has been used to treat venomous bites,
throats for decades [19]. Thus, the current research was framed to the synthesis of chitosan tuberculosis, and sore
throats for decades
nanoparticles [19]. Thus,
(MAL-CNPs) withthe currentannua
Martynia research
extractwastoframed
evaluatetotheir
the synthesis of chi-
in vitro antioxi-
tosan nanoparticles (MAL-CNPs) with Martynia annua
dant, cytotoxic, α- amylase, α-glucosidase, and mutagenicity properties (Scheme 1). vitro
extract to evaluate their in
antioxidant, cytotoxic, α-amylase, α-glucosidase, and mutagenicity properties (Scheme 1).

Scheme 1. Schematic representation of green synthesized chitosan nanoparticles (MAL-CNPs) using

Martynia annua extract and its biomedical applications.
Crystals 2022, 12, 1540 3 of 16

2. Materials and Methods

2.1. Materials
The healthy leaves of the Martynia annua sample were collected from East Rajapalayam,
Salem District, Tamil Nadu, India. The collected plant was identified and recognized
as Martynia annua by Professor P. Jayaraman, Director, Plant Anatomy Research Centre
Chennai, Tamil Nadu (Reg. No. of Certificate: PARC/2020/4375). The collected leaf sample
was rinsed with clean tap water to remove dirt and shadow-dried until it was completely
dehydrated. The well-dried leaf sample was pulverized using an electric mixer and sieved
through a standard flour filter. Chitosan, ethanol, acetic acid, and other solvents used in
this work were purchased from Sigma Aldrich, St. Louis, MO, USA.

2.2. Leaf Extract Preparation

The standard hot plate extraction approach was maintained to attain extreme yield
with vital phytochemicals [20]. Approximately 5 g of fine powdered Martynia annua
leaf sample was added separately to 25 mL of ethanol, hexane, chloroform, methanol,
and water. The sample comprising solvent blends was heated at 70 ◦ C for 1 h. After the
extraction procedure, the solvent extract was filtered and then concentrated using a vacuum
evaporator (Heidolph vacuum evaporator, Model:G3; Schwabach, Germany). Each solvent
extract yield was assessed using the following principle:

(Weight of beaker with extract(g)) − (Weight of beaker without extract (g)

Extract yield(%) =
Sample weight (g)

2.3. Synthesis of Chitosan Nanoparticles

Approximately 10 mL of 1% organic chitosan (dissolved in acetic acid: v/v) was mixed
with 10 mL of ethanol extract and incubated for 1 h at 50 ◦ C in an orbital shaker at 100 rpm.
The turbidity of the reaction mixture was centrifuged after incubation at 10,000 rpm for
10 min. The supernatant was discarded, and subsequent centrifugation and rinsing of
the pellets with an acetic acid solution (to remove un-synthesized nanoparticles) was
performed. After subsequent centrifugation, the chitosan nanoparticles (MAL-CNPs) were
freeze-dried and subjected to further characterization techniques [21].

2.4. Characterization of MAL-CNPs

The freeze-dried MAL-CNPs were suspended in acetic acid (1%), and their spectral
absorbance was recorded on a UV-visible spectrophotometer (ASK Lab Instruments, Hyder-
abad, India). The functional groups (responsible for reduction, capping, and stabilization)
present over the surface of MAL-CNPs were analyzed by following typical FT-IR protocol,
and the frequency band was scanned in the range 400–4000 cm−1 using Nicolet (iS50)
FT-IR (Thermo Fisher, St. Louis, MO, USA). Dynamic light scattering (DLS) was performed
on the Malvern panalytical instrument, Malvern, Worcestershire, United Kingdom. The
HRTEM analysis of MAL-CNPs was carried out using JEOL Model JEM2100F at an oper-
ating voltage of 200 kV, Tokyo, Japan. SEM analysis was performed using SEM (ZEISS),
Jena, Germany.

2.5. Phytochemical Qualitative Study

The ethanol extract of Martynia annua was examined for phytochemical investigation
using extract yield and thin layer chromatography (TLC) analyses. The typical phytochem-
ical properties, such as alkaloids (Dragendorff’s), terpenoids (Salkowski test), carbohy-
drates (Benedict’s), glycosides (Keller-Kilani test), flavonoids (Zinc-HCl reduction test),
protein and amino acids (Millon’s test), tannin and phenol (FeCl2 test), saponin (Froth test),
quinones (Borntrager’s test), fixed oil (paper test), resins (Acetone test), coumarins (Fluores-
cence test), and carotenoids were qualitatively examined with typical procedures [22,23].
Crystals 2022, 12, 1540 4 of 16

2.6. Antioxidant Activity Competence Analysis

2.6.1. DPPH Assay
The free radicals scavenging potential of Martynia annua extract-mediated CNPs (MAL-
CNPs) was investigated by the following methodology of Narayanan et al. [20] with minor
changes. Briefly, 100 µL of freshly prepared DPPH (0.1 mM) solution was mixed with 300 µL
of different concentrations (500, 250, 100, 50, and 10 µg/mL) of chitosan nanoparticles (in
triplicate) and shaken vigorously, then kept at room temperature for 30 min. After 30 min
of incubation, the absorbance of each concentration reaction blend was recorded at 517 nm
using a UV-vis spectrophotometer, and ascorbic acid was used as control. The following
formula was applied to calculate the DPPH radicals scavenging percentage, and linear
regression analysis was performed to calculate the IC50 values.

(Absorbance of control − Absorbance of reaction mixture)

DPPH scavenging (%) = × 100
Absorbance of control

2.6.2. H2 O2 Scavenging Assay

The typical methodology was followed to evaluate the H2 O2 scavenging potential of
MAL-CNPs [24]. Concisely, 0.6 mL of 43 mM of H2 O2 (1 M of phosphate buffer: pH 7.4)
was mixed with 1.4 mL of various dosages, such as 500, 400, 300, 200, 100, 80, 60, 40, 20,
and 10 µg/mL (individually) of MAL-CNPs. Subsequently, each reaction mixture was
kept undisturbed for 15 min at room temperature, and the absorbance of each of them
was measured with a UV-visible spectrophotometer at 230 nm. Furthermore, the H2 O2
scavenging percentage and IC50 were calculated through the standard formula and linear
regression analyses.

(Absorbance of control − Absorbance of reaction mixture)

H2 O2 scavenging (%) = × 100
Absorbance of control

2.7. In Vitro Cytotoxic Assay

The cytotoxic property of MAL-CNPs was determined by following the standard
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay on RIN-
m5F (Rat Islet Cells) cell line (procured from NCCS, Pune, India). Briefly, 200 µL of
(1 × 105 cells/mL/well) RIN-m5F cells were placed into a 96-well plate containing a
DMEM medium. After incubation, the wells were rinsed with PBS and then treated with
various concentrations (500, 400, 300, 200, 100, 80, 60, 40, 20, and 10 µg/mL) of MAL-CNPs
in triplicate and then incubated at 37 ◦ C in a 5% CO2 (humidified) incubator for 24 h. After
incubation, approximately 20 µL of MTT (5 mg/mL) was added to each well and again
incubated for 4 h until the formations of purple color precipitated. Next, fluids from the
wells were completely aspirated and washed with 200 µL of 1X PBS, then formazan crystals
were dissolved with 100 µL of DMSO and shaken for 5 min. Subsequently, the absorbance
of each well was recorded using a microplate reader (Thermo Fisher Scientific, Franklin,
MA, USA) at 570 nm, and the percentage cell viability and IC50 values were determined
with standard formulas.
Test OD
Cell viability (%) = × 100 (1)
Control OD

2.8. α-Amylase Inhibitory Assay

The α-amylase inhibitory activity competence of MAL-CNPs was evaluated through
the standard protocol of Shao et al. [25] with slight modifications. Briefly, approximately
100 µL of different dosages (500, 250, 100, 50, and 25 µg/mL) of MAL-CNPs were individ-
ually blended with 200 µL of freshly prepared α-amylase solution and kept undisturbed
for 35 min at 25 ◦ C. Subsequently, 400 µL of newly prepared 0.25% starch solution was
mixed with each sample tube and left undisturbed for 5 min at 37 ◦ C. Next, 1.0 mL of the
DNS solution was blended to inhibit the reaction and then left in a water bath (10 min)
Crystals 2022, 12, 1540 5 of 16

and chilled to room conditions. The acarbose was used as a positive control, and the
absorbance of each sample reaction was read at 540 nm. The following formula and linear
regression analysis were applied to calculate the α-amylase inhibitory percentage and IC50
values, respectively.

A control − (A test − A background)

α-Amylase Inhibition (%) = × 100
Control

2.9. α-Glucosidase Inhibitory Assay

The standard protocol [25] with slight modifications was followed to assess the α-
glucosidase inhibitory efficiency of MAL-CNPs. Briefly, approximately 60 µL of various
dosages (500, 250, 100, 50, and 25 µg/mL) of MAL-CNPs were individually blended with
50 µL of α-glucosidase solution (0.2 U/mL: prepared in 0.1 M of phosphate buffer: pH 6.8) in
96 well plates. These reaction mixtures were incubated at 37 ◦ C for 30 min. After incubation,
approximately 50 µL of 5 mM ρ-nitrophenyl-α-D-glucopyranoside (PNPG) solution was
added to each reaction mix and incubated at 37 ◦ C for 20 min. Next, approximately 160 µL
of 0.2 M NaCO3 was added to each well to inhibit the reaction. Subsequently, the absorbance
of each well was measured at 405 nm using a microplate reader and compared to positive
control acarbose. The α-glucosidase inhibitory efficiency was calculated following the
formula, and the IC50 value was determined by linear regression analysis.

Aco − At
α-Glucosidase Inhibition (%) = × 100
Aco

2.10. Mutagenicity Assay

The mutagenicity properties of MAL-CNPs were assessed by a typical Ames assay
according to Organization for Economic co-operation and Development (OECD 471) [26].
Trial I (plate incorporation technique) and trial II (pre-incubation technique) were fol-
lowed with various strains (TA 1535, TA 1537, TA 98, TA 100, and WP2) (trp pKM101) of
Salmonella typhimurium and E. coli in the presence and absence of metabolic activator (+S9
and −S9) in triplicate. The different concentrations (0.125, 0.625, 1.25, 2.5, and 5 mg/plate)
of MAL-CNPs were studied for both trial-I and trial-II as per OECD 471.

2.11. Statistical Analysis

All the data were analyzed with mean and standard deviation.

3. Results and Discussion

3.1. Plant Extract Yield and TLC Analysis
The several solvents (methanol, ethanol, hexane, chloroform, and water) hot plate-
based extract preparation approach delivers diverse yield extents. Among these solvent
extracts, ethanol provides an extreme yield in comparison with the other solvents. The
ethanol solvent extract delivered an extreme yield of 0.43 g, and in percentage, it was
measured as 8.68%; water extract yielded 0.39 g (7.98%), hexane 0.23 g (4.64%), methanol
0.27 g (5.42%), and chloroform 0.21 g (4.12%), as shown in Figure S1 (Supplementary File).
The maximum yield of ethanol extracts proposes that they might comprise valuable photo-
chemical compounds in comparison with other solvents. The TLC outcomes also showed
that the ethanol extract displayed a superior number of visible spots than the other solvent
extract under numerous visualization methods and had extreme Rf values with numerous
colors visualized under various factors 255 nm and 365 nm, iodine, DPPH, 10% ferric
chloride, visible light, Dragendorff’s reagent, and concentrated sulfuric acid. The results
report that the ethanol solvent efficiently extracts several bioactive phytochemicals from
the Martynia annua Linn. leaf sample. According to a previous report, Kaushik et al. [27]
attained approximately 19% of ethanol extract yield from the Martynia annua fruit sam-
ple. The plant extract increased yield for a specific solvent designates that the solvent
Crystals 2022, 12, 1540 6 of 16

precise phytochemicals are enhanced in that plant sample, and, in addition, its strength is
associated with the polarity interaction of solvent properties and phytochemicals [28].

3.2. Screening of Qualitative Phytochemicals

Based on the outcomes attained from different solvent yields and TLC investigation,
Martynia annua leaf ethanol extract phytochemical contents were qualitatively examined.
Remarkably, the ethanol extract comprises pharmaceutically valuable phytochemicals,
including alkaloid, tannin, phenol, protein, amino acids, saponin, glycosides, quinones,
fixed oil, resins, and carotenoids, as exhibited in Table 1.

Table 1. Qualitative phytochemical screening of Martynia annua ethanol extract.

Serial No. Phytochemicals Test Ethanol Extract

1 Carbohydrate Benedict’s Test −
2 Protein and Amino Acids Millions Test +
3 Alkaloid Dragendorff’s Test +
4 Tannin and Phenol Ferric Chloride Test +
5 Flavonoids Zn-HCl Test −
6 Terpenoids Salkowski Test −
7 Saponin Froth Test +
8 Glycosides Keller-Kilani Test +
9 Quinons NaOH Test +
10 Fixed Oil Paper/spot Test +
11 Resins Acetone Test +
12 Coumarins Fluorescence Test −
13 Carotenoids NA +
Note: (+) means presence of phytochemical and (−) absence of phytochemical.

3.3. Chitosan Nanoparticle Synthesis and Characterization

3.3.1. UV-Visible Analysis of MAL-CNPs
The ethanol leaf extract of Martynia annua displayed substantial chitosan nanoparticle
(MAL-CNPs) fabricating potential. The Chitosan reducing the potential of this ethanol
extract was primarily at peaks at 221, 232, and 319 nm (Figure 1). The peaks observed at
these nanometer ranges were associated with the nanometer of MAL-CNPs. The UV-visible
spectrum of MAL-CNPs are in the range 200–325 nm. These broad absorption bands
are due to the existence of the CO group [29]. Oh et al. [30] reported that the UV-visible
spectrum of MAL-CNPs using the ionic gelation approach noted the band at 320 nm and
stated their antibacterial activity against phytopathogenic bacteria. The pure chitosan
particle’s UV-visible spectrum is 339 nm [31]. The UV-visible spectrum disparities of MAL-
CNPs are directly associated with the process and synthesizing constraints followed for the
MAL-CNPs synthesis approach [32].

3.3.2. FT-IR Analysis

The functional groups involved in the reduction, capping, and stabilization of MAL-
CNPs were inspected through FT-IR analysis (Figure 2). The numbers of major peaks at
3941, 3789, 3435, 2920, 2852, 2065, 1630, 1270, 1110, 1060, 1035, 875, 617, and 564 cm−1
corresponded to various functional groups. The occurrence of peaks between 3940 and
3430 cm−1 is associated with–NH2 and –OH stretching vibrations, and it results in extra
molecular bioactive molecules of H bonding. Correspondingly, the band noticed between
2920 and 2065 cm−1 associated with the C-H stretching vibrations is related to aldehyde
and alkane groups. The peaks noticed at 1635 cm−1 correlated to the stretch of amide
I, 1270 cm−1 is attributed to –NH2 bending, 1110–1035 cm−1 is attributed to the amide
III stretching vibrations, and 870–565 cm−1 C-H bending connected to stretching of keto
groups. A related FT-IR spectrum has been found for chitosan nanoparticles synthesized
using Achyranthes aspera plant extract [33]. These outcomes indicate that the ethanol extract
of Martynia annua comprises the most active compounds, which have the probability to
 Crystals 2022, 12, 1540 7 of 16

Crystals 2022, 12, x FOR PEER REVIEW 7 of 17

reduce the chitosan original form into chitosan nanoparticles. These phytochemicals play a
significant role in the reduction, stabilization, and capping of chitosan nanoparticles [34].

Crystals 2022, 12, x FOR PEER REVIEW 8 of 17

UV-visiblespectrum
Figure1.1.UV-visible
Figure spectrum analysis
analysis of
of MAL-CNPs.
MAL-CNPs.

3.3.2. FT-IR Analysis

The functional groups involved in the reduction, capping, and stabilization of MAL-
CNPs were inspected through FT-IR analysis (Figure 2). The numbers of major peaks at
3941, 3789, 3435, 2920, 2852, 2065, 1630, 1270, 1110, 1060, 1035, 875, 617, and 564 cm−1 cor-
responded to various functional groups. The occurrence of peaks between 3940 and 3430
cm−1 is associated with–NH2 and –OH stretching vibrations, and it results in extra molec-
ular bioactive molecules of H bonding. Correspondingly, the band noticed between 2920
and 2065 cm−1 associated with the C-H stretching vibrations is related to aldehyde and
alkane groups. The peaks noticed at 1635 cm−1 correlated to the stretch of amide I, 1270
cm−1 is attributed to –NH2 bending, 1110–1035 cm−1 is attributed to the amide III stretching
vibrations, and 870–565 cm−1 C-H bending connected to stretching of keto groups. A re-
lated FT-IR spectrum has been found for chitosan nanoparticles synthesized using Achy-
ranthes aspera plant extract [33]. These outcomes indicate that the ethanol extract of
Martynia annua comprises the most active compounds, which have the probability to re-
duce the chitosan original form into chitosan nanoparticles. These phytochemicals play a
significant role in the reduction, stabilization, and capping of chitosan nanoparticles [34].

Figure 2. FT-IR analysis of MAL-CNPs.

Figure 2. FT-IR analysis of MAL-CNPs.

3.3.3. HR-TEM Analysis

The size and morphology of as-synthesized chitosan nanoparticles (MAL-CNPs)
with ethanolic leaf extract of Martynia annua plant are evaluated by using high-resolution
transmission electron microscopic analysis (HR-TEM). The HR-TEM analysis showed the
morphological properties and surface appearance of chitosan nanoparticles as being
spherical in shape (Figure 3a, smooth surface, and size range of approximately 60–130
nm). The selected area electron diffraction (SAED) pattern illustrates characteristic rings
Crystals 2022, 12, 1540 8 of 16
Figure 2. FT-IR analysis of MAL-CNPs.

3.3.3. HR-TEM Analysis

3.3.3. HR-TEM Analysis
The size and morphology of as-synthesized chitosan nanoparticles (MAL-CNPs)
The size and
with ethanolic leaf morphology of as-synthesized
extract of Martynia annua plantchitosan nanoparticles
are evaluated by using(MAL-CNPs) with
high-resolution
ethanolic leaf extract of Martynia annua plant are evaluated by using high-resolution
transmission electron microscopic analysis (HR-TEM). The HR-TEM analysis showed the trans-
mission electron
morphological microscopic
properties andanalysis
surface(HR-TEM).
appearance Theof HR-TEM
chitosan analysis showedasthe
nanoparticles mor-
being
phological properties and surface appearance of chitosan nanoparticles as being spherical in
spherical in shape (Figure 3a, smooth surface, and size range of approximately 60–130
shape (Figure 3a, smooth surface, and size range of approximately 60–130 nm). The selected
nm). The selected area electron diffraction (SAED) pattern illustrates characteristic rings
area electron diffraction (SAED) pattern illustrates characteristic rings (Figure 3b), which
(Figure 3b), which indicate that these chitosan nanoparticles are highly crystalline in na-
indicate that these chitosan nanoparticles are highly crystalline in nature. The average
ture. The average particle size of the chitosan nanoparticle is ⁓100–120 nm.
particle size of the chitosan nanoparticle is ~100–120 nm.

(a)HR-TEM
Figure3.3.(a)
Figure HR-TEMimage
imageand
and(b)
(b)SAED
SAEDpattern
patternofofMAL-CNPs.
MAL-CNPs.
Crystals 2022, 12, x FOR PEER REVIEW 9 of 17
3.3.4. SEM and DLS Analysis
3.3.4. SEM and DLS Analysis
The scanning electron microscopic (SEM) analysis of as-synthesized chitosan nanopar-
The
ticles scanning electron
(MAL-CNPs) microscopic
is further (SEM)
carried out to analysis of the
investigate as-synthesized
surface chitosan nano-
morphology of the
MAL-CNPs
particles (Figure
(MAL-CNPs) 4a,b). It is
is furtherrevealed that
carried out relatively
to spherical
investigate and uniform
the surface MAL-CNPs
morphology of the
MAL-CNPs (Figure 4a,b). It is revealed that relatively spherical and uniform
are formed. The SEM images suggest the existence of organic moieties on the surface ofMAL-CNPs
are formed. The SEM images suggest the existence of organic moieties on the surface of
nanoparticles as stabilizing agents. The accumulation of phytomolecules possibly occurs
nanoparticles as stabilizing agents. The accumulation of phytomolecules possibly occurs
due to the hydrogen bonding and/or electrostatic interactions between the functional
due to the hydrogen bonding and/or electrostatic interactions between the functional
groups of active phytomolecules and the surface of chitosan nanoparticles. Furthermore,
groups of active phytomolecules and the surface of chitosan nanoparticles. Furthermore,
the DLS analysis exhibited that the average size of MAL-CNPs was 53 nm, maximum di-
the DLS analysis exhibited that the average size of MAL-CNPs was 53 nm, maximum
ameter was 130.7 nm, and the polydispersity index was found to be 0.315 (Figure 5, Sup-
diameter was 130.7 nm, and the polydispersity index was found to be 0.315 (Figure 5,
plementary File Table S1).
Supplementary File Table S1).

(a,b)Scanning
Figure4.4.(a,b)
Figure Scanningelectron
electronmicroscopy
microscopyof
ofthe
theMAL-CNPs
MAL-CNPsatatdifferent
differentmagnifications.
magnifications.
Crystals 2022, 12, 1540 9 of 16

Figure 4. (a,b) Scanning electron microscopy of the MAL-CNPs at different magnifications.

Figure5.5.DLS
Figure DLSanalysis
analysisofofMAL-CNPs.
MAL-CNPs.

3.4.
3.4.Antioxidant
AntioxidantActivity
ActivityAnalysis
Analysis
The
Thefree
freeradicals
radicalsscavenging
scavengingefficiency
efficiencyofofMAL-CNPs
MAL-CNPswaswasevaluated
evaluatedwith
withDPPH
DPPHandand
HH22O
O22 scavenging
scavenging assays.
assays. Figures
Figures 66 and
and 77 demonstrate
demonstratethe
theOptical
OpticalDensity
Densityand
andpercentage
percentage
of DPPH scavenging activities, respectively. A considerable DPPH radical scavenging rate
of approximately 66.78% was found at a 50 µg/mL concentration of MAL-CNPs. The IC50
value was found to be 2.431 µg/mL. This scavenging percentage was better than the DPPH
scavenging efficiency of ascorbic acid (34.62%) (Figure 6). Similarly, Figure 7 represents
H2 O2 scavenging percentages of various concentrations of MAL-CNPs. Obtained results
revealed that the 50 µg/mL concentration of MAL-CNPs showed approximately 91.65%
of H2 O2 scavenging potential. Interestingly, it was considerably more significant than
the H2 O2 scavenging percentage (90.91%) of the positive control (Figure 7). These results
strongly suggest that the 50 µg/mL concentration of MAL-CNPs is the optimal value to
donate the electron to convert the unstable radicals into stable radicals [35]. The bioactive
compounds, which are involved in the synthesis, capping, and stabilization of MAL-CNPs
and coated over their surface, might possess fine antioxidant activity by acting as electron
donors. Such MAL-CNPs can improve free radical scavenging activity and also amplify
the antioxidant activity of particles coated over their surface [36]. A similar pattern of
antioxidant activity was reported by Kumar et al. [37] against DPPH and nitrate radicals.
The antioxidants that donate electrons can convert the violet-colored DPPH into the yellow-
colored diphenylpicryl hydrazine [38]. The MAL-CNPs can neutralize reactive oxygen
species (ROS) in the micro-environments where it is incorporated, lowering cell-induced
oxidative stress [39].
 donors. Such MAL-CNPs can improve free radical scavenging activity and also amplify
the antioxidant activity of particles coated over their surface [36]. A similar pattern of an-
tioxidant activity was reported by Kumar et al. [37] against DPPH and nitrate radicals.
The antioxidants that donate electrons can convert the violet-colored DPPH into the yel-
low-colored diphenylpicryl hydrazine [38]. The MAL-CNPs can neutralize reactive oxy-
Crystals 2022, 12, 1540 10 of 16
gen species (ROS) in the micro-environments where it is incorporated, lowering cell-in-
duced oxidative stress [39].

Crystals 2022, 12, x FOR PEER REVIEW 11 of 17

Figure 6.
Figure 6. DPPH
DPPH scavenging
scavengingpercentage
percentageofofMAL-CNPs.
MAL-CNPs.Note:
Note:positive
positivecontrol:
control:ascorbic
ascorbic acid;
acid; nega-
negative
tive control:
control: DPPH.DPPH.

Figure 7.
Figure 7. H
H22O22 scavenging
scavenging percentage
percentage of MAL-CNPs.

3.5. Cytotoxic Property Analysis

The results obtained from the MTT assay are presented in Figures 8 and 9, which
reveal the viability percentage and corresponding absorbance values of MAL-CNPs on
RIN-m5F. The results suggest that the cell viability and cytotoxicity were dose-dependent.
At minimum concentration (1–25 μg/mL), the cell viability was not affected by MAL-
CNPs; therefore, cytotoxicity was recorded at increasing concentrations from 50 μg/mL.
Crystals 2022, 12, 1540 11 of 16

3.5. Cytotoxic Property Analysis

The results obtained from the MTT assay are presented in Figures 8 and 9, which
reveal the viability percentage and corresponding absorbance values of MAL-CNPs on
RIN-m5F. The results suggest that the cell viability and cytotoxicity were dose-dependent.
At minimum concentration (1–25 µg/mL), the cell viability was not affected by MAL-
CNPs; therefore, cytotoxicity was recorded at increasing concentrations from 50 µg/mL.
According to this, IC50 was found to be 39.93 µg/mL. A report stated that chitosan-based
nanoparticles showed a considerable level of cytotoxicity and yielded identical IC50 and
IC20 values against the A549 cells. The cytotoxicity of chitosan nanoparticles was achieved
by reducing the degree of polymer deacetylation, but it was less affected by decreasing
the molecular weight [40]. Loutfy et al. [41] reported that chitosan-based nanoparticles
showed cytotoxicity at increased concentration against the HepG2 cell, and they found that
the IC50 value was 239 µg/mL. Detailed analysis of the cytotoxic activity of the MAL-CNPs
revealed a considerable impact on the apoptotic gene (caspase 3, p 53, and Bak) expression
of mRNA [42]. The caspase 3 gene after the cell is exposed indicates the participation
Crystals 2022, 12, x FOR PEER REVIEW 12 of 17
of an
Crystals 2022, 12, x FOR PEER REVIEW 12 of 17
apoptotic caspase-independent route besides raising the exposure of the MAL-CNPs to a
certain concentration [43].

Figure 8. Cell viability percentage analysis by MTT assay with MAL-CNPs. Note: control: un-
Figure 8.8.Cell
Figure Cellviability
treated cells. viabilitypercentage
percentageanalysis byby
analysis MTT assay
MTT with
assay MAL-CNPs.
with Note:
MAL-CNPs. control:
Note: un- untreated cells.
control:
treated cells.

Figure 9. MTT assay with MAL-CNPs on treated cells.

Figure9.9.MTT
Figure MTTassay
assaywith
withMAL-CNPs
MAL-CNPson
ontreated
treatedcells.
cells.

3.6.α-Amylase
3.6. α-AmylaseInhibitory
Inhibitoryand
andα-Glucosidase
α-GlucosidaseInhibitory
InhibitoryAssays
Assays
The substances α-amylase and α-glucosidase are
The substances α-amylase and α-glucosidase are the themost
mostessential
essentialinhibitory
inhibitorytargets
targets
to control diabetic type II disease. Figure 10 depicts (OD and percentage of inhibition,
to control diabetic type II disease. Figure 10 depicts (OD and percentage of inhibition,
respectively) the α-amylase inhibiting potential of MAL-CNPs. The obtained results sug-
Crystals 2022, 12, 1540 12 of 16

3.6. α-Amylase Inhibitory and α-Glucosidase Inhibitory Assays

Crystals 2022, 12, x FOR PEER REVIEW 13 of 17
The substances α-amylase and α-glucosidase are the most essential inhibitory targets
to control diabetic type II disease. Figure 10 depicts (OD and percentage of inhibition,
respectively) the α-amylase inhibiting potential of MAL-CNPs. The obtained results
gradually
suggest thatincreased from 9.01
the MAL-CNPs to 33.88%
showed for 10–500
moderate μg/mLactivity
inhibitory concentration
(3.38%)ofatMAL-CNPs.
250 µg/mL
Similarly, Pterocarpus
concentration The IC50 marsupium extract-mediated
value was found CNPs demonstrated
to be 1.981 µg/mL. This inhibitory a considerable
activity was
percentage comparable
reasonably of dose-dependent
with theα-amylase
α-amylaseas well as α-glucosidase
inhibitory inhibitory
activity of acarbose (9.05 activities
µg/mL).
[34]. Inhibiting
Similarly, such hydrolytic
the MA-CNPs enzymes can
also demonstrated help with type
considerable 2 diabetes inhibitory
α-glucosidase treatment activity
by low-
ering postprandial hyperglycemia [44].
(Figure 11). The MAL-CNPs showed dose-dependent α-glucosidase inhibitory activity. An
The bioactive
increased compounds
concentration coated demonstrated
(500 µg/mL) over the surface of MAL-CNPs
maximum are up
inhibition thought to be
to 33.88%.
promising
The IC50 valueand was
efficient
foundinhibitors
to be 161.8of α-amylase as well as α-glucosidase.
µg/mL. Interestingly, it was comparable This study re-
with the
vealed thatactivity
inhibitory the MAL-CNPs inhibited
of the positive α-amylase
control and 50.76%).
(acarbose: α-glucosidase in a concentration-de-
This inhibition percentage
pendent [45]
gradually manner.
increased The9.01
from α-glucosidase
to 33.88% for had a more
10–500 substantial
µg/mL inhibitoryofeffect
concentration than α-
MAL-CNPs.
amylase [46].
Similarly, The apparent
Pterocarpus variation
marsupium in the inhibition
extract-mediated CNPseffect of α-amylase
demonstrated and α-gluco-
a considerable
percentage
sidase might of lead
dose-dependent
to undigested as well the
sugars reaching
α-amylase as α-glucosidase
colon, resultinginhibitory activities
in intestinal [34].
microbial
Inhibiting suchsuccessive
digestion and hydrolyticintestinal
enzymes illnesses,
can help with type 2abdominal
including diabetes treatment by lowering
discomfort, constipa-
postprandial hyperglycemia
tion, and diarrhea [47]. [44].

Figure10.
Figure 10. Percentage
Percentage of
of α-amylase
α-amylase inhibitory
inhibitoryactivity
activityassay
assaywith
withMAL-CNPs.
MAL-CNPs.

The bioactive compounds coated over the surface of MAL-CNPs are thought to be
promising and efficient inhibitors of α-amylase as well as α-glucosidase. This study
revealed that the MAL-CNPs inhibited α-amylase and α-glucosidase in a concentration-
dependent [45] manner. The α-glucosidase had a more substantial inhibitory effect than α-
amylase [46]. The apparent variation in the inhibition effect of α-amylase and α-glucosidase
might lead to undigested sugars reaching the colon, resulting in intestinal microbial diges-
tion and successive intestinal illnesses, including abdominal discomfort, constipation, and
diarrhea [47].
Crystals 2022,
Crystals 2022, 12,
12, 1540
x FOR PEER REVIEW 14
13 of 17
of 16

Figure 11. Percentage

Percentage of
of α-glucosidase
α-glucosidaseinhibitory
inhibitoryactivity
activityassay
assaywith
withMAL-CNPs.
MAL-CNPs.Note:
Note:positive
positive
control: alphamylase + substrate; negative control: acarbose.
control: alphamylase + substrate; negative control: acarbose.

3.7. Mutagenicity Analysis

The mutagenicity
mutagenicityproperty
property of of
MAL-CNPs
MAL-CNPs against Salmonella
against typhimurium
Salmonella TA100TA100
typhimurium strain
and Escherichia
strain coli WP2
and Escherichia coli(trp
WP2 pKM101) strain (out
(trp pKM101) of five
strain (outstrains
of five from
strainseachfromspecies) demon-
each species)
strated slight toxicity.
demonstrated In theIn
slight toxicity. presence or absence
the presence of metabolic
or absence activation,
of metabolic there
activation, waswas
there no
decrease in the revertant number of colonies inhibition compared
no decrease in the revertant number of colonies inhibition compared to the negative con- to the negative control
at any
trol at of theof
any tested concentrations
the tested in eitherinstrain.
concentrations eitherAccording to the findings,
strain. According to the 5findings,
mg/plate5
was
mg/plate was chosen as the highest proportion for such main study trials (trials I both
chosen as the highest proportion for such main study trials (trials I and II), in
and II),
the
bothpresence and absence
in the presence of metabolic
and absence activation.
of metabolic The plate
activation. incorporation
The plate incorporation method was
method
performed with five concentrations separated by a factor of two in
was performed with five concentrations separated by a factor of two in triplicate of test triplicate of test items
and
itemsthe
and negative and positive
the negative controls
and positive with the
controls withthree strains,
the three i.e., TA
strains, i.e.,1537, TA1535,
TA 1537, and
TA1535,
TA98. For the remaining two tester strains, TA100 and WP2 (trp
and TA98. For the remaining two tester strains, TA100 and WP2 (trp pKM101), cytotoxi- pKM101), cytotoxicity
results werewere
city results incorporated
incorporated in Trial-I up toup
in Trial-I five
toconcentrations.
five concentrations.
Neither
Neither biologically substantial increaseinin
biologically substantial increase revertant
revertant counts
countswas wasnoted
notedfrom anyany
from of the
of
five test strains pre-incubated with the test item in the absence or presence of metabolic
the five test strains pre-incubated with the test item in the absence or presence of metabolic
activation. Either precipitation or reduction in background lawn was evidenced from any
activation. Either precipitation or reduction in background lawn was evidenced from any
of the dosages investigated. The positive controls demonstrated an unambiguous rise in
of the dosages investigated. The positive controls demonstrated an unambiguous rise in
revertant counts, including all five test strains and corresponding controls, affirming the test
revertant counts, including all five test strains and corresponding controls, affirming the
system’s sensitivity [48]. Based on these results, Martynia annua-mediated MAL-CNPs may
test system’s sensitivity [48]. Based on these results, Martynia annua-mediated MAL-CNPs
indeed be non-mutagenic, as they did not stimulate gene mutation in any of the test strains
may indeed be non-mutagenic, as they did not stimulate gene mutation in any of the test
at the concentrations tested. De Lima et al. [49] reported that the CNPs demonstrated no
strains at the concentrations tested. De Lima et al. [49] reported that the CNPs demon-
substantial changes in the mitosis process on human lymphocyte cells, and this suggests
strated no substantial changes in the mitosis process on human lymphocyte cells, and this
that the CNPs are non-toxic. The technique used demonstrates great potential to be used in
suggests that the CNPs are non-toxic. The technique used demonstrates great potential to
nanoparticle safety checks, implying that these materials will be used in various biological
be used in nanoparticle safety checks, implying that these materials will be used in various
and commercial applications in the future [50].
biological and commercial applications in the future [50].
4. Conclusions
4. Conclusions
The present study aimed to evaluate the antioxidant, cytotoxicity, and mutagenic
Theofpresent
activity study aimed MAL-CNPs.
green-synthesized to evaluate the antioxidant,
The cytotoxicity,
obtained results conclude and that
mutagenic
the MAL-ac-
tivity exhibited
CNPs of green-synthesized MAL-CNPs.
antioxidant activities, The was
and this obtained results
confirmed by conclude
DPPH andthat H2 Othe
2 MAL-
radicals
CNPs exhibited
scavenging assay.antioxidant
The MTT assay activities, andrevealed
results this wasthat
confirmed by DPPHcytotoxicity
the MAL-CNPs and H2O2 radicals
activi-
Crystals 2022, 12, 1540 14 of 16

ties were dose-dependent from the concentration of 50 µg/mL. The MAL-CNPs showed
significant α-glucosidase and α-amylase inhibitory activity. These results indicate that
these green synthesized MAL-CNPs may be considered as a valuable material for type 2
diabetic treatment. Furthermore, the Ames test results indicated that the MAL-CNPs were
non-toxic, as they did not induce mutagenesis on bacterial test strains. These MAL-CNPs
could be considered as therapeutic nanomaterials for several biomedical applications in
the near future in medical fields. However, in-vivo studies need to be performed to ensure
their efficiency and safety for future therapeutic applications.

Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/cryst12111540/s1, Figure S1: Different solvent extracts yield of Martynia an-
nua Linn. stated values are mean and standard error of triplicates; Table S1: Volume Distribution
Table of DLS Results.
Author Contributions: Conceptualization, N.D. and S.D.; methodology, N.D. and S.D.; formal
analysis, M.R.S., A.H.S., J.P.S., and B.S.; investigation, N.D., S.D., and M.R.S.; resources, S.D.; data
curation, N.D. and S.D.; writing—original draft preparation, N.D., S.D., and M.R.S.; writing—review
and editing, N.D., S.D., and M.R.S.; supervision, S.D.; project administration, S.D.; funding acquisition,
A.H.S. All authors have read and agreed to the published version of the manuscript.
Funding: The authors extend their appreciation to the Researchers Supporting Program for funding
this work through Researchers Supporting Project number (RSP-2021/371), King Saud University,
Riyadh, Saudi Arabia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article and supplementary file.
Acknowledgments: The authors extend their appreciation to the Researchers Supporting Program
for funding this work through Researchers Supporting Project number (RSP-2021/371), King Saud
University, Riyadh, Saudi Arabia.
Conflicts of Interest: The authors declare no conflict of interest.

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