Open Access Original

Article DOI: 10.7759/cureus.46003

Evaluation of the Anti-inflammatory,

Antimicrobial, Antioxidant, and Cytotoxic Effects
Review began 09/20/2023
of Chitosan Thiocolchicoside-Lauric Acid Nanogel
Review ended 09/21/2023
Published 09/26/2023 Ameena M 1 , Meignana Arumugham I 2 , Karthikeyan Ramalingam 1 , Rajeshkumar S 3

© Copyright 2023
M et al. This is an open access article 1. Oral Pathology and Microbiology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and
distributed under the terms of the Creative Technical Sciences, Saveetha University, Chennai, IND 2. Public Health Dentistry, Saveetha Dental College and
Commons Attribution License CC-BY 4.0., Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, IND 3. Pharmacology,
which permits unrestricted use, distribution, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University,
and reproduction in any medium, provided Chennai, IND
the original author and source are credited.

Corresponding author: Karthikeyan Ramalingam, karthikeyanr.sdc@saveetha.com

Abstract
Aim: The present study explored the anti-inflammatory, antimicrobial, antioxidant, and cytotoxic effects of
a combination of chitosan thiocolchicoside and lauric acid (CTLA) nanogel.

Materials and methods: A nanogel formulation of thiocolchicoside and lauric acid was developed and tested
for potential applications. The antimicrobial activity was assessed using the well diffusion method, while the
antioxidant activity was evaluated using the 2,2-diphenyl-1-picryl hydrazyl (DPPH) free radical scavenging
assay and hydrogen peroxide (H2O2) antioxidant assay methods. The anti-inflammatory activity was
determined through the egg albumin denaturation method, the bovine serum albumin denaturation method,
and the membrane stabilization assay. A brine shrimp lethality assay was used to study the cytotoxic effect
of the nanogel.

Results: We identified significant positive outcomes for the CTLA nanogel. The results showed a percentage
of inhibition of 81% at 50μg/mL, which showed the nanogel’s significant anti-inflammatory activity by
inhibiting bovine serum albumin denaturation. The anti-inflammatory properties of the nanogel were
comparable to the standard diclofenac sodium at all tested concentrations. The egg albumin denaturation
assay results revealed a percentage inhibition of 76% at 50 μg/mL. In the membrane stabilization assay, a
percentage inhibition of 86% was obtained at a concentration of 50 μg/mL against 89% for the standard
drug. The nanogel exhibited a zone of inhibition of 20 mm against Streptococcus mutans and 22 mm with a
dilution of 100 µg/mL of CTLA nanogel against Staphylococcus aureus. The antioxidant activity was studied
by using the DPPH method, 50 μg/ml has an 89% inhibition, which was similar to the standard. The
inhibitory activity of CTLA nanogel at 50 μg/ml was 81.6% in the hydroxyl free radical scavenging assay,
which was comparable to the standard drug. At 5 μg/mL concentration of CTLA nanogel, approximately 90%
of the nauplii remained alive after 48 hours.

Conclusion: The CTLA nanogel showed excellent anti-inflammatory and antioxidant properties suggesting
its potential for managing inflammatory conditions and oxidative stress-related disorders.

Categories: Pathology, Dentistry, Therapeutics

Keywords: lethality assay, cytotoxicity, antimicrobial, lauric acid, thiocolchicoside, chitosan, ctla, antioxidant, anti-
inflammatory, nanogel

Introduction
Nanogels are hybrid materials that blend the properties of nanomaterials with hydrogels [1]. Hydrogels,
known for their high water content, offer tunability regarding their physical and chemical structures, and
excellent mechanical and nontoxic properties [2]. In nanomedicine, nanogel-based formulations have shown
great potential in various applications, including imaging, anticancer therapy, and drug delivery [3].

Chitosan, a deacetylated chitin is a biopolymer that is a significant component of the cell wall of fungi, the
exoskeleton of insects, and the shells of crustaceans. It is a linear copolymer containing β-(1 to 4)-2-amino-
d-glucose units and β-(1 to 4)-2-acetamido-d-glucose units, known to have excellent properties like
biodegradability and biocompatibility with the least immune response even after implantation or
application due to its nontoxic nature [4].

Thiocolchicoside is a semi-synthetic derivative of colchicoside, which is obtained from plants like Gloriosa
superba and Colchicum autumnale [5]. It is an anti-inflammatory drug used as a muscle relaxant in the
treatment of musculoskeletal disorders [6]. The muscle relaxant activity of thiocolchicoside is attributed to
its inhibition of the glycine receptor in the brain stem and spinal cord [7]. In previous studies,

How to cite this article

M A, I M, Ramalingam K, et al. (September 26, 2023) Evaluation of the Anti-inflammatory, Antimicrobial, Antioxidant, and Cytotoxic Effects of
Chitosan Thiocolchicoside-Lauric Acid Nanogel. Cureus 15(9): e46003. DOI 10.7759/cureus.46003
 thiocolchicoside has been formulated in various semi-solid forms such as ointments, gels, and creams, with
ointments showing higher drug release compared to other formulations [8].

Lauric acid, on the other hand, is a saturated fatty acid commonly used in nutritional and cosmetic
applications. It is a 12-carbon chain fatty acid found in certain plants, particularly coconut oil and palm
kernel oil [9]. Lauric acid has been recognized for its broad spectrum of antimicrobial activity against
bacteria and viruses. Although the exact mechanism of its action against bacteria is not fully understood, it
is believed to disrupt the cell membrane and is thus helpful in protecting against microbial infection and can
control human microbiota balance in our body [10,11]. Due to its biological activity and strong antiviral
properties, lauric acid is considered one of the most active components in coconut oil [12].

Inflammaging is a term used to denote a systemically developing chronic inflammation that is of low-grade
type in the absence of infection in elderly people. As most age-related disorders are linked with
inflammation, inflammaging is a significant risk factor for morbidity and mortality in old age [13].
Developing new anti-inflammatory drugs is always of interest to limit the chronic inflammatory process in
the human body which includes arthritis, colitis, dermatitis, neurodegenerative disorders, and malignancy.
Since the anti-inflammatory drugs to relieve inflammation are always non-steroidal anti-inflammatory
drugs (NSAID) or corticosteroids, which develop a lot of adverse drug reactions like gastric irritation, and
liver, and renal disorders on long-term usage [14].

Mitochondrial metabolism plays a powerful role in the induction of carcinogenesis by increasing the levels of
reactive oxygen species (ROS) production from oncogene transformation to cancer progression. An increase
in ROS production results in structural damage to cellular components which leads to cancer, inflammation,
and different disorders. There is a recent trend to study natural compounds with significant antioxidant
activity that could affect the redox reactions taking place in a cell and prevent and control free radical-
mediated reactions [4].

The objective of the present study was to assess the antimicrobial activity, antioxidant activity, anti-
inflammatory activity, and cytotoxic effects of a chitosan nanogel formulation containing thiocolchicoside
and lauric acid (CTLA nanogel). By combining these two active components, the researchers aimed to
explore the potential synergistic effects and broaden the biomedical applications of the nanogel.

Materials And Methods

Preparation of chitosan thiocolchicoside and lauric acid (CTLA) nanogel was reported by Ameena et al. [15].
This method allowed for the integration of the lauric acid and thiocolchicoside into a nanogel formulation.
The chitosan served as a medium to facilitate the incorporation of the active components and stabilize the
nanogel structure.

Anti-inflammatory activity
Bovine Serum Albumin (BSA) Denaturation Assay

The anti-inflammatory activity of the CTLA nanogel was evaluated as described by Das et al. [16]. To assess
the anti-inflammatory activity, 0.05 mL of the CTLA nanogel was taken, and various concentrations ranging
from 10µg/mL, 20µg/mL, 30µg/mL, 40µg/mL, and 50µg/mL were added to 0.45 mL of a 1% aqueous solution
of bovine serum albumin. The pH of the solution was corrected to 6.3 using a small amount of 1N
hydrochloric acid. These samples were then incubated at room temperature for 20 minutes, followed by
heating at 55°C for 30 minutes in a water bath. After the heating process, the samples were allowed to cool
down, and the absorbance was measured using a spectrophotometer at 660 nm. Diclofenac sodium was the
standard drug for comparison. Dimethyl sulfoxide (DMSO) was used as a control in this experiment.

The percentage of protein denaturation was determined using the following equation: % Inhibition =
(Absorbance of control - Absorbance of sample/Absorbance of control) x 100.

Egg Albumin Denaturation Assay

The anti-inflammatory activity of the CTLA nanogel was determined. The samples used for this assay
include 0.2 mL of egg albumin (fresh), 2.8 mL of phosphate-buffered saline (PBS) at pH 6.4, and 0.6 mL of the
nanogel at various concentrations dissolved in 0.2% DMSO. The concentrations of the nanogel in the total
reaction solution ranged from 10-50 µg/mL. The samples were incubated for 10 minutes at 37°C and then
heated at 70°C in a water bath for an additional 20 minutes to induce denaturation of the egg albumin. After
cooling the mixture, the absorbance was measured at 660 nm. Negative controls consisting of 0.2 mL of
fresh egg albumin, 0.6 mL of 0.2% DMSO, and 2.8 mL of PBS were included in the experiment. Diclofenac
sodium was used as a positive control for the study.

The percentage of protein denaturation inhibition, which indicates the anti-inflammatory activity of the
compound, was calculated by the following equation: % Inhibition = (As/Ac - 1) × 100 (As = absorbance of

2023 M et al. Cureus 15(9): e46003. DOI 10.7759/cureus.46003 2 of 10

 sample, Ac = absorbance of control).

Membrane Stabilization Assay

The in vitro membrane stabilization assay is a commonly employed technique for evaluating the membrane
stabilizing properties of natural and synthetic drugs. This assay measures the ability of a drug to stabilize
the cell membrane by preventing its disruption and subsequent release of intracellular contents. The
materials include human red blood cells (RBCs), Tris-hydrochloride (Tris-HCl) buffer (50 mM at pH 7.4), and
PBS. Different concentrations of CTLA nanogel (10-50 µg/mL) were prepared. Saline solution and distilled
water were used as controls in this study.

Fresh human blood was collected in a sterile tube containing anticoagulants. The blood was centrifuged for
10 minutes at 1000 g at room temperature to separate the RBCs from other blood components. The
supernatant was slowly removed and the RBCs left behind were washed three times using PBS. Then RBCs
were resuspended in Tris-HCl buffer to obtain a 10% (v/v) RBC suspension.

1mL of the RBC suspension was pipetted into each centrifuge tube and different concentrations of CTLA
nanogel were added to each tube, gently mixed, and incubated for 30 minutes at 37°C. The centrifuge tubes
were then centrifuged at 1000 g for 10 minutes at room temperature to pellet the RBCs. The absorbance of
the supernatant obtained was measured at 540 nm using an ultraviolet spectrophotometer.

The percentage inhibition of hemolysis was calculated using the following formula: % Inhibition = {(OD
control - OD sample)/OD control} x 100. OD control in the formula is the absorbance of the RBC suspension
without the test compound and OD sample is the absorbance of the RBC suspension with the test
compound.

Anti-microbial activity
The antibacterial activity of the CTLA nanogel was investigated against bacterial strains including
Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, Lactobacillus and Candida albicans. For
this experiment, a 24-hour freshly prepared culture of the bacteria was used. To determine the zone of
inhibition, Muller-Hinton agar (MHA) was prepared and sterilized by autoclaving at 121°C for 30 minutes.
The sterilized MHA was poured into sterile Petri plates and was allowed to solidify. The wells were then
created using a Well cutter and the fresh bacterial culture of Streptococcus mutans, Staphylococcus aureus,
Pseudomonas aeruginosa, Lactobacillus and Candida albicans was evenly spread on the Petri plates. Different
concentrations of the CTLA nanogel (25 µg, 50 µg, 100 µg) were loaded into separate wells on the agar plate
in triplicates. Additionally, an antibiotic Amoxyrite was used as a standard for bacteria, and for candida,
Fluconazole was used as a standard and placed in the fourth well for comparison. The plates were incubated
for 24 hours and 48 hours for fungal cultures at 37°C. The antimicrobial activity of the compound was
assessed by measuring the diameter of the zone of inhibition around the wells. The zone of inhibition was
measured using a ruler and recorded in millimeters (mm). This measurement indicated the antibacterial
effectiveness of the nanogel.

Anti-oxidant activity
2,2-Diphenyl-1-Picryl Hydrazyl (DPPH) Free Radical Scavenging Assay

The antioxidant activity of CTLA nanogel was analyzed using the DPPH assay. Various concentrations
(10μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, and 50 μg/mL) of the nanogel were mixed with 1 mL of DPPH (0.1
mM) in methanol and 450 μg/mL of 50 mM Tris-HCl buffer at pH 7.4. The mixture was then incubated in a
dark room for 30 minutes. The reduction in the quantity of the DPPH free radical was assessed by measuring
the absorbance at 517 nm. This measurement indicated the antioxidant capacity of the nanogel. Ascorbic
acid was used as standard in this assay. By evaluating the absorbance at 517 nm, this assay determined the
antioxidant activity of the CTLA nanogel.

The percentage of the inhibition was determined from the following equation: % inhibition of sample =
(Absorbance of control - Absorbance of sample/Absorbance of control) x 100.

Hydroxyl Free Radical Scavenging Assay

The hydroxyl free radical scavenging assay was conducted. Freshly prepared solutions were used for the
experiment. In a reaction mixture of 1.0 mL, the following components were added: 100 µL of a 28 mM
solution of 2-deoxy-2-ribose dissolved in phosphate buffer at a pH 7.4, 500 µL of a solution containing
different concentrations of the CTLA nanogel (ranging from 10 to 50 µg), 200 µL of a 200 µM ferric chloride
(FeCl3) and 1.04 mM ethylenediaminetetraacetic acid (EDTA) mixture in a 1:1 volume ratio, 100 µL of H 2O2
(1.0 mM), and 100 µL of ascorbic acid (1.0 mM). The reaction mixture was incubated for 1 hour at 37°C. The
extent of deoxyribose degradation after the incubation period, was determined by thiobarbituric acid (TBA)
reaction. The mixture was further incubated for 1 hour at 37°C, and the optical density at 532 nm was

2023 M et al. Cureus 15(9): e46003. DOI 10.7759/cureus.46003 3 of 10

 measured against a blank solution. For comparison, vitamin E served as the positive control, and ascorbic
acid was used as the standard. By measuring the optical density at 532 nm, the hydroxyl radical scavenging
activity of the CTLA nanogel was assessed.

Cytotoxic effect
Brine Shrimp Lethality Assay

To prepare the solution, 2 grams of iodine-free salt was weighed and dissolved in 200 mL of distilled water.
Six enzyme-linked immunosorbent Assay (ELISA) well plates were used for the experiment, and each well
was filled with 10-12 mL of prepared saline water. Subsequently, 10 nauplii were added slowly to each well,
with different concentrations of the CTLA nanogel (5 µg/mL, 10 µg/mL, 20 µg/mL, 40 µg/mL, and 80 µg/mL)
added to the respective wells. The sixth well served as a control and did not receive the nanogel. The plates
were then incubated at room temperature for 24 hours, allowing the desired effects of the nanogel on the
nauplii to take place.

After 24 hours, the ELISA plates were carefully observed and counted for the number of live nauplii present
and calculated by using the following formula: Number of dead nauplii/Number of dead nauplii + Number of
live nauplii × 100.

Results
The anti-inflammatory activity of CTLA nanogel was evaluated using the bovine serum albumin
denaturation assay. The nanogel was tested at different concentrations, and its inhibitory effects were
compared to standard values. The results showed a percentage of inhibition of 47% at a concentration of 10
μg/mL, 53% at 20 μg/mL, 69% at 30 μg/mL, 72% at 40 μg/mL, and 81% at 50 μg/mL. These values indicate
that the nanogel exhibits significant anti-inflammatory activity by inhibiting bovine serum albumin
denaturation. Moreover, the anti-inflammatory properties of the nanogel were comparable to the standard
diclofenac sodium at all tested concentrations (Table 1).

10 μg/mL 20 μg/mL 30 μg/mL 40 μg/mL 50 μg/mL

CTLA nanogel 44 58 70 75 80

Standard 47 60 72 78 84

TABLE 1: Anti-inflammatory activity with BSA assay

Table showing the anti-inflammatory activity of chitosan thiocolchicoside lauric acid (CTLA) nanogel with bovine serum albumin (BSA) denaturation assay

The anti-inflammatory activity of CTLA nanogel was assessed using the egg albumin denaturation assay.
The nanogel was tested at various concentrations and compared to standard values. The results revealed a
percentage of inhibition of 53% at a concentration of 10 μg/mL, 58% at 20 μg/mL, 61% at 30 μg/mL, 69% at
40 μg/mL, and 76% at 50 μg/mL. These findings demonstrate that the nanogel exhibits significant anti-
inflammatory activity in the egg albumin denaturation assay. Furthermore, the anti-inflammatory properties
of the nanogel were found to be comparable to the standard diclofenac sodium at all tested concentrations
like 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, and 50 μg/mL. Therefore, the CTLA nanogel shows promising
anti-inflammatory activity in the context of the egg albumin denaturation assay (Table 2).

10 μg/mL 20 μg/mL 30 μg/mL 40 μg/mL 50 μg/mL

CTLA nanogel 53 62 66 70 80

Standard 55 64 69 72 81

TABLE 2: Anti-inflammatory activity with egg albumin assay

Table showing the anti-inflammatory activity of chitosan thiocolchicoside lauric acid (CTLA) nanogel with egg albumin denaturation assay

The anti-inflammatory activity of CTLA nanogel was assessed using the human red blood cell membrane
stabilization assay. The nanogel was tested at different concentrations and compared to standard values.
The results revealed a percentage of inhibition of 56% at a concentration of 10 μg/mL, 67% at 20 μg/mL, 75%

2023 M et al. Cureus 15(9): e46003. DOI 10.7759/cureus.46003 4 of 10

 at 30 μg/mL, 80% at 40 μg/mL, and 86% at 50 μg/mL against 58%, 70%, 77%, 82%, and 89% for the standard,
diclofenac sodium at same concentrations. These findings show a significant anti-inflammatory activity of
CTLA nanogel in the membrane stabilization assay (Table 3).

10 μg/mL 20 μg/mL 30 μg/mL 40 μg/mL 50 μg/mL

CTLA nanogel 56 67 75 80 86

Standard 58 70 77 82 89

TABLE 3: Red blood cell membrane stabilization assay

Table showing the human red blood cell membrane stabilization assay of chitosan thiocolchicoside lauric acid (CTLA) nanogel

The antimicrobial activity of CTLA nanogel was evaluated using the agar well diffusion method. The
nanogel exhibited a zone of inhibition of 20 mm with a dilution of 100 µg/mL of CTLA nanogel against
Streptococcus mutans while 13 mm was the zone of inhibition for the standard. The nanogel developed a zone
of inhibition of 22 mm with a dilution of 100 µg/mL of CTLA nanogel against Staphylococcus aureus while 11
mm was the zone of inhibition for the standard. The zone of inhibition of CTLA nanogel at 100 µg/mL
against Candida albicans was 16 mm and fluconazole was 20 mm. The nanogel exhibited a zone of inhibition
of 9 mm against both Lactobacillus species and Pseudomonas aeruginosa at three different concentrations,
indicating similar levels of inhibition for both bacterial strains. In comparison, the fourth well, which
contained the commercial antibiotic Amoxyrite, showed a higher zone of inhibition of 14 mm against
Lactobacillus species and a lower area of inhibition of 9 mm against Pseudomonas aeruginosa at the same
diluted concentration (Figure 1).

FIGURE 1: Antimicrobial activity of CTLA nanogel

Images showing the anti-microbial activity of chitosan thiocolchicoside lauric acid (CTLA) nanogel against
Streptococcus mutans (A), Lactobacillus species (B), Staphylococcus aureus (C), Pseudomonas aeruginosa (D),
and Candida albicans (E)

The antioxidant activity was done by using the DPPH method. DPPH assay was compared from a lower
concentration to a higher concentration of the CTLA nanogel. In the CTLA nanogel, a different
concentration was added. In the different concentrations of nanogel, 10μg/ml had a 68% inhibition, 20
μg/ml had a 76% inhibition, 30 μg/ml had a 79% inhibition, 40 μg/ml had an 85% of inhibition and 50 μg/ml
had an 89% of inhibition. The antioxidant activity of CTLA nanogel was slightly similar when compared to
the standard (Figure 2).

2023 M et al. Cureus 15(9): e46003. DOI 10.7759/cureus.46003 5 of 10

 FIGURE 2: Graph of DPPH assay
Graphical representation of the activity of chitosan thiocolchicoside lauric acid (CTLA) nanogel estimated by 2,2-
diphenyl-1-picryl hydrazyl (DPPH) free radical scavenging assay

The antioxidant activity of CTLA nanogel was evaluated using the H 2O2 method. Different nanogel
concentrations were tested, ranging from lower to higher concentrations. The results showed that the
nanogel exhibited antioxidant activity, with increasing levels of inhibition as the concentration of the
nanogel increased. At 10 μg/ml, the nanogel demonstrated 50.6% inhibition, while at 20 μg/ml, it showed
54.7% inhibition. The inhibitory activity further increased at 30 μg/ml with 65.12% inhibition, at 40 μg/ml
with 74.3% inhibition, and at 50 μg/ml with 81.6% inhibition. The CTLA nanogel displayed similar
antioxidant activity to the standard (Figure 3).

FIGURE 3: Graph of H2O2 assay

Graphical representation of the activity of chitosan thiocolchicoside lauric acid (CTLA) nanogel estimated with
hydrogen peroxide (H2O2) assay

The cytotoxic effects of CTLA nanogel were evaluated using the brine shrimp lethality assay. This assay is
commonly employed to assess the cytotoxicity of substances by measuring their impact on the survival of
the brine shrimp nauplii. In the present study, a control group without any drug was maintained to establish
a baseline for calculating the percentage of live nauplii. The results of the cytotoxicity assessment indicated
that different concentrations of the nanogel exhibited varying effects on nauplii survival. At a concentration

2023 M et al. Cureus 15(9): e46003. DOI 10.7759/cureus.46003 6 of 10

 of 5 μg/mL of thiocolchicoside-lauric acid nanogel, approximately 90% of the nauplii remained alive.
Similarly, at concentrations of 20 μg/mL and 40 μg/mL, the nanogel resulted in the preservation of
approximately 70% of live nauplii. However, at a higher concentration of 80 μg/mL, only 60% of the nauplii
survived (Figure 4).

FIGURE 4: Graph showing the cytotoxic effect of CTLA nanogel

Graphical representation of the cytotoxic effect of chitosan thiocolchicoside lauric acid (CTLA) nanogel estimated
by brine shrimp lethality assay

Discussion
Anti-inflammatory properties of various plant extracts are available in the literature [17]. Overall, our study
highlights the ability of the CTLA nanogel and the herbal formulation extract to effectively inhibit
inflammation by preventing protein denaturation.

The lysosomal membrane stabilization in activated neutrophils prevents the leakage of lysosomal contents
like protease and other bactericidal enzymes, thereby playing a pivotal role in anti-inflammatory response
in the human body. Red blood cells and lysosomal membranes of neutrophils have a similar structure, so
stabilization of one membrane may limit the destruction of the other. This principle is the basis for the
human red blood cell membrane stabilization assay which uses hypotonicity-induced lysis to evaluate the
anti-inflammatory activity of drugs [14]. In the present study, it was found that the CTLA nanogel had
excellent anti-inflammatory action using the human red blood cell membrane stabilization assay, and the
process of membrane stabilization could be directly related to its anti-inflammatory properties.

Kyene et al. using the agar well diffusion method studied the antimicrobial activity of the zinc oxide nano
particles against Staphylococcus aureus, Escherichia coli, Salmonella typhi, and Candida albicans [18].
Shanmugam et al. reported that silver nanoparticles with the addition of curcumin-assisted chitosan
nanocomposite had remarkable antibacterial activity against gram-positive bacteria when compared to
gram-negative bacteria in their study [19].

Furthermore, the individual component of the nanogel, lauric acid, demonstrated significant inhibitory
effects against various clinical isolates. In previous research works, the inhibitory effect varied based on the
concentration of the acid, with the highest zone of inhibition observed against Staphylococcus aureus (10
mm), Streptococcus species (10 mm), and Lactobacillus species (10 mm). On the other hand, the lowest
inhibitory effect was found against Escherichia coli (4 mm) at the same concentration of dilution [12].
Previous studies have also reported the antimicrobial effects of lauric acid, specifically against Gram-
positive streptococci but not as effective against Gram-negative bacilli such as Escherichia coli, Klebsiella
oxytoca, Klebsiella pneumoniae, and Serratia marcescens [11]. Nagase et al. compared the bactericidal activity
of ovirgin coconut oil and lauric acid using an antibacterial disk diffusion test and reported that the
bacteria-inhibiting zone was 0.17 μmol /40 μL or 0.085 μmol / 40 μL of Lauric acid on plates inoculated with
Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mutans, and Streptococcus sanguinis and was
greater than that of the paper disk containing 0.17 μ mol40 μL of virgin coconut oil [20]. They found that
lauric acid has significant antimicrobial activity against Staphylococcus aureus and Streptococcus salivarius
and organic virgin coconut oil had weaker antimicrobial activities against several Streptococcus species than
lauric acid.

Bhardwaj et al. studied the antibacterial activity of coconut oil by agar well diffusion method using
ciprofloxacin as a standard antibiotic [21]. They found that Streptococcus species were susceptible to coconut

2023 M et al. Cureus 15(9): e46003. DOI 10.7759/cureus.46003 7 of 10

 oil while Escherichia coli were not. They confirmed that the presence of lauric acid is responsible for the
bactericidal action of coconut oil. These findings highlight the antimicrobial potential of lauric acid,
particularly against certain bacterial strains, as demonstrated by various studies.

Chitosan nanoparticles exhibited redox-regulatory activity due to inhibition of free radical production,
decreasing serum free fatty acids, and malondialdehyde, and increasing intracellular antioxidant enzymes in
vitro as well as in vivo studies [4]. The antioxidant activity of CTLA nanogel was evaluated using the DPPH
method. Different nanogel concentrations were tested, ranging from lower to higher concentrations. The
results indicated that the nanogel exhibited antioxidant activity, with varying degrees of inhibition at each
concentration. Comparatively, the CTLA nanogel showed a slight similarity to the standard antioxidant.

Virgin coconut oil reduced the DPPH free radical concentration by 50% and EC50 was found to be 5.07 ± 0.19
mg/L in a study by Ahmad et al. [22]. The hydrogen-donating capacity of virgin coconut oil makes it a good
antioxidant, but the level of free-radical scavenging activity depends on the processing condition of virgin
coconut oil [22]. The free radical scavenging activity (%) of green tea-loaded with chitosan nanoparticles,
green tea, and ascorbic acid was reported by Piran et al. showed the high antioxidant activity of green tea
and green tea-loaded chitosan nanoparticles [23]. The range of green tea and green tea-loaded chitosan
nanoparticles scavenging was found to be 32.07-91.0 μg/ml and 46.75-96.1 μg/ml respectively [23]. Wen et
al. observed the effects of chitosan nanoparticles in H2O2-induced oxidative damage in murine macrophage
cells and found that viability loss in cells induced by H2O2 was significantly replaced by chitosan
nanoparticles [24]. It suppressed the production of malondialdehyde, restored superoxide dismutase and
glutathione peroxidase, and increased total antioxidant capacity.

Marina et al. reported that virgin coconut oil, due to its antioxidant properties, reduced the initial
concentration of DPPH radicals by 50%, with an EC50 value of approximately 5.07 ± 0.19 mg/L [25]. Virgin
coconut oil can donate hydrogen ions and thus can act as an antioxidant. The processing conditions of virgin
coconut oil may influence the free radical scavenging activity of its phenolic compounds [25].

Thyme essential oil encapsulated in chitosan nanoparticles proved to have greater antioxidant activity than
free thyme essential oil [26]. Similarly, green tea-loaded chitosan nanoparticles and green tea itself
demonstrated significant scavenging activity, indicating high antioxidant capacity. The scavenging activity
ranged from 32.07% to 91.034% for green tea and from 46.75% to 96.12% for green tea-loaded chitosan
nanoparticles at various concentrations [27]. Overall, the CTLA nanogel, exhibited promising antioxidant
properties, as demonstrated by their ability to scavenge free radicals and inhibit oxidative processes.

Overall, the cytotoxic activity results revealed a relatively low toxicity rate of the CTLA nanogel, which
aligns with the findings of the current study. This indicates that the nanogel formulation exhibited minimal
cytotoxic effects on brine shrimp nauplii, suggesting its potential safety for future applications.

Limitations
We performed various in-vitro analyses in the present study to assess the activity of CTLA nanogel. Testing
with Fourier-transform infrared spectroscopy (FTIR) or nuclear magnetic resonance (NMR) will help to
decipher the active ingredients of our nanogel formulation. Further in vivo studies including animal studies
and clinical trials will help in a better understanding of its effects.

Conclusions
In conclusion, combining thiocolchicoside and lauric acid as chitosan nanogel showcases a versatile
therapeutic agent with multifaceted properties. Its antimicrobial activity, eco-friendly nature, and
biocompatibility highlight its promising role in combating infections. Moreover, its anti-inflammatory and
antioxidant properties suggest its potential for managing inflammatory conditions and oxidative stress-
related disorders. These characteristics position thiocolchicoside-lauric acid as a promising candidate for
future biomedical applications, paving the way for further exploration and development in the field of
medicine.

Additional Information
Author Contributions
All authors have reviewed the final version to be published and agreed to be accountable for all aspects of the
work.

Concept and design: Karthikeyan Ramalingam, Ameena M, Meignana Arumugham I

Drafting of the manuscript: Karthikeyan Ramalingam, Ameena M, Meignana Arumugham I, Rajeshkumar

S

Critical review of the manuscript for important intellectual content: Karthikeyan Ramalingam,

2023 M et al. Cureus 15(9): e46003. DOI 10.7759/cureus.46003 8 of 10

 Ameena M, Meignana Arumugham I, Rajeshkumar S

Supervision: Karthikeyan Ramalingam, Rajeshkumar S

Acquisition, analysis, or interpretation of data: Ameena M, Meignana Arumugham I, Rajeshkumar S

Disclosures
Human subjects: All authors have confirmed that this study did not involve human participants or tissue.
Animal subjects: Institutional Animal Ethics Committee, Saveetha Institute of Medical and Technical
Sciences Issued protocol number SDC/PhD/PHD-2118. Conflicts of interest: In compliance with the ICMJE
uniform disclosure form, all authors declare the following: Payment/services info: All authors have
declared that no financial support was received from any organization for the submitted work. Financial
relationships: All authors have declared that they have no financial relationships at present or within the
previous three years with any organizations that might have an interest in the submitted work. Other
relationships: All authors have declared that there are no other relationships or activities that could appear
to have influenced the submitted work.

References
1. Hamidi M, Azadi A, Rafiei P: Hydrogel nanoparticles in drug delivery . Adv Drug Deliv Rev. 2008, 60:1638-49.
10.1016/j.addr.2008.08.002
2. Ahmed EM: Hydrogel: preparation, characterization, and applications: a review . J Adv Res. 2015, 6:105-21.
10.1016/j.jare.2013.07.006
3. Molina M, Asadian-Birjand M, Balach J, Bergueiro J, Miceli E, Calderón M: Stimuli-responsive nanogel
composites and their application in nanomedicine. Chem Soc Rev. 2015, 44:6161-86. 10.1039/c5cs00199d
4. Ivanova DG, Yaneva ZL: Antioxidant properties and redox-modulating activity of chitosan and its
derivatives: biomaterials with application in cancer therapy. Biores Open Access. 2020, 9:64-72.
10.1089/biores.2019.0028
5. Mahendran D, Kavi Kishor PB, Sreeramanan S, Venkatachalam P.: Enhanced biosynthesis of colchicine and
thiocolchicoside contents in cell suspension cultures of Gloriosa superba L. exposed to ethylene inhibitor
and elicitors. Ind Crops Prod. 2018, 120:123-30. 10.1016/j.indcrop.2018.04.040
6. Cimino M, Marini P, Cattabeni F: Interaction of thiocolchicoside with [3H]strychnine binding sites in rat
spinal cord and brainstem. Eur J Pharmacol. 1996, 318:201-4. 10.1016/s0014-2999(96)00884-9
7. Shrivastav J, Shah K, Mahadik M, et al.: Application of HPTLC in the simultaneous estimation of
thiocolchicoside and diclofenac in bulk drug and pharmaceutical dosage form. Bull Pharm Res. 2011, 1:34-7.
8. Jiang L, Liang X, Liu G, et al.: The mechanism of lauric acid-modified protein nanocapsules escape from
intercellular trafficking vesicles and its implication for drug delivery. Drug Deliv. 2018, 25:985-94.
10.1080/10717544.2018.1461954
9. Rozanna D, Chuah TG, Salmiah A, et al.: Fatty acids as phase change materials (PCMs) for thermal energy
storage: a review. Int J Green Energy. 2005, 1:495-513. 10.1081/GE-200038722
10. Ogbolu DO, Oni AA, Daini OA, et al.: In vitro antimicrobial properties of coconut oil on Candida species in
Ibadan, Nigeria. J Med Food. 2007, 10:384-7. 10.1089/jmf.2006.1209
11. Matsue M, Mori Y, Nagase S, et al.: Measuring the Antimicrobial Activity of Lauric Acid against Various
Bacteria in Human Gut Microbiota Using a New Method. Cell Transplant. 2019, 28:1528-41.
10.1177/0963689719881366
12. Abbas A, Assikong EB, Akeh M, Upla P: Antimicrobial activity of coconut oil and its derivative (lauric acid)
on some selected clinical isolates. Int J Med Sci Clin Invent. 2017, 30:4.
13. Shayganni E, Bahmani M, Asgary S, Rafieian-Kopaei M: Inflammaging and cardiovascular disease:
management by medicinal plants. Phytomedicine. 2016, 23:1119-26. 10.1016/j.phymed.2015.11.004
14. Qamar M, Akhtar S, Ismail T, et al.: Syzygium cumini (L.), skeels fruit extracts: in vitro and in vivo anti-
inflammatory properties. J Ethnopharmacol. 2021, 271:113805. 10.1016/j.jep.2021.113805
15. Ameena M, Meignana Arumugham I, Ramalingam K, Rajeshkumar S, Perumal E: Cytocompatibility and
Wound Healing Activity of Chitosan Thiocolchicoside Lauric Acid Nanogel in Human Gingival Fibroblast
Cells. Cureus. 2023, 15:e43727. 10.7759/cureus.43727
16. Das P, Ghosal K, Jana NK, Mukherjee A, Basak P: Green synthesis and characterization of silver
nanoparticles using belladonna mother tincture and its efficacy as a potential antibacterial and anti-
inflammatory agent. Mater Chem Phys. 2019, 228:310-7. 10.1016/j.matchemphys.2019.02.064
17. Pranati T, Rajasekar A, Rajeshkumar S: Anti-inflammatory and cytotoxic effect of clove and cinnamon
herbal formulation. Plant Cell Biotechnol Mol Biol. 2020, 21:69-77.
18. Kyene MO, Droepenu EK, Ayertey F, et al.: Synthesis and characterization of ZnO nanomaterial from Cassia
sieberiana and determination of its anti-inflammatory, antioxidant and antimicrobial activities. Sci African.
2023, 19:01452. 10.1016/j.sciaf.2022.e01452
19. Shanmugam R, Subramaniam R, Kathirason SG, et al.: Curcumin-chitosan nanocomposite formulation
containing Pongamia pinnata-mediated silver nanoparticles, wound pathogen control, and anti-
inflammatory potential. Biomed Res Int. 2021, 2021:3091587. 10.1155/2021/3091587
20. Nagase S, Matsue M, Sugitani K, et al.: Comparison of the antimicrobial spectrum and mechanisms of
organic virgin coconut oil and lauric acid against bacteria (Article in Japanese). J Wellness Heal Care. 2017,
41:87-95. 10.24517/00048862
21. Bhardwaj V: Antimicrobial potential of Cocos nucifera (coconut) oil on bacterial isolates . Adv Exp Med Biol.
2023, 10.1007/5584_2023_786
22. Ahmad Z, Hasham R, Nor NF, Sarmidi MR: Physico-chemical and antioxidant analysis of virgin coconut oil
using West African tall variety. J Adv Res in Materials Science. 2015, 13:1-10.

2023 M et al. Cureus 15(9): e46003. DOI 10.7759/cureus.46003 9 of 10

 23. Piran F, Khoshkhoo Z, Hosseini SE, Azizi MH: Controlling the antioxidant activity of green tea extract
through encapsulation in chitosan-citrate nanogel. J Food Qual. 2020, 1-9. 10.1155/2020/7935420
24. Wen ZS, Liu LJ, Qu YL, Ouyang XK, Yang LY, Xu ZR: Chitosan nanoparticles attenuate hydrogen peroxide-
induced stress injury in mouse macrophage RAW264.7 cells. Mar Drugs. 2013, 11:3582-600.
10.3390/md11103582
25. Marina AM, Man YB, Nazimah SA, Amin I: Antioxidant capacity and phenolic acids of virgin coconut oil . Int
J Food Sci Nutr. 2009, 60 Suppl 2:114-23. 10.1080/09637480802549127
26. Ghaderi GM, Barzegar M, Sahari MA, Azizi MH.: Enhancement of thermal stability and antioxidant activity
of thyme essential oil by encapsulation in chitosan nanoparticles. J Agric Sci Technol. 2016, 8:1781-1792.
27. Kedare SB, Singh RP: Genesis and development of DPPH method of antioxidant assay . J Food Sci Technol.
2011, 48:412-22. 10.1007/s13197-011-0251-1

2023 M et al. Cureus 15(9): e46003. DOI 10.7759/cureus.46003 10 of 10