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Original Article
2025
:18;
852025
doi:
10.25259/AJC_85_2025

Silver nanoparticles embedded chitosan-gelatin polymeric composite: Green synthesis, characterization, and investigation of its catalytic activity and protective effect on cerebral ischemia-reperfusion injury in rats

, , ,
 Department of Neurosurgery, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Third Hospital of Shanxi Medical University, Tongji Shanxi Hospital, Taiyuan, Shanxi, 030032, China

*Corresponding author: E-mail address: renbinlc@126.com (R. Bin)

Received: , Accepted: ,
© 2025 Arabian Journal of Chemistry - Published by Scientific Scholar
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

After an ischemic stroke, mechanical recanalization or thrombolysis can be used to restore blood flow. Reperfusion, however, may worsen the ischemia-induced harm in certain patients, resulting in a condition known as “cerebral reperfusion injury.” This injury is caused by various pathogenic events, including postischemic hyperperfusion, leukocyte infiltration, platelet and complement activation, and blood-brain barrier disruption. Formulating a current remedial drug for stroke is a research priority in the world. Integrating nanoscience and medicine is essential as the nano revolution develops. The creation of biogenic technologies to produce sophisticated nanomaterials is the result of the secret discovered in nature. The present study describes the sustainable synthesis of polymeric nanocomposite-based Ag nanoparticles (NPs) after specifying its features and biological investigations. We employed chitosan-gelatin (CS-Gel) compound hydrogel as the template. The biopolymers had the potential to play the role of an environmentally friendly metric-reducing agent of the approaching silver ions as well as containing a stabilizer of the silver NPs through activating and using their electron-loaded organo-functions. Fourier transform infrared spectroscopy (FT-IR) Spectroscopy, UV-Vis spectroscopy, scanning electron microscope (SEM), transmission electron microscopy (TEM), energy dispersive X ray (EDX), elemental mapping, and X-ray diffraction (XRD) constituted analytical tools for assessing and recognizing the physicochemical properties of the substance. In the water solvent and ambient temperature, the performance of the CS-Gel/Ag NPs nanocomposite had to be evaluated in the reduction of methyl orange (MO) as a harmful water contaminant. The reactions were appraised via a time-based UV-Vis spectroscopic investigation demonstrating the great capability of the catalyst. The CS-Gel/Ag NPs nano catalyst was separated using the centrifuge and became so constant that it could be reused for nine successive periods with no significant harm to the action. The middle cerebral artery was blocked for 90 mins and then reperfused for 48 hours to cause cerebral ischemia-reperfusion damage in rats. For three days prior to the ischemia reperfusion, the rats were given intravenous injections of 40, 80, 160, and 320 µg/kg of CS-Gel/Ag NPs nanocomposite once daily. The cell damage index was determined by measuring the infarct volume, serum malondialdehyde (MDA) level, lactate dehydrogenase activity, learning and memory performance, and IgG extravasation into the cerebral parenchyma. Rats’ cerebral ischemic stroke-reperfusion injury was found to be protected by pretreatment with CS-Gel/Ag NPs nanocomposite. Pretreatment with CS-Gel/Ag NPs nanocomposite inhibited the learning and memory deficits induced by an ischemia stroke-reperfusion injury. Additionally, the CS-Gel/Ag NPs nanocomposite significantly blocked the extravasation of IgG and decreased the infarct volume and lactate dehydrogenase activity.

Keywords

Ag nanoparticles
Cerebral ischemia-reperfusion injury
Chitosan-gelatin thin film
Methyl orange

1. Introduction

Stroke is a major factor in fatalities, permanent impairment, and financial burdens, underscoring the critical importance of finding successful therapies [1-3]. In the critical stage, the only treatments that have received food and drug administration (FDA) approval to restore blood flow to the brain include the use of endovascular thrombectomy, a drug that breaks down blood clots, the direct injection of recombinant tissue plasminogen activator, and a procedure that physically removes clots from blood vessels [3-6]. Because of the brief time available for treatment and the significant risk of brain bleeding, only a small number of patients with acute stroke can safely receive tissue plasminogen activator therapy [4,5]. Although endovascular thrombectomy can be done over a longer period, specialty centers only execute this procedure on patients who have occlusion in a bigger, anatomically more proximal vasculature. Regardless of the technique, ischemia-reperfusion damage poses a further difficulty in the event of successful recanalization [3-5]. Additionally, tissue plasminogen activator is neurotoxic and impairs the integrity of the blood-brain barrier (BBB), exacerbating reperfusion injury [5,7]. A nanoparticle (NP)-based approach may be able to get around some of the aforementioned problems and provide a thrombolytic drug that can be safely used after the window for tissue plasminogen activator treatment has closed [6-8]. A variety of NP characteristics are also being investigated to create a multifunctional thrombolytic drug that can also contain therapies, such as neuro/vasoprotective, antioxidant, anti-inflammatory, or imaging agent—also known as a theragnostic agent [7-10]. Furthermore, depending on their composition, certain nanomaterials have demonstrated promise in scavenging reactive oxygen species (ROSs) in strokes [7,9]. The clinical translation and antioxidants application greatly depend on the medicines’ capacity to be delivered using nanomaterials. To shield cells from oxidative stress-induced death, the NPs target the ischemic brain and raise the drug’s blood concentration and half-life [8-10].

Environmental pollution and the increasing demand and consumption of energy are among the most pressing global challenges. Nanomaterials have emerged as a new opportunity to deal with this concern and to control the adverse effects of wastewater on the environment through treating pollutants and energy generation by organic materials within the context of sustainable development [11-13]. The excessive depletion of waste poisonous organic coloring agents (hues), like methyl orange (MO), into soils and water pools hampers environment protection. Therefore, it is essential to handle the dyeing wastewater in safe and innocuous ways [14,15].

In the past few years, material science has experienced significant progress resulting from the emergence of nanoscience and nanotechnology. Concerning practical usage in associated areas, nanomaterials are increasingly demonstrating engrossing features in comparison with their numerous competing chemicals. The traditional synthetic methods of metallic NPs with fine manufacturing and structures or elegant construction requiring physical and chemical procedures frequently face large and varied challenges [16-20]. Within such a framework, the synthesis notion stimulated by biology while comprising Mother Nature became eminent in the semi-transparently formed NPs, supposed to remove many barriers by using practical and promising manners. The biogenic or bio-inspired approach seems more beneficial and frugal compared with other ways concerning eco-friendly aspects: consuming safe water solvent, abundance of resources, cost-effectiveness, and livability to be adjusted and improved with various biomolecules [21-24]. The latter ones have indications of demonstrating enhanced biological features. Specially, the bio-motivated and biopolymer made original metal NPs practical and caused them to obtain immense significance in academic circles thanks to their massive imputations in several areas such as bio and electrochemical sensing, chemical catalysis, preventing environment pollution, forensics, hologram printing, food, textile, hue industries and so on [25-29]. Furthermore, these biocompatible original metal NPs are extensively connected with remedial sphere including cancer treatment, targeted drug release, besides exhibiting excellent antioxidant, antibacterial, and cytotoxic features due to the extremely fine scale, vast plane zone, many more various plane active types, and notably, the potential to generate ROS [30].

Consequently, we were promoted to explore biomotivated Ag NPs based on a compound hydrogel-made bio-macromolecules, chitosan-gelatin (CS-Gel). In the nanomaterial, the polysaccharides (CS and Gel) are provided full of abundant electron organo-roles such as amines and hydroxyls [31,32], and they also operate as original main metabolites productively accomplishing the eco-friendly decrease of Ag+ ions into the related bioNPs (Scheme 1) [33-36]. The prepared CS-Gel/Ag NPs nanocatalyst was applied for the catalytic reduction of MO as a harmful water contaminant (Scheme 1). After successfully synthesizing and characterizing their characteristics, the CS-Gel/Ag NPs nanocomposite was evaluated for its therapeutic activities on cerebral ischemia-reperfusion injury in rats.

Schematic manufacturing CS-Gel/Ag NPs nanocomposite and its catalytic application in the reduction of methyl orange.
Scheme 1.
Schematic manufacturing CS-Gel/Ag NPs nanocomposite and its catalytic application in the reduction of methyl orange.

2. Materials and Methods

2.1. Synthesis of CS-Gel/Ag NPs bio-nanocomposite

We prepared the CS-Gel hydrogel by shaking the two macromolecules (0.1 g of either of them) into a 1% CH3COOH aqueous mixture (v/v) for 12 hours. Then we added aqueous AgNO3 (0.01% w/V, 10 mL) solution (the precursor of Ag) dropwise into it and knocked it up at 80°C for 30 mins. The color altering of the solution from colorless to deep brown confirmed the formation of Ag NPs by environmentally friendly electronic reduction. Ultimately, we collected the final CS-Gel/Ag NPs nanocomposite using a centrifuge, rinsed it several times with an EtOH/H2O mixture, and subsequently dried it at aerial conditions, and used inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis to specify the Ag condensation in the material as 0.036 mmol/g.

2.2. The catalytic activity of the CS-Gel/Ag NPs in the reduction of MO

In MO degradation, a similar procedure was followed, where we obtained an aqueous mixture of MO (5.0 ppm, 3 mL) in the cuvette along with the catalyst. Afterward, we added NaBH4 aqueous solution (25 mM, 1.0 mL) to it and monitored it. Here too, the reaction progress was monitored as a decrease in absorbance at 464 nm for MO, respectively.

We applied the ensuing Eq. (1) to calculate the % decrease of MO:

(1)
% Reuction  =   A 0   −  A t A 0 × 100

In which:

  • A0 refers to the primary absorbance of the mixture prior to the reaction.

  • At stands for absorbance of the mixture after a given reaction time.

The pseudo-first-order kinetic model’s linear form, which is represented by the following Eq. (2), was used to assess the reaction kinetics:

(2)
ln ( C t / C 0 ) =  ln  ( A t / A 0 ) = − kt

In which:

  • Ct, and at represent the condensation and absorbency for reactant at time t.

  • C0, and A0 are the primary condensation and absorbency.

  • kapp is the reduction rate constant.

  • t is the reaction time.

By drawing the natural logarithm of the proportion of Ct to C0 against time (t), we acquired a linear relationship, and the resultant slope determines the constant rate (k).

2.3. Animals

Male Wistar rats weighing between 220 and 230 g were used in this investigation. After a 12:12 hour D:L illumination cycle, the animals were housed in a chamber with a regulated temperature of 25°C. Every single one of the rats was fed a commercial meal and had unlimited access to water.

2.4. Stroke design

The rats were split up into six groups, each of which included fifteen rats. These groups were divided into the following categories: untreated control group (I), treatment group with 40 μg/kg of CS-Gel/Ag NPs nanocomposite (II), treatment group with 80 μg/kg of CS-Gel/Ag NPs nanocomposite (III), treatment group with 160 μg/kg of CS-Gel/Ag NPs nanocomposite (IV), and treatment group with 320 μg/kg of CS-Gel/Ag NPs nanocomposite (V). The rats were then given a 1.5-hour middle cerebral artery closure and two days of reperfusion to cause a stroke [37].

The animals were placed on a jumping device floor composed of parallel stainless steel bars following a 48-hour reperfusion period. A 36V electric foot shock was administered to the animals after they had adapted for five mins. The rat’s learning latency and learning mistakes were determined by timing how long it took it to leap onto the elevated platform and how often it jumped from the platform to the floor while getting electric shocks within 5 mins. The rat was put directly on the platform the following day, and the amount of time it took to jump to the floor and receive electric foot shocks within five mins was recorded to assess the memory latency and error rate. A computerized video tracking system was used to follow the rat’s travel around the maze. The trials were divided into 2 phases: navigation probe and place spatial. For the place navigation trial, each rat took part in 4 trials daily for 4 days in a row. After being submerged in water in each of the 4 quadrants, the rats were allowed to swim freely until they stayed and were found on the platform. The escape latency is the amount of time it took the rat to find and stay. The platform was taken down, and the rats were submerged in the water in the northeast quadrant for the probing testing. They had 60 seconds to swim as they pleased. The number of entries made into the southwest quadrant (where the platform was first placed) and the length of time and distance the rat spent walking inside that quadrant were noted. To calculate the infarct volume percentage, a brain matrix was used to dissect the brain horizontally. Each animal’s blood was drawn after the previously described tests were finished, and it was centrifuged for ten mins at 4,000 gravity. After that, the resultant serum was moved to another tube. Using a biochemistry test kit, the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH) were measured. Additionally, the brain parenchyma IgG level was measured using a commercially available rat IgG enzyme-linked immunosorbent assay (ELISA) kit. Since the brain parenchyma IgG presence might be affected by a damaged BBB, this assessment indirectly reveals the permeability of the barrier [37].

2.5. Statistical analysis

Three duplicates of each stroke therapy were administered, along with a control group. Using Statistics 8.1 software, factorial analysis of variance (ANOVA) was used to evaluate the data about stroke outcomes. A Tukey test was used to compare the means to evaluate the impact of the therapies on stroke.

3. Results and Discussion

3.1. Chemical characterization analysis

The CS-Gel/Ag NPs nanocomposite was prepared by using an environmentally friendly method employing CS-Gel polymers, as revealed in Scheme 1. The mechanism for the CS-Gel/Ag NPs formation requires the Ag ions mechanistic adsorption into CS-Gel compound hydrogel [38-41] due to the present alcoholic functions of the backbone, which acted to reduce Ag+ ions. Then, as a capping and stabilizing agent, the amino and hydroxyl groups of CS-Gel envelop the Ag NPs. A variety of cutting-edge methods were then used to extensively describe the resultant CS-Gel/Ag NPs nanocomposite.

The bio-produced Ag NPs in the presence of CS-Ge were discovered by seeing the manifestation of a dark brown mixture, confirmed by UV-Vis investigation, as shown in Figure 1. The data ascertained that Ag NPs absorbance was a chain at ∼445-450 nm (λmax). The result was confirmed by other studies containing the phyto-interceded procurement of Ag NPs using the Pistacia bark extract that the Ag NPs band was seen near 445 nm [42].

UV-Vis spectra of CS-Gel/Ag NPs nanocomposite at different times.
Figure 1.
UV-Vis spectra of CS-Gel/Ag NPs nanocomposite at different times.

The morphological structure of the as-synthesized CS-Gel/Ag NPs nanocomposite was analyzed using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) pictures (Figures 2 and 3). The obtained NP appeared in polymorphic dimensions with implanted globular Ag nanoparticles (Figure 2). The coverage factors available in CS-Gel because of the presence of hydroxyl and amino groups, can powerfully cover the NPs, shaping a reinstating layer, which, in turn, creates particles with adjusted dimensions [43,44]. As illustrated in Figure 3, the silver NPs are monotonously distributed in the birthplace with great crystallinity. It also indicates that the Ag NPs in the CS-Gel/Ag NPs dimension is 30-40 nm with global forms. The same results for Ag NPs made from Ducrosia anethifolia [45] and leaves of Welsh onion are similarly described [46].

FE-SEM image of CS-Gel/Ag nanoparticles.
Figure 2.
FE-SEM image of CS-Gel/Ag nanoparticles.
TEM image of CS-Gel/silver nanoparticles.
Figure 3.
TEM image of CS-Gel/silver nanoparticles.

We applied Energy dispersive X ray (EDX) investigation to the material to achieve an out-knowledge of its chemical composition. We can infer from Figure 4 that silver engrosses the main part or symptom of 86.7% of the profile. Nonetheless, another potent sign, because of Au (2.1 keV), can be noticed as well, rising from the gold steam deposition over the sample before our investigation. We can also notice a few feeble signals in the bottom area of the profile attributed to non-metals like C, N, and O. They certainly originate from the CS-Gel composite hydrogel. We accentuated more on the EDX results by elemental mapping investigation. Figure 5 depicts the X-ray scanning profile of the FE-SEM picture. The colored dots symbolize the atomic species of substance ingredients, which are Ag, C, N, and O. Likewise, Veisi et al, [47,48] characterized by approximately the same conclusion for Ag NPs synthesis utilizing Panax ginseng root extract.

EDX spectrum of the CS-Gel/Ag NPs.
Figure 4.
EDX spectrum of the CS-Gel/Ag NPs.
Elemental mapping of CS-Gel/Ag NPs.
Figure 5.
Elemental mapping of CS-Gel/Ag NPs.

The essence of the crystal, deflection climaxes, and purity of the bio-nanocomposite were affirmed over XRD studies. As the XRD profile shows in Figure 6, the existence of an integrated stage confirms the sample as a highly limpid and tightly intermixed one. The primary perspective reveals a non-crystalline broad signal in the 2θ regions up to 2θ = 20o, connected with fruit phytochemicals. The remaining area looks extremely crystalline as discovered from refraction climaxes at 2θ = 38.2°, 44.1°, 64.3°, and 78.4°, related to Ag crystalline (111), (200), (220), and (311) surfaces, respectively. The apexes were carefully coincident with the modulus Ag NP fcc model.

XRD pattern of CS-Gel/Ag nanoparticles.
Figure 6.
XRD pattern of CS-Gel/Ag nanoparticles.

3.2. Catalytic application of CS-Gel/Ag NPs catalyst

In the UV-Vis spectrum, there is a noticeable absorption at 464 nm when examining the MO aqueous solution. Thermodynamically, it is advantageous to reduce MO only with NaBH4, but earlier reports indicate that the reaction rates are extremely slow and the related λmax remains almost unchanged in the absence of an appropriate catalyst [46,47]. Experimentally, the MO aqueous solution (5.0 ppm, 3 mL) was filled in the quartz cuvette, and subsequently, the freshly prepared NaBH4 solution was added (1.0 mL). Under catalyst-free conditions, the corresponding UV-visible spectra exhibited λmax at 464 nm. Nevertheless, after adding 1.0 mg (otherwise, 2.0 mg and 3.0 mg) of CS-Gel/Ag NPs nano catalyst, the peak intensity started to diminish immediately. Figures 7(a-f) represent the corresponding output monitored at different intervals, where the peak gradually flattened, indicating the full reduction of MO. Mechanistically, when the active catalyst exists, NaBH4 decreases the MO molecule azo function to its hydrazine derivative having free amino groups. We can apply Eq. (2) to obtain the % decrease in MO from the UV-visible perspective scheme. The rate constant kapp, which corresponds to the reaction pseudo-first-order kinetics, could be derived from the slope after depicting the proportion natural logarithm of At to A0 against the decrease time, illustrated in Figures 7(b), 7(d), and 7(f). In the present paper, the kapp values obtained on the catalyst load of 1.0, 2.0, and 3.0 mg were 0.007, 0.031, and 0.051 s-, respectively.

(a,c,e) Time-dependent UV-Vis spectra in the MO reduction (1.0 mg, 2.0 mg and 3.0 mg catalyst respectively); (b,d,f) The plot of ln(At/A0) vs. t (1.0 mg, 2.0 mg and 3.0 mg catalyst respectively).
Figure 7.
(a,c,e) Time-dependent UV-Vis spectra in the MO reduction (1.0 mg, 2.0 mg and 3.0 mg catalyst respectively); (b,d,f) The plot of ln(At/A0) vs. t (1.0 mg, 2.0 mg and 3.0 mg catalyst respectively).

Figure 8(a) displays the recyclability of the CS-Gel/Ag NPs nano catalyst for the diminution of MO through NaBH4. The outcome of this survey manifested that the efficiency of the CS-Gel/Ag NPs nano catalyst in decreasing MO stayed the same with no significant lose in catalytic activity post the 9th cycle. These results and the TEM image (Figure 8b) from the reused catalyst after 9th run indicated that the CS-Gel/Ag NPs are not only a stable but a heterogeneous catalyst as well.

(a) Recyclability of the CS-Gel/Ag nanoparticles nano catalyst in the reduction of MO, and (b) TEM image after 9th cycle.
Figure 8.
(a) Recyclability of the CS-Gel/Ag nanoparticles nano catalyst in the reduction of MO, and (b) TEM image after 9th cycle.

3.3. Treatment of stroke by CS-Gel/Ag NPs nanocomposite

According to recent studies, cerebral ischemia-reperfusion considerably increased the likelihood of memory and learning mistakes, as well as the amount of time needed to learn. However, as seen in Table 1, these effects were much lessened when rats received several doses of the CS-Gel/Ag NPs nanocomposite (40, 80, 160, and 320 µg/kg).

Table 1. The efficacy of CS-Gel/Ag NPs nanocomposite on several parameters (*p≤0.01).
Parameters Groups (n=10)
Control CS-Gel/Ag NPs-40 CS-Gel/Ag NPs-80 CS-Gel/Ag NPs-160 CS-Gel/Ag NPs-320
Proportion of swim distance (%) 18±2 27±2* 31±4* 35±2* 39±4*
Proportion of time (%) 17±1 25±2* 29±3* 31±2* 35±3*
Number of entries 1±0 3±0* 3±0* 3±0* 3±0*
Escape latency (Sec) 39±5 24±4* 23±4* 20±2* 15±3*
Memory latency (Sec) 125±9 173±6* 177±11* 185±8* 197±7*
Learning latency (Sec) 53±4 38±5* 34±2* 31±1* 30±3*

Every animal showed a similar level of proficiency in finding the escape platform on the first training day (Table 1). All rats’ average escape latencies dramatically dropped on the fourth day of training. When rats were given 40, 80, 160, and 320 µg/kg of the CS-Gel/Ag NPs nanocomposite, the escape latency lengthening was successfully prevented.

Both the time spent and the entries made during the crossing of the distance to the platform decreased for the control animal group, as reported in Table 1. However, the animals showed a markedly enhanced spatial probe capacity when given 40, 80, 160, and 320 µg/kg of CS-Gel/Ag NPs nanocomposite, suggesting a protective effect.

The findings shown in Table 2 indicate that the percentage of cerebral infarcts increased in the control animal group. However, the rats showed a markedly improved cerebral infarct percentage when given 40, 80, 160, and 320 µg/kg of CS-Gel/Ag NPs nanocomposite, suggesting a protective effect.

Table 2. The efficacy of CS-Gel/Ag NPs nanocomposite on the cerebral infarct percentage (%) (*p≤0.01).
Parameters Groups (n=10)
Control CS-Gel/Ag NPs-40 CS-Gel/Ag NPs-80 CS-Gel/Ag NPs-160 CS-Gel/Ag NPs-320
Cerebral infarct percentage (%) 42±3 29±3* 22±4* 20±1* 16±3*

The control group showed substantial increase in serum LDH concentration and MDA amount, as seen in Table 3. However, the increase in serum LDH concentration and MDA amount was successfully inhibited by administering 40, 80, 160, and 320 µg/kg of CS-Gel/Ag NPs nanocomposite.

Table 3. The efficacy of CS-Gel/Ag NPs nanocomposite on the MDA content (nmol/ml) and LDH activity (U/L) (*p≤0.01).
Parameters Groups (n=10)
Control CS-Gel/Ag NPs-40 CS-Gel/Ag NPs-80 CS-Gel/Ag NPs-160 CS-Gel/Ag NPs-320
MDA content (nmol/mL) 17±3 10±1* 8±1* 6±1* 5±1*
LDH activity (U/L) 6574±132 5621±103* 5372±122* 5005±94* 4763±114*

Only when the blood-brain barrier (BBB) is disrupted may the brain parenchyma exhibit low serum IgG levels. Table 4 presents the results, which show that administering 40, 80, 160, and 320 µg/kg of CS-Gel/Ag NPs nanocomposite significantly reduced IgG levels in the ipsilateral cortex, ipsilateral subcortex, contralateral cortex, and contralateral subcortex after cerebral ischemia-reperfusion.

Table 4. The efficacy of CS-Gel/Ag NPs nanocomposite on brain parenchyma serum IgG (*p≤0.01).
Parameters Groups (n=10)
Control CS-Gel/Ag NPs-40 CS-Gel/Ag NPs-80 CS-Gel/Ag NPs-160 CS-Gel/Ag NPs-320
Contralateral subcortex IgG 29±2 17±3* 15±2* 12±2* 8±1*
Ipsilateral subcortex IgG 174±12 109±6* 96±8* 91±5* 82±4*
Contralateral cortex IgG 225±14 152±11* 138±9* 117±10* 103±8*
Ipsilateral cortex IgG 164±14 115±9* 100±8* 83±6* 76±7*

Numerous studies have shown how beneficial herbal NPs are in reducing the negative effects of reperfusion ischemia. According to this research, plants can successfully lower the incidence of sensory and neurological problems, brain lesions, neuronal damage, and cerebral edema [48-52]. Many processes, including the decrease in astrocyte and microglia activity, the avoidance of DNA fragmentation and oxidative damage, the reduction of lipid peroxidation, and the relief of oxidative and nitrative stress, are thought to be responsible for the protective effects of silver nanoparticles [53-55]. Furthermore, it has been discovered that silver nanoparticles lower the eicosanoids levels, such as leukotriene, prostaglandins, and tromyoxan, boost the expression of mitochondrial genes, block the apoptotic proteins development, and decrease the expression of inflammatory mediators [53,54]. The majority of the research examining the protective benefits of silver nanoparticles against stroke has not evaluated their possible toxic effects or calculated the safety margin between toxic and therapeutic effects [53-56]. Animals were given Occium basalicum extract at concentrations ranging from 10–2000 mg/kg in a research reported by Chandrashekhar et al. The fatal dose for 50% of the population, or LD50, was calculated 24 hours later. The extract was next examined using the ischemia model at doses of 10, 20, and 30% LD50 [57]. The study by Buch et al. examined the maca extract toxicity in rats at a dose of 500 mg/kg. The extract was also evaluated at 10% and 20% LD50 against ischemia [56]. Nevertheless, additional indications, including body weight, organ abnormalities, and pathological modifications, were not determined in the two aforementioned trials. It is recommended to investigate the poisonous qualities of medicinal herbs, as well as their remedial activities, to guarantee consumer safety [58-60]. Most of the research on the protective qualities of herbal nanoparticles on stroke has been done in vivo, without the use of human participants, according to a study of the literature. Clinical studies should be carried out in conjunction with preclinical research because the biochemical constituents of herbal NPs are metabolized in the liver and enzymatic processes of the body, which may change their structure and effects.

4. Conclusions

Finally, we can conclude that the present investigation demonstrated a sustainable approach towards a bio-inspired method for the synthesis of a new eco-friendly material, namely CS-Gel/Ag NPs, through creating silver nanoparticles in situ over a dual biopolymeric hydrogel made of chitosan and gelatin. We employed a wide range of polar hydroxyl and amino functionalities of the two bio-macromolecules to reduce Ag+ ions in an ecologically safe manner without the usage of dangerous chemicals or additional inherent stability. Some advanced characterization techniques were employed to investigate the substance’s physical form and other inimitable features. The as-prepared CS-Gel/Ag NPs nanocomposite showed good catalytic activity in the reduction of MO as a harmful water contaminant. In the in vivo experiments, many parameters were evaluated, such as the learning and memory function, serum MDA level, infarct volume, LDH activity, cerebral parenchyma IgG extravasation, and cell damage index. According to the results, the CS-Gel/Ag NPs nanocomposite showed protective qualities against stroke-reperfusion damage caused by cerebral ischemia. In particular, they prevented learning and memory impairments, inhibited IgG extravasation, and decreased serum MDA levels, LDH activity, and infarct volume. To validate this fact, clinical trial research is required on humans. Pharmacological studies that concentrate on additional genes connected to stroke are necessary to elucidate the action mechanism of the CS-Gel/Ag NPs nanocomposite.

CRediT authorship contribution statement

Ren Bin, Wei Xiaocong, Yang Leifang, Guo Min: Visualization, Writing original draft, Formal analysis, Yang Leifang, Guo Min: Funding acquisition, Methodology, Supervision. Ren Bin, Wei Xiaocong: Writing original draft, Formal analysis, Writing-review and editing. All authors reviewed the manuscript.

Declaration of competing interest

The authors report no conflicts of interest in this work.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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