Effect of Crotalaria medicaginea-based silver nanoparticles on morphological and molecular inflammatory markers

Saima Bashir; Sumera Javad; Zeb Saddiqe; Muhammad Shahzad; Usman Aftab; Anis Ali Shah; Ozair Chaudhry; Mohamed El-Sheikh

doi:10.25259/JKSUS_381_2024

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Research Article

2025

:37;

3812024

doi:

10.25259/JKSUS_381_2024

Effect of Crotalaria medicaginea-based silver nanoparticles on morphological and molecular inflammatory markers

Saima Bashir^a, Sumera Javad^a,*, Zeb Saddiqe^b, Muhammad Shahzad^c, Usman Aftab^c, Anis Ali Shah^d,*, Ozair Chaudhry^e, Mohamed El-Sheikh^f

aDepartment of Botany, Lahore College for Women University, Lahore, 54700, Punjab, Pakistan

bDepartment of Botany, Queen Marry College, Lahore, 54700, Punjab, Pakistan

cDepartment of Pharmacology, University of Health Sciences, Lahore, 54700, Punjab, Pakistan

dDepartment of Botany, Division of Science and Technology, University of Education, Township Campus, Lahore, 54770, Pakistan

eDepartment of Planning and Economic Development Department, Durham HQ. Regional Municipality of Ontario, 605 Rossland Road East, Fourth Floor, Whitty Ontario, 91761, Canada

fDepartment of Botany and Microbiology Department, College of Science, King Saud University, Riyadh, 12271 Saudi Arabia

*Corresponding authorE-mail address: anisalibot@gmail.com (A.A. Shah); zif_4@yahoo.com (S. Javad)

Received: 2024-11-23, Accepted: 2025-06-04, Published: 2025-07-21

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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

There is a growing need for novel natural anti-inflammatory agents due to the side effects posed by existing anti-inflammatory compounds. Therefore, the present study aimed to prepare silver nanoparticles (AgNPs) from Crotalaria medicaginea and check their antioxidant potential. After characterizing these AgNPs (using a particle size analyzer, scanning electron microscopy-SEM, and Fourier transform infrared-FTIR analysis), their antioxidant and anti-inflammatory potential was assessed with the help of standard methods, both in vivo and in vitro. Furthermore, mRNA expression for inflammatory markers was determined using qPCR. Results showed that Crotalaria-fabricated silver nanoparticles (C-AgNPs) had strong in vitro antioxidant and anti-inflammatory activities. In vivo studies corroborated the results, demonstrating significant activity as compared with disease and drug control groups. C-AgNPs showed maximum inhibition of inflammation in rats as 69.46, 29.65, and 57.58% in carrageenan-induced paw edema (CPE), cotton pellet-induced granuloma (CPG), and xylene-induced ear edema (XEE), respectively, as compared with other treatments and control. C-AgNPs also upregulated anti-inflammatory markers such as IL-4 (64.22%, 54.41%, and 68.44%) and IL-10 (74.36%, 92.19%, and 92.56%) in all three groups of inflammation studies, respectively. Therefore, the present study concludes that C-AgNPs have potent antioxidant and anti-inflammatory potential. They can also upregulate the antioxidant system and inflammation markers of the living body to boost the immune system.

Keywords

Antioxidants

Nanoparticle synthesis

Natural anti-inflammatory agents

Plant secondary metabolites

Silver nanoparticles

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1. Introduction

Inflammation is an essential response of the body to various factors, including any damage to cells by pathogens or irritants. Despite its importance, inflammation is potentially harmful. Various reactions of the body, such as molecular and cellular events, resulting in the activation of inflammatory markers, constitute an inflammatory response. Physiologically, it is recognized by redness, warmth, pain, swelling, etc. At the cellular level, there may be fluid or protein leakage from cells, or extravascular movements of leucocytes (Azab et al., 2016). Inflammation is necessary for tissue repair and activation of the immune system. But when it goes unregulated and becomes chronic, it can lead to various diseases of the heart, lungs, kidneys, liver, and immune system. Anti-inflammatory drugs are used to resolve such issues. Some examples of such drugs are ibuprofen, aspirin, diclofenac, prednisone, dexamethasone, naproxen, etc (Zappavigna et al., 2020). Most of the present-era anti-inflammatory drugs are accompanied by acute or chronic side effects including weight gain, genotoxic effects, immunodeficiency, disturbed hormonal levels, etc. These side effects necessitate the search for new anti-inflammatory agents that can regulate inflammation with the least number of side effects (Nunes et al., 2020). In medicinal herbs the common anti-inflammatory phytoconstituents are polyphenols, flavonoids, steroids, terpenoids, alkaloids, glycosides, curcumins, aliphatic alcohols, and phenolic diterpenes. (Shahrajabian and Sun, 2023; Akhlaq et al., 2024).

Nanoparticles (NPs) are another viable option offering higher efficiency. In this global age, Nanotechnology is playing diverse roles, while offering miraculous solutions to many research problems. Agriculture, the environment, medicine, and material engineering, among others, are all greatly influenced by advancements in nanotechnology. NPs have also shown pronounced medicinal potential for developing new therapeutic agents (Haleem et al., 2023). Particularly, silver nanoparticles (AgNPs) have emerged as strong antibacterial, antifungal, antioxidant, and anti-inflammatory agents. They possess unique properties due to their shape and size, which give them a broad spectrum of medicinal activities. Their green synthesis also adds to their quality (Menichetti et al., 2023). In green synthesis, plant-based bio-chemicals are added to the surface of NPs, which increases their bioactivity and biocompatibility. Green-synthesis method is a cost-effective, sustainable, nature-friendly, and eco-safe method for the synthesis of NPs (Ghaffar et al., 2024). To increase acceptance of NPs in the field of medicine, there is a dire need to evaluate their toxic effects along with the benefits.

The selection of plant type, genus, and parts is a very crucial step for the green synthesis of NPs. The bioactive compounds present in plants and in their parts not only facilitate the synthesis of NPs but can also enhance their biological activity in a synergistic way. Crotolaria medicaginea is a well-known plant of the family Fabaceae, and it possesses significant antioxidant and anti-inflammatory activities. This plant has been traditionally used in Unani and Ayurvedic medicines (Kusar et al., 2024). Owing to its strong reducing and anti-inflammatory potential, it provides a platform for the synergistic activity of silver and plant metabolites.

Therefore, the present study was conducted to understand the effect of Crotolaria medicaginea-based silver nanoparticles (C-AgNPs) on the activation of various inflammatory markers at molecular and morphological levels.

2. Materials and Methods

2.1 Materials and chemicals

Aerial parts of C. medicaginea were obtained from areas in Teer and Jattipind, Abbottabad, Pakistan. The plant specimens were washed, air-dried, and ground into a fine powder. This powdered plant material was stored in airtight jars for further analysis. Silver nitrate (AgNO₃) was used as a raw silver source for synthesizing AgNPs. Analytical-grade chemicals were purchased and used for all stages of this research.

2.2 Preparation of plant extract

Microwave-assisted extraction (MAE) was used as a fast and efficient extraction tool for extracting phytochemicals of interest from C. medicaginea. Plant material (10 g) (C. medicaginea) was added to 100 mL of water. Independent parameters were set for MAE: a power level of 300 W and an extraction time of 1 min. After 1 min of extraction, it was filtered, and the filtrate was used for NP synthesis.

2.3 Green synthesis of NPs

A slightly modified method of Ghaffar et al. (2024) was applied to synthesize C-AgNPs (Ghaffar et al., 2024). AgNO₃(0.1 M) was prepared in 30% EtOH and mixed with the filtrate of the MAE. This mixing was done in the ratio of 1:3 plant filtrate:salt solution. A magnetic stirrer was used to thoroughly mix the two solutions dropwise and slowly. After 1 h of mixing, the filtrate was centrifuged for 12 min, and the rpm was set at a value of 10,000. After centrifugation, the pellets were carefully separated and washed, dried at ambient temperature, and stored as C-AgNPs for future use.

2.4 Characterizing C-AgNPs

2.4.1 Visual and UV-visible analysis

When the plant material and salt solution were mixed, any change in the color of mixture was carefully observed. An absorption spectrum of the mixture was also run on a UV-Visible spectrophotometer (Cary 60 Agilent) between 200-800 nm wavelengths. The preparation of the NP sample was done by dissolving 3 mg dried pellets in EtOH (10 mL). The sonication of the mixture was carried out for 30 min to make a fine dispersion. In the cuvette, a suspension aliquot was taken and loaded into the spectrophotometer. A spectrum was obtained in a wavelength range of 200-800 nm.

2.4.2 Fourier transform infrared spectroscopy (FT-IR)

This technique was adopted to estimate functional groups that are present on the surface of newly synthesized NPs. These functional groups are related to the secondary metabolites responsible for the synthesis of NPs. Powdered plant material (20 mg) was loaded with the help of a spatula onto the IRT Racer Cary 630 Agilent FTIR spectrophotometer. To study the functional groups responsible for producing NPs, their spectrum was generated and observed in the wavelength range of 500 - 4500 cm^-1(Ghaffar et al., 2024).

2.4.3 Particle size analysis (PSA)

The C-AgNPs pellet (2 mg) was suspended in EtOH (2 mL) for sample preparation. Before analysis, the solution was sonicated for 20 min. Later, a Litesizer^TM 500 Anton Paar (PSA) was used for the estimation of particle size (Ghaffar et al., 2024).

2.4.4 Scanning electron microscopy (SEM)

SEM was used to characterize the size, structure, and morphology of NPs. For this analysis, the SEM model no. ZEISS-EVO/LS10 was used. The voltage of the instrument was set at 20 kV. Furthermore, the elemental nature of NPs was confirmed using energy-dispersive X-ray spectroscopy (EDX) (Ghaffar et al., 2022).

2.5 In vitro antioxidant activity

Four different assays were used to assess the in vitro antioxidant activity of the synthesized C-AgNPs, including 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, hydroxyl radical scavenging assay (HRSA), ferric reducing antioxidant power (FRAP) assay, and ABTS radical scavenging assay. There are different radicals and mechanisms of reduction in all the above methods. Therefore, various assays are used here with a different mechanism of action. A series of dilutions with different strengths of C-AgNPs (1000 to 31.25 µg/mL) was made and standard antioxidant selected was ascorbic acid. The overall formula used to calculate the % reduction/inhibition of radicals was as follows $% inhibition = \frac{A b s - A b t}{A b s} \times 100$

Where, Ab_s = control absorbance and Ab_t = test sample absorbance.

2.5.1 DPPH assay

The ability of green synthesized C-AgNPs to scavenge free radicals was estimated by DPPH assay as described by Ashraf et al. (2015). However, antioxidant values of the highest concentrations of the C-AgNPs are reported in the present research. To perform the assay, C-AgNPs sample (0.5 mL) was mixed in 0.05 mM DPPH solution (2.5 mL). The prepared mixture was incubated at room temperature for 40 min. Later, absorbance was measured at 517 nm. Inhibition (%) was calculated by using the following formula $% inhibition = \frac{A c - A s}{A c} \times 100$

Where A_c = DPPH solution absorbance and A_s = sample + DPPH absorbance.

2.5.2 Hydroxyl radical scavenging activity (HRSA)

Hydroxyl radical (OH) is known as a strong reactive oxygen species (ROS) in biological systems; therefore, HRSA was also carried out by a slightly modified method by Tijani et al. (2018). The reaction blend included 1000 µL of different concentrations of NPs, Fe-EDTA solution (1 mL), 0.018% EDTA (0.5 mL), and DMSO (1 mL). Tubes were tightly capped and heated at 80-90^°C for 15-16 min. This heating was carried out in a water bath. The reaction was ended by adding 1 mL of chilled TCA. Then, 3.5 mL of Nash reagent was added, and the mixture was incubated at room temperature. After 15 min, as the color was being developed during this incubation period, absorbance was noted at 412 nm, which gave an estimation of the intensity of the yellow color.

2.5.3 FRAP Assay

The activity was calculated by using the potassium ferricyanide-ferric chloride method (Chen et al., 2010). Plant extract (1 mL) was mixed with 2.5 mL of phosphate buffer and 1% potassium ferricyanide, followed by a 20-min incubation at 55^°C. Then, 2.5 mL of TCA was added. After adding TCA, each tube was diluted with dist. water in a 1:1 ratio. Finally, ferric chloride (FeCl₃) solution (500 µL) was poured into each test tube, and the experiment was set aside for an incubation period of 30 min. After this incubation time, the antioxidant activity of C-AgNPs was calculated by taking the values of absorbance at 700 nm.

2.5.4 ABTS radical scavenging assay

This activity was estimated following a slightly modified method of Gorinstein (2009). Aqueous ABTS and potassium persulfate solution in the ratio of 1:1 were used to make a radical solution and stored in the dark. This mixture was stored in the dark for at least the whole day (24 h). For each bioassay, fresh working solutions were prepared. This was done by diluting 1 mL ABTS⁺ radical solution with EtOH, targeting the absorbance value of 0.700 at a wavelength of 745 nm. Afterwards, different concentrations of AgNPs (100 µL) were added to the ABTS⁺ solution (2 mL). The solutions were placed in the dark for 10 min at ambient temperature. The readings of absorbance were taken without much exposure of the samples to light. EtOH was used as a blank with ascorbic acid as a standard. The % inhibition was calculated using the formula $% Inhibition = \frac{1 - A c}{A s} \times 100$

Where, A_c = control absorbance and A_s = sample absorbance.

2.6 Anti-inflammatory action (in vitro)

2.6.1 Inhibition of heat-induced protein denaturation

The inhibition potential of C-AgNPs in reducing the heat-induced denaturation of protein was estimated. In this assay, egg albumin was used as substrate as described by Carter & Thornburg (2000). The reaction mixture contained egg albumin (20 µL), phosphate buffer saline (3.28 mL), and a series of dilutions with different strengths of C-AgNPs (1000 to 31.25 µg/mL). Aspirin (acetylsalicylic acid) at the same dose served as a standard drug and distilled water as a control. The incubation of the mixture was done at room temperature for 10 min. Heating of the mixture-containing tubes was done at 70^°C for 5 min. The mixture was then allowed to cool. Absorbance of each test tube sample was measured on spectrophotometer at 660 nm and following formula was used. $% Inhibition = \frac{A b s - A b t}{A b s} \times 100$

Where, Ab_s = control absorbance and Ab_t = test sample absorbance.

2.6.2 Inhibition of hypotonicity-induced hemolysis

Fresh human blood was collected from a healthy person and its centrifugation was done for 12 min, and the rpm was set at 3000 rpm. Normal saline was used for the pellet of packed cells. Human red blood cells (HRBC) (10%) suspension was obtained using phosphate buffer saline. The final reaction mixture contained phosphate buffer saline (1 mL), hyposaline (2 mL), HRBC suspension (0.5 mL) with 0.5 mL of dilutions of C-AgNPs separately, and the same concentrations of aspirin were utilized as a standard anti-inflammatory drug. All samples were incubated at ambient temperature for 30 min and after that, each sample was centrifuged for 20 min with rpm set at 3000. The supernatant was separated and taken as a sample to be checked for absorbance at 560 nm (Anosike et al., 2012). $% Inhibition = \frac{A b s - A b t}{A b s} \times 100$

Where, Ab_s = control absorbance and Ab_t = test sample absorbance.

2.7 In-vivo assays

2.7.1 Pre-experimental animals’ conditioning

Male albino rats were chosen in the present research work and weight of each rat ranged between 180-200 g. Animals were taken from The Animal House, University of Veterinary and Animal Sciences (UVAS), Lahore. All rats were placed in plastic cages. These cages had cover lids made from stainless steel. As bedding material, wheat straws were used. Animals were placed in optimized laboratory conditions including temperature (25 ± 2°C) and dark-light cycle of 14-10 h. All the animals were provided with commercial feed and water according to animal requirements. Animal care protocols were followed strictly as per criteria outlined in guide for the care and laboratory animal usage. For the present study, ethical authorization was issued by the Institutional Ethical Committee, Lahore College for Women University, Lahore (Protocol approval number DEC/LCWU/2023-03).

2.7.2 Animal grouping for in-vivo studies

Animals were divided into five groups (n=6) for in-vivo studies. The first group was vehicle-treated control and received only DMSO (1%). The second group was the disease control group treated with inflammatory substances (carrageenan, xylene, or cotton pellets). The third group was a standard drug control group treated with diclofenac sodium (25 mg/kg body weight). The fourth group was treated with C-AgNPs (400 µg/kg body weight), prepared from an aqueous extract of C. medicaginea. The fifth group was the healthy group, which didn’t receive any treatment throughout study. At the end of the experiment, to get the sera, deep anesthesia was given to animals (60 mg ketamine/ 4 mg xylazine per kg). The blood was obtained by cardiac puncture and collected in heparin tubes. For obtaining serum, blood samples were centrifuged for 15 min, and rpm was set at 3000 rpm. Serum storage was done at −20^°C and was used for studying antioxidant potential as described in the next points. Absorbance of all assays was measured by using Synergy HTX multimode reader (BioTek).

2.7.3 In vivo antioxidant activity

The activity of four antioxidant enzymes from the treated and non-treated rat blood samples was treated. These four antioxidant enzymes were catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and lipid peroxidase by standard methods. Whereas the formula used was $% Inhibition = \frac{A b s - A b t}{A b s} \times 100$

Where Ab_s = control absorbance and Ab_t = test sample absorbance.

2.7.3.1 Determination of SOD activity

In serum samples, SOD activity was assessed by following the method given by Zhang et al. (2017). The reaction blend had 2500 μL of 50 mM Tris-HCl buffer, 1 mM EDTA (100 μL), serum (100 μL), 0.4 mM pyrogallol (200 μL), and distilled water (100 μL). The final volume was made 3000 µL by using distilled water. Blank contained all reagents except pyrogallol. The activity was performed in triplicate by using a 96-well microtiter plate. The wavelength of 420 nm was set to check absorbance.

2.7.3.2 Determination of CAT activity

CAT activity in serum samples was studied and calculated by using a little modified method of Atawodi et al. (2013). The reaction blend consisted of serum (10 µL) and 50 mM potassium phosphate buffer (2800 µL). The addition of 100 µL freshly prepared H₂O₂ (30 mM) initiated the reaction. The H₂O₂ decomposition rate was calculated at 240 nm for 5 min.

2.7.3.3 Determination of GPx activity

Glutathione (GSH) levels in serum were evaluated using a method by Kaltalioglu (2022). In this method, DTNB reacts with reduced GSH, and a yellow color is developed. For determining the activity, 25 µL serum was diluted in phosphate buffer solution (5000 µL). From dilution of DTNB, 3000 µL of phosphate buffer was taken and 20 µL DTNB (0.01 M) was added. The activity was accomplished on a 96-well microtiter plate and treatments were done in triplicate. OD was measured against a blank at a wavelength of 412 nm (without serum) that was prepared under same conditions.

2.7.3.4 Determination of lipid peroxidase activity (LPO)

LPO activity in serum samples was determined by using a method of Draper and Hadley (1990). Serum (50 µL), 14% TCA (1000 µL), and 0.6% TBA (1000 µL) were added. The reagent, as prepared above, was heated in a water bath (30 min) and afterward cooled on ice for a duration of 5 min. Centrifugation was done at 2000 × g for 10 min. The activity was performed in triplicate on a 96-well microtiter plate. The absorbance was recorded at 535 nm.

2.7.4 Anti-inflammatory activity (In-vivo)

2.7.4.1 Carrageenan-induced paw edema assay (CPE)

A standard protocol previously described by Kedi et al. (2020) was adopted where paw size was assessed by a digital vernier caliper. Briefly, after one week of acclimatization, there was a random division of experimental animals into four groups. Inflammation was initiated in the right paw of each rat by injecting 0.1 mL of carrageenan. After injecting carrageenan, the rats were orally administered various treatments i.e., DMSO, diclofenac sodium, and NPs. Readings of rat paw size were taken after 0.5, 1, 2, 3, and 4 h of carrageenan injection. Anti-inflammatory activity was calculated as percentage edema inhibition in the treated and control groups in various treatments using the equation: $% edema Inhibition = \frac{(R t - R O) c o n t r o l - (R t - R O) t r e a t d}{(R t - R o) c o n t r o l} \times 100$

Where R_t and R_o are the average paw diameters for each group after and before treatment.

2.7.4.2 Xylene-induced ear edema (XEE)

In this part of study, all doses were administered by oral intubation. Test and standard drug with the same doses were given to the animals 1 h before xylene application where xylene application was done in the right ear of each animal. After 15 min, animals were sacrificed by dislocating the spinal cord from neck and a 16 cm circular area was cut from each ear of the animal. Weight of the inflamed ear was compared with the control (left) ear. Anti-inflammatory activity of standard drug and C-AgNPs was tested for ear edema percentage inhibition in non-treated and treated groups by using the following equation (Soliman et al., 2023): $% Edema degree = M_{(r i g h t)} - M_{(l e f t)}$ $% Inhibition = \frac{E d e m a d e g r e e_{(c o n t r o l)} - E d e m a d e g r e e_{(t r e a t e d)}}{E d e m a d e g r e e_{(c o n t r o l)}} \times 100$

Where M_(right) = right ear weight and M_(left) = left ear weight.

2.7.4.3 Cotton pellet-induced granuloma (CPG) in rats

This method provides another measure to assess the inhibition of chronic inflammation by test compounds (Aziz et al., 2020). The animals were placed in an animal house for 7 days and served with a normal water ad libitum diet. On the 8^th day, rats were lightly anesthetized with 60 mg/kg ketamine plus 4 mg/kg xylazine, and two sterilized cotton pellets (20 mg) were implanted (subcutaneously) in the dorsal region. After implantation of cotton pellets, animals were given all test doses by oral intubation for eight days. On the 9^th day, rats were sacrificed as described earlier and cotton pellets were dissected out without affecting adjacent granuloma tissues. Weight of wet and dried cotton pellets was recorded where pellets were dried at 60°C for 48 h. The weight of granuloma formation was calculated and percent inhibition of granuloma by test samples and standard drug was calculated as per following formula $Granuloma inhibition (%) = \frac{1 - G r a n u l o m a i n t r e a t e d g r o u p}{G r a n u l o m a i n C o n t r o l} \times 100$

2.8 Determination of mRNA expression for inflammatory markers using qPCR

RNA was isolated from treated and non-treated rats’ sera using Trizol reagent and a Nano-drop spectrophotometer was used for its quantification. RNA was mixed with a cDNA synthesis kit reagent (Zokeyo, China) for preparation of cDNA. For quantification of mRNA expression, 7.5 µL of SYBR Green PCR Master Mix (Bioshop, Canada) was mixed with 0.5 µL of reverse and forward primers each, with 3.5 µL of water (nuclease-free). Half a microliter of cDNA and 1.5 µL of above said mixture of primers were transferred to the well of PCR plate. To check the amplification of genes the plate was placed in qPCR (Koleva et al., 2022). GAPDH was the reference gene, and the sequence of used primers has been presented in Table 1.

Table 1. Designed primers with known sequence for identification of inflammatory cytokines

Markers	Forward primer/Reverse primer	Annealing temp
TNF-α	5´-GGACACCATGAGCACGGAA-3´ 5´-GGGCCATGGAACTGATGAGA-3´	59.3^°C
IL-1β	5´-GCTGTCCAGATGAGAGCATC-3´ 5´-GTCAGACAGCACGAGGCATT-3´	56.9^°C
IL-4	5´-AACACCACGGAGAACGAGCTCATC-3´ 5´-AGTGAGTTCAGACCGCTGACACCT-3´	60.1^°C
IL-6	5´-AGACTTCCAGCCAGTTGCCT-3´ 5´-CTGACAGTGCATCATCGCTG-3´	58.5^°C
IL-10	5´-AGAAGAGGGAGGAGCCTTTG-3´ 5´-GCCTTTGCTGGTCTTCACTC-3´	58.3^°C
GAPDH	5´-TCTCTGCTCCTCCCTGTTCT-3´ 5´-CTTGCCGTGGGTAGAGTCAT-3´	58.6^°C

2.9 Statistical analysis

SPSS version 20.0 was utilized as a statistical instrument. Statistical significance of data was checked at a 5% level of significance where a comparison of means was conducted using One-way analysis of variance and Duncan’s Multiple Range Test (DMRT) was applied as a post hoc test for estimating the significance.

3. Results and Discussion

3.1 Synthesizing and characterizing C-AgNPs

3.1.1 UV-Visible Spectroscopy

Visual observation showed a color change of plant extract from green to dark brown. It is the first indication of a reaction for synthesis of NPs. Further, in the current study, spectroscopy was used for monitoring and confirming the NP’s synthesis because it is the most accessible tool for quantifying metal ions reduction at preliminary stages. This characterization of NPs is based on their optical properties. Metallic NPs have a characteristic surface plasmon resonance, due to which they show specific peaks in the absorption spectrum. The peak exhibited by C-AgNPs at 435 nm showed the synthesis of stable NPs as it was a single peak (Fig. 1a). These absorption spectra also correspond to the previous literature. Ahmed et al. (2016), Albert et al. (2024), and Ghaffar et al. (2024) reported peaks of AgNPs at 420, 472, and between 400-450 nm respectively.

(a) UV-visible spectroscopy analysis, (b) FT-IR analysis, (c) Particle size analysis, (d) SEM micrograph and (e) EDX spectrum of biosynthesized C-AgNPs synthesized from C. medicaginea .

3.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)

FTIR analysis gives insights into the functional groups of plant metabolites which are present on the surface of NPs. These metabolites help in the synthesis and stabilization NPs. In the present study, the FTIR spectrum of synthesized C-AgNPs using C. medicaginea aqueous extract detected various peaks at 3220, 2922, 2109, 1908, 1535, 1371, 1244, and 976 cm⁻¹ (Fig. 1b). These peaks corresponded to different functional groups like alcohols, phenolics, flavonoids, amines, and carbonyls (Alkhathlan et al., 2020) of plant secondary metabolites.

These phenolics and carbonyl groups are considered responsible for the reduction of silver into its nanoform (Nunes et al., 2020). Thus, phenolics present in the extract of C. medicaginea may play a significant role in reducing, capping, and stabilizing AgNPs. These secondary metabolites of C. medicaginea enhanced the stability of green synthesized NPs and prevented their agglomeration, thus confirming the functional integrity of nascent NPs.

3.1.3 Particle Size Analysis (PSA)

A nano-laser particle size analyzer is a rapid, reliable, and practical technique to have a size range estimate of synthesized NPs. We can use this instrument for analyzing the sizes of NPs and macroparticles. It can quantify the distribution of average particle size on both the macro and nanoscale. In the current study, the results of particle size analysis showed two sharp peaks at 80 and 145 nm. It shows that C-AgNPs were present in the reaction mixture (80-85 nm size range), while a smaller peak of a little bit larger NPs was also present (Fig. 1c). It also indicates the monodispersed nature of the green-synthesized NPs formed in the present study. Earlier, Ahmad et al. (2020) reported a size of 92 nm on average for the synthesized AgNPs using ethanolic C. burhia whereas, this is the first report of synthesis of AgNPs using water extract of C. medicaginea. Water extract makes this protocol eco-friendlier. Water is a polar solvent and a very good choice for microwave-based extractions. It can extract a good number of metabolites from plant material depending upon the parameters selected in the microwave extraction. This is also a well-known fact that type, quality, and quantity of metabolites in plant extract can determine the shape and size of NPs.

3.1.4 SEM Analysis

This analysis was used to visualize green synthesized C-AgNPs surface morphology. Size, shape, distribution, aggregation, and topography were calculated by high-resolution micrographs of NPs. Fig. 1d shows the SEM micrograph of the green synthesized C-AgNPs. Round-shaped AgNPs were revealed by the SEM analysis and their size was calculated to be 70.26 nm on average. These NPs are present individually, but they also formed a degree of agglomeration attributed to higher surface energy. These results are also correlated with the results of PSA analysis. Minor differences in size readings of both techniques are usually reported as in the present study (Netam et al., 2021). The size of synthesized C-AgNPs and their shape reinforces the affectivity of the synthesizing method and the role of metabolites of C. medicaginea in capping and stabilizing newly formed silver NPs. Results also conclude that SEM is a reliable tool for studying the morphology of synthesized NPs. Functions and significance of NPs are determined by studying their shape and size. Similarly, NPs’ anti-inflammatory activity also depends on size and shape. Infact, these characters determine the NPs’ interaction with an inflammatory surface, responsible for their mode of action (Varghese et al., 2024).

3.1.5 EDX Analysis

EDX analysis is a strong tool for determining the elemental composition of the formed NPs. It provides fair proof of the presence of green synthesized NPs in the solution, as can be seen from Fig. 1e where the peak of silver element (61.58%) is visible at 3 KeV. While other peaks indicate the presence of elements of plant metabolites present at the surface of NPs. These peaks of silver are specific to the energy levels of the silver atoms. These findings support the facts that AgNPs are formed by the extract of C. medicaginea with the organic molecules at the surface as capping agents. EDX analysis with other characterization techniques as described in previous sections provide valid proof of green synthesized C-AgNPs. EDX analysis is a meaningful and strong test for determining the elemental composition of NPs formed (Gahffar et al., 2024).

3.2 In vitro antioxidant activities

The in-vitro antioxidant activity of synthesized C-AgNPs was assessed by four assays (Fig. 2). These four assays were a source of a comprehensive understanding of the antioxidant mechanism of C-AgNPs. AgNPs showed a variation in the antioxidant profile as compared to the standard antioxidant, used in the study, i.e., ascorbic acid. Results showed that C-AgNPs have a lower DPPH radical scavenging activity, and higher OH scavenging and ferric-reducing activity when compared with ascorbic acid. It shows that C-AgNPs showed a lower potential to neutralize DPPH radicals. OH radicals are one of the most toxic radicals produced in living organisms because of stress. These radicals can impose a severe threat to living cells and their components where we see that C-AgNPs showed a strong anti-OH radical activity (84.47%), significantly higher than standard. Furthermore, C-AgNPs also showed strong ferric-reducing antioxidant power (55.78%) which is even higher than the standard antioxidant. Results showed that C-AgNPs have strong antioxidant activity as well as electron donating or reducing power. C-AgNPs show a promising antioxidant potential with various mechanisms in which two characteristics of C-AgNPs help them enhance antioxidant potential. One of them is the presence of secondary metabolites of C. medicaginea and the other one is characters of synthesized NPs including shape, and size. Plant metabolites have antioxidant activity because of their redox property that enables them to scavenge and neutralize free radicals (Jaisankar and Arivarasu, 2020).

In vitro antioxidant activity of C-AgNPs synthesized from C. medicaginea in comparison with the standard (Ascorbic acid).

3.3 In vitro anti-inflammatory activities

Two main key assays were used for the estimation of anti-inflammatory potential of the synthesized C-AgNPs (Fig. 3). These main assays included inhibition of protein denaturation and membrane stabilization potential for human RBCs and aspirin was used as a standard anti-inflammatory drug. Results showed that C-AgNPs possess a higher inhibition potential against protein denaturation (95.87%). Inflammation causes the denaturation of proteins and provokes autoimmune responses. The present study suggests that C-AgNPs help proteins to stabilize and prevent denaturation during inflammation. This also suggests that AgNPs can inhibit the inflammation markers at cellular and molecular levels.

(a) In vitro anti-inflammatory activity of C-AgNPs.

HRBC membrane stabilization potential of C-AgNPs is also higher (96.52%) as compared to aspirin. If membranes are stabilized, it leads to lesser rupture of membranes including the membranes of lysosomes. This results in reduced release of lysosomal enzymes under stress (Bussi et al., 2023). Thus C-AgNPs can be used to treat inflammation disorders because of these characteristics, playing their role in the integrity of cellular membranes. These can also help to stabilize the structure of proteins in case of inflammation.

The parameters studied were inhibition of heat-induced protein denaturation, inhibition of hypotonicity-induced hemolysis, and hemolytic activity of synthesized C-AgNPs at different concentrations (dilutions from 1000-31.25 µg/mL) but the highest values are reported here. In all the assays, the highest inhibition rate was observed at the highest concentration 1000 µg/mL. The general aspect of higher activity and efficiency of NPs is attributed to their smaller size. The smaller size of NPs gives them a larger surface area and can make them more interactive. Silver particles’ penetration also increases due to their nano size, which aids better utilization. Based on the size, NPs have also shown the capacity to enter the human circulatory system and to cross the blood–brain barrier, making them more effective and potent anti-inflammatory agents (Parvez et al., 2022). Increased drug dosage increases its efficiency, but also leads to a higher risk of side effects. Therefore, it is necessary to develop new agents that can be used to lessen the inflammation that is associated with systemic conditions which are more effective and natural with lesser side effects (Mohd Zaid et al., 2023).

It is well known that AgNPs have strong anti-inflammatory activity, and many researchers have tried to apply NPs as anti-inflammatory agents. The AgNPs’ inhibitory activity can be considered a good candidate for management of excessive inflammation. This inflammation response of the body is produced usually by multiple defense pathways of the immune system. Few studies have evaluated the influence of Ag ions on the inflammatory activity of drugs (Sriramulu and Sumathi, 2017). These drugs are extensively used in the treatment of inflammatory states. However, their usage may have side effects like abdominal pain, ulcers, and nausea. All these can be prevented by production of NPs using herbal formulation that can eliminate these side effects because they have higher activity due to the additional effect of plant secondary metabolites and their lower doses are required due to highly active AgNPs (Jyoti et al., 2016; Ahmad et al., 2024).

The ability of synthesized C-AgNPs to inhibit protein denaturation and hypotonicity-induced hemolysis has been investigated in this study. Earlier research has shown that during inflammation, protein denaturation is one of the main causes of immune responses, tissue damage, and arthritic reactions (Chopade et al., 2012). Agents that are responsible for preventing protein denaturation can be used in the development of anti-inflammatory drugs. This study can be improved further by knowing the benefits of these plants and the mechanism behind their action in future studies. This study, therefore, shows the anti-inflammatory properties of AgNPs. This potential of C-AgNPs can be used for exploring its therapeutic potency in future.

3.4 In vivo antioxidant activity

The living body has developed a complex defense strategy to reduce the damaging effects of several oxidants. In the present work, the effects of C-AgNPs were determined on the oxidative stress parameters in rat serum. It was done to assess which oxidative stress appears to play a fundamental role in inflammation. In all three treatments, results in the serum of inflamed rats (Fig. 4a-c) revealed an increase in CAT activity in the presence of C-AgNPs (400 µg/kg body weight) with values 77.72, 64.46, and 62.58% for paw edema, ear edema, and granuloma formation, respectively, as compared with the disease control group treated with carrageenan, xylene and cotton pellet with the values of 50.35, 48.59, and 66.62%, respectively. The activity of SOD was significantly increased in the blood serum samples of inflamed animals supplied with AgNPs (53.89, 80.16, and 80.65% for paw edema, ear edema, and granuloma formation, respectively) as compared with the disease control group (47.48, 77.01, and 75.13%) and standard drug treated group (60.44, 85.21, and 84.63%). The results of the LPO assay (Fig. 4a-c) revealed a decrease in lipid peroxidation in the C-AgNPs group with the value of 45.05, 36.58, and 37.49% for paw edema, ear edema, and granuloma formation, respectively. It was closer to standard drug values which were 45.17, 44.39, and 32.83%. These values were also significantly lower than the disease control group (57.24, 53.82, and 54.59% for paw edema, ear edema, and granuloma formation, respectively). The serum levels of GSH increased in the inflamed animals treated with AgNPs with values 76.00% (for CPE), 82.57% (xylene-induced edema), and 84.57% (CPG) as compared with the corresponding disease control group (64.96, 76.22, and 75.55%). The results were also comparable to the diclofenac sodium treated group (75.54, 82.24, and 83.06 for paw edema, ear edema, and granuloma formation, respectively).

In vivo antioxidant activity of C-AgNPs synthesized from C. medicaginea and their comparison with standard drug in rats with various sources of inflammation (a) CPE, (b) XEE, and (c) CPG where T1-T15 are rat groups with T1= Carrageenan, T2= Carr+ 1% DMSO, T3= Carr + diclofenac sodium (25 mg/kg b.w), T4= Carr + AgNPs (400 µg/kg b.w), T6= Xylene, T7= Xyl + 1% DMSO, T8= Xyl + diclofenac sodium (25 mg/kg b.w), T9= Xyl + AgNPs (400 µg/kg b.w), T11= cotton pellet, T12= CP + 1% DMSO, T13= CP + diclofenac sodium (25 mg/kg b.w), T14= CP + AgNPs (400 µg/kg b.w) whereas T5, 10, and 15 are healthy rat groups without inflammation (negative control)

This potential increase in the activity of C-AgNPs triggered CAT, SOD, LPO, and GSH content of rat serum in three various inflammatory events. These results of stress enzymes show that NPs have a strong potential for reducing oxidative stress from living cells and protecting cells from damage. CAT is the main enzyme that helps in the neutralization of H₂O₂and converts it into hydrogen and oxygen (Ighodaro and Akinloye, 2018). SOD is particularly active against superoxide radicals. H₂O₂is produced in this way and is undertaken by other antioxidant enzymes for neutralization. The synergistic effects of CAT and SOD can be one of the reasons for the enhanced action of C-AgNPs in the inflammatory condition of rats in the present study. It is reported that if superoxide radical is not regulated in the body system, it causes damage to the body in multiple ways and it is a common byproduct of oxygen metabolism (Hayyan et al., 2016). The H₂O₂ produced during SOD activity is also toxic for healthy cells. Therefore, it is broken down into smaller fragments by the activity of other enzymes like CAT. GSH is a non-enzymatic, tripeptide and is a biological antioxidant. Cellular proteins are protected by this compound against ROS (Chakravorty et al., 2020). Lipid peroxidation is a free-radical mediated, auto-catalytic, and destructive process, in which the degradation of polyunsaturated fatty acids (cell membranes) occurs, forming lipid hydroperoxides (Clemente et al., 2020). ROS also degrades polyunsaturated lipids and form malondialdehyde. Malondialdehyde is a highly reactive aldehyde that acts as a reactive electrophile species and causes oxidative stress leading to the formation of advanced lipid peroxidation end products. The content of this aldehyde serves as a biomarker to detect the level of oxidative stress in living organisms (Jadoon and Malik, 2017). The current study revealed that AgNPs significantly decreased levels of lipid peroxidase enzyme which prevented lipid peroxidation. Reduction in GSH level relates to increased lipid peroxidation, which was confirmed in this study. The crude plant extract increased the GSH level with a corresponding decrease in lipid peroxidation. These results agree with previous reports of Bhandarkar and Khan, (2004), Jackie et al. (2011), and Ekechi et al. (2023). These results revealed that AgNPs have significant in vivo antioxidant activities as was confirmed from in vitro oxidant results of present studies.

3.5 In vivo anti-inflammatory activity

Fig. 5a reflects the anti-inflammatory action of C-AgNPs in rats. Inflammation was initiated in the rat paws by carrageenan, a sulfated polysaccharide. The control group of rats developed edema within 30 min of the injection of carrageenan. Edema increased gradually and reached its maximum value within 3 h of carrageenan exposure. But the rat group, that was treated with oral C-AgNPs doses, experienced a significant reduction in edema. The maximum inhibition percentage in C-AgNPs treated rats was 69.46% after 4 h for the dose of 400 mg/kg body weight. Diclofenac sodium was selected for comparison of the anti-inflammatory action of C-AgNPs as a standard anti-inflammatory drug. Results of edema reduction by diclofenac (77.30%) and C-AgNPs (69.46%) were comparable, showing that C-AgNPs are a strong candidate as an anti-inflammatory drug.

In vivo anti-inflammatory activity of C-AgNPs synthesized from C. medicaginea and their comparison with standard drug in rats with various sources of inflammation (a) CPE, (b) XEE and (c) CPG where Carr is Carrageenan and CP cotton pellet.

This method of anti-inflammation action is an extensively studied and highly reproducible experimental model that is often opted to study inflammation. The typical puffiness that happens in rat paws, is because of edema formation and increased vascular permeability. Carrageenan causes Edema in three main phases. In the first phase, mediators like serotonin and histamine are released (within one hour). Kinins are released in the second phase (within 2 h of initiation), whereas prostaglandins and cyclooxygenase products are released during the third hour, which is recognized as the third phase and can last for 3 to 5 h (Mancipe et al., 2023). When animals are treated orally with C-AgNPs, the edema rate is inhibited significantly in all inflammation phases as compared to the control group. The results suggest that NPs containing Ag may reduce the production of inflammatory mediators and cause a balance in the system to avoid the inflammatory response of the body. Furthermore, it can also be suggested that C-AgNPs have a higher and persistent anti-inflammatory activity as they can enter more easily to the edema region due to their smaller size (Moldovan et al., 2016).

Fig. 5b shows the percentage inhibition of ear edema in different experimental groups. All groups were compared with the disease control group. C-AgNPs strongly inhibited the ear edema with the inhibition value of 57.58% which was closer to the inhibition caused by standard drug (inhibition value of 58.23%). Xylene-induced inflammation is partly connected with “P” substance that is extensively distributed in the nervous system (both central and peripheral) (Baluk et al., 1992). Neuromodulator release causes plasma extravasations and vasodilation, which involves neurogenic inflammation and causes swelling in rat ears (Agbaje and Fageyinbo, 2012). In both processes, diclofenac sodium has been reported to have greater involvement in the suppression of mediators. C. medicaginea based AgNPs was also found to possess potential activity against the mediators involved.

Furthermore, C. medicaginea C-AgNPs also reduced the inflammation process in rats through granuloma inhibition in comparison with control rats (Fig. 5c). This was indicated by a reduction of dry and wet weights of cotton pellets. Diclofenac sodium, used as standard drug, was also effective in reducing granuloma formation compared to the control groups. The highest inhibition (56.03%) of granuloma tissue formation was observed for the standard drug. The inhibition potential of C-AgNPs was found to be 29.65%.

In this method, subcutaneous implantation of cotton pellets induces the formation of granuloma that is observed as undifferentiated connective tissue composed of giant cells besides fluid infiltration. The amount of granuloma tissue formed was determined by measuring the weight of desiccated pellets. This model replicates the proliferative phase of inflammation (Odabasoglu et al., 2011). The proliferation of macrophages, neutrophils, and fibroblasts involved in inflammation are the major sources of the formation of granuloma. Hence, the decrease in granuloma weight specifies that the proliferative phase was suppressed by the C-AgNPs that is linked with inhibition of collagen, fibronectin and glycosaminoglycan synthesis. In CPG, the WBC count is increased. There are many reasons for this increase including neutrophil activation, adhesion of molecules on endothelial cells, lymphocytes, and monocytes proliferation, by increase in binding and leukocytes transmigration (Sarma et al., 2019). Earlier Buabeid et al. (2022) also showed an anti-inflammatory effect of AgNPs loaded with statins.

3.6 C-AgNPs and inflammatory markers

In rats’ serum with edema, caused by carrageenan, an increase was noticed in the expression intensities of pro-inflammatory markers (79.66% in TNF-α, 78.47% in IL-1β, and 64.71% in IL-6) as compared with the healthy group. Treatment of these rats with diclofenac and C-AgNPs led to a significant decrease (p <0.05) in the expression of these pro-inflammatory markers. In the group of rats treated with diclofenac sodium, a decrease of 33.88% TNF-α, 47.16% in IL-1β, and 44.65% IL-6 is seen whereas for C-AgNPs it was 49.32, 60.21, and 49.18%, respectively. The expression of anti-inflammatory markers (IL-4 and IL-10) decreased (p <0.05) in the disease control group (37.00 ± 1.72% and 37.83 ± 1.48%), while a significant up-regulation in IL-4 and IL-10 was determined in diclofenac sodium (53.68 and 68.64%) and C-AgNP (64.22 and 54.41%) treated groups (Table 2).

Table 2. Effect of C. medicaginea based AgNPs on expression levels of pro-inflammatory and anti-inflammatory markers in rats affected by CPE

Markers	Control group	Diseased group (Carrageenan)	Treatment group with AgNPs	Treatment group with DS
TNF-α	1 ± 0	79.66^{^a} ± 2.19^{^A}	49.32^{^c} ± 0.78^{^B}	33.88^{^d} ± 2.35^{^C}
IL-1β	1 ± 0	78.47^{^a} ± 1.52^{^A}	60.21^{^a} ± 2.36^{^B}	47.16^{^c} ± 1.33^{^C}
IL-6	1 ± 0	64.71^{^b} ± 1.02^{^A}	49.18^{^c} ± 1.31^{^B}	44.65^{^c} ± 0.96^{^B}
IL-4	1 ± 0	37.00^{^c} ± 1.72^{^C}	64.22^{^a}± 1.56^{^A}	53.68^{^b} ± 1.79^{^B}
IL-10	1 ± 0	37.83^{^c} ± 1.48^{^C}	54.41^{^b} ± 2.82^{^B}	68.64^{^a} ± 1.74^{^A}

Values are presented as mean ± SD for six animals per group. Within the same row and same column, values denoted by different superscript letters are significantly different (p < 0.05).

In the XEE method, expression levels of the studied inflammatory markers (TNF-α, IL-1β, and IL-6) increased significantly (p< 0.05) in the control treatment by 54.49, 56.26, and 62.83%, respectively. There was a significant decrease in the pro-inflammatory markers’ expression in diclofenac sodium (30.57, 37.43, and 33.53%) and C-AgNPs (34.65%, 35.29, and 43.64%) treated groups. Furthermore, a significant up-regulation in anti-inflammatory markers was observed in drug-treated (56.71 and 56.86%) and C-AgNPs (68.44 and 74.36 %) were recorded (Table 3).

Table 3. Effect of C. medicaginea based AgNPs on expression levels of pro-inflammatory and anti-inflammatory markers in rats affected by XEE

Markers	Control group	Diseased group (Xylene)	Treatment group with AgNPs	Treatment group with DS
TNF-α	1 ± 0	54.49^{^b} ± 1.81^{^A}	34.65^{^d} ± 1.27^{^B}	30.57^{^b} ± 0.91^{^B}
IL-1β	1 ± 0	56.26^{^b} ± 1.97^{^A}	35.29^{^d} ± 2.36^{^B}	37.43^{^b} ± 2.19^{^B}
IL-6	1 ± 0	62.83^{^a} ± 1.02^{^A}	43.64^{^c}± 1.31^{^B}	33.53^{^b} ± 0.96^{^C}
IL-4	1 ± 0	33.28^{^c} ± 1.72^{^C}	68.44^{^b} ± 1.56^{^A}	56.71^{^a} ± 1.79^{^B}
IL-10	1 ± 0	35.46^{^c} ± 0.91^{^C}	74.36^{^a} ± 2.82^{^A}	56.86^{^a} ± 1.74^{^B}

Values are presented as mean ± SD for six animals per group. Within the same row and same column, values denoted by different superscript letters are significantly different (p < 0.05).

In the case of granuloma formation in rats (cotton palette method), expression levels of pro-inflammatory gene markers were upregulated in disease control group (58.09, 67.31, and 77.84%). Significant attenuation in pro-inflammatory marker levels was observed in diclofenac sodium (27.01, 35.65, and 31.39%) and C-AgNPs (33.82, 34.12, and 43.89%) treated groups. For anti-inflammatory markers, a significant up-regulation was observed in drug treated (63.85 and 64.18%) and C-AgNPs (92.19 and 92.56%). The results have been summarized in Table 4.

Table 4. Effect of C. medicaginea based AgNPs on expression levels of pro-inflammatory and anti-inflammatory markers in rats affected by CPG

Markers	Control group	Diseased group (Cotton pellet)	Treatment group with AgNPs	Treatment group with DS
TNF-α	1 ± 0	58.09^{^c} ± 1.11^{^A}	33.82^{^c} ± 1.27^{^B}	27.01^{^c} ± 2.53^{^B}
IL-1β	1 ± 0	67.31^{^b} ± 2.02^{^A}	34.12^{^c} ± 2.17^{^B}	35.65^{^b} ± 3.63^{^B}
IL-6	1 ± 0	77.84^{^a} ± 1.46^{^A}	43.89^{^b} ± 2.65^{^B}	31.39^{^b} ± 1.16^{^C}
IL-4	1 ± 0	44.34^{^d} ± 2.07^{^C}	92.19^{^a} ± 1.97^{^A}	63.85^{^a} ± 2.30^{^B}
IL-10	1 ± 0	42.84^{^d} ± 1.91^{^C}	92.56^{^a} ± 1.02^{^A}	64.18^{^a} ± 0.96^{^B}

Values are presented as mean ± SD for six animals per group. Within the same row and same column, values denoted by different superscript letters are significantly different (p < 0.05).

It has been shown previously that AgNPs show immunogenicity properties (Engin and Hayes, 2018). Production of pro-inflammatory molecules is well controlled and inhibited by IL-4. IL-4 is produced in a normal reaction of inflammation with other markers. It is known for the elimination of intermediates of ROS, thus eliminating the effects of stress caused, like inflammation. It is known to inhibit the activation of macrophages which results in a controlled level of tissue damage occurring because of immune responses (Psachoulia et al., 2016). In the present study, exposure of animals to C-AgNPs caused an increase in IL-4 levels, thus demonstrating their anti-inflammatory potential. An increase in the expression of IL-10 was also noted indicating the suppression of kynurenine pathway (Boros and Vecsei, 2019). From the results of the present study, it is indicated that C-AgNPs showed an additive type of antioxidant potential and differential immunomodulatory potential. These characters of synthesized AgNPs lowered the level of pro-inflammatory cytokines and raised the levels of anti-inflammatory cytokines (Tu et al., 2005).

4. Conclusion

The present study concludes that the conjugate of plant extract of C. medicaginea and AgNPs have strong antioxidant and anti-inflammatory activity that is comparable to the standard drugs used in the study. C-AgNPs increased the production of anti-inflammatory markers, thus helping in the control of inflammation, and decreasing the chances of other diseases. Experiments on various inflammation studies of rats showed that C-AgNPs synthesized in the present study effectively reduced the inflammation. This activity is due to the smaller size and larger surface area of C-AgNPs along with the presence of secondary metabolites (phenolics, flavonoids, saponins, and tannins) of plants’ origin on their surface. C-AgNPs increased the production of anti-inflammatory markers, thus helped in controlling all types of inflammation induced in rats and decreasing the chances of other diseases. C-AgNPs caused the upregulation of important anti-inflammatory genes, that caused an increase in the concentration of anti-inflammatory markers in the blood serum of rats. Whereas these NPs actively showed antioxidant activity against ROS species that helped the whole system to reduce the stress from rats. These findings highlight the dual therapeutic potential of plant-derived AgNPs as robust antioxidants and anti-inflammatory agents, suggesting their promising application in biomedical and pharmaceutical fields. Future studies should explore the detailed mechanisms of action and assess the long-term safety of these NPs for potential clinical applications.

Acknowledgment

The authors would like to extend their sincere appreciation to the Ongoing Research Funding Program, (ORF-2025-182), King Saud University, Riyadh, Saudi Arabia.

CRediT authorship contribution statement

Saima Bashir: Investigation, Formal Analysis. Sumera Javad: Conceptualization, Supervision, Validation. Zeb Saddiqe: Conceptualization, Supervision, Validation. M. Shahzad: Statistical Analysis, writing-original draft. Usman Aftab: Statistical Analysis, writing-original draft. Anis A. Shah: Data Curation, Formal analysis. Ozair Chaudhry: Resource acquisition, Investigation. Mohamed El. Sheikh: Resource acquisition, Formal Analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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.

Data availability

Data generated or analyzed during this study are provided in full within the published article.

Ethical approval and consent to participate

Animal care and experimental protocols were followed strictly as per criterion outlined in guide for care and laboratory animal usage. For study, ethical authorization was issued by Institutional Ethical Committee Lahore College for Women University, Lahore (Protocol approval number DEC/LCWU/2023-03).

Our experiment follows with the relevant institutional, national, and international guidelines and legislation.

References

Agbaje A., Fageyinbo M.S.. Evaluating anti-inflammatory activity of aqueous root extract of Strophanthus hispidus DC. (Apocynaceae) Int. J. Appl. Res. Nat. Prod.. 2012;4:7-14.
[Google Scholar]
Ahmad I., Khan M.N., Hayat K., Ahmad T., Shams D.F., Khan W., Tirth V., Rehman G., Muhammad W., Elhadi M., Alotaibi A., Shah S.K.. Investigating the antibacterial and anti-inflammatory potential of polyol-synthesized silver nanoparticles. ACS Omega. 2024;9:13208-13216. https://doi.org/10.1021/acsomega.3c09851
[Google Scholar]
Ahmad K., Noor R., Younus M., Chohan A., Habiba U., Asif H.M., Chishti A.W., Saifullah S.. Green synthesis and characterization of silver nanoparticles using crude extract of Crotalaria burhia. RADS J. Pharm. Pharm. Sci.. 2021;8 https://doi.org/10.37962/jpps.v8i4.464
[Google Scholar]
Ahmed S., Saifullah, Ahmad M., Swami B.L., Ikram S.. Green synthesis of silver nanoparticles using Azadirachta indica aqueous leaf extract. J. Radiat. Res. Appl. Sci.. 2016;9:1-7. https://doi.org/10.1016/j.jrras.2015.06.006
[Google Scholar]
Akhlaq M., Alum M.K., Alam M.M.. Anti-inflammatory potential of medicinal plants. Med. J. Pharm. Sci.. 2024;2:13-21. http://dx.doi.org/10.5281/zenodo.6399381
[Google Scholar]
Albert H.M., Mendam K., Bansod P.G., Rao M.S.S., Asatkar A., Chakravarthi M.K., Mallesh M.P.. Biosynthesis, spectroscopic, and antibacterial investigations of silver nanoparticles. J. Fluoresc.. 2024;34:2009-2017. https://doi.org/10.1007/s10895-023-03398-7
[Google Scholar]
Alkhathlan A.H., AL-Abdulkarim H.A., Khan M., Khan M., AlDobiy A., Alkholief M., Alshamsan A., Alkhathlan H.Z., Siddiqui M.R.H.. Ecofriendly synthesis of silver nanoparticles using aqueous extracts of Zingiber officinale (Ginger) and Nigella sativa L Seeds (Black Cumin) and comparison of their antibacterial potential. Sustainability. 2020;12:10523. https://doi.org/10.3390/su122410523
[Google Scholar]
Anosike C.A., Obidoa O., Ezeanyika L.U.. Membrane stabilization as a mechanism of the anti-inflammatory activity of methanol extract of garden egg (Solanum aethiopicum) Daru. 2012;20:76. https://doi.org/10.1186/2008-2231-20-76
[Google Scholar]
Ashraf A., Sarfraz R.A., Mahmood A., Din M., ud. Chemical composition and in vitro antioxidant and antitumor activities of Eucalyptus camaldulensis Dehn. leaves. Industrial Crops and Products. 2015;74:241-248. https://doi.org/10.1016/j.indcrop.2015.04.059
[Google Scholar]
Atawodi S.E., Liman M.L., Onyike E.O.. Antioxidant effects of Tamarindus indica following acute and chronic carbon tetrachloride induced liver injury. Int. J. Agr. Biol. Sci.. 2013;15:60-65.
[Google Scholar]
Azab A., Nassar A., Azab A.N.. Anti-inflammatory activity of natural products. Molecules. 2016;21:1321. https://doi.org/10.3390/molecules21101321
[Google Scholar]
Aziz T.A., Kareem A.A., Othman H.H., Ahmed Z.A.. The anti-inflammatory effect of different doses of aliskiren in rat models of inflammation. Drug Des. Devel. Ther.. 2020;14:2841-2851. https://doi.org/10.2147/DDDT.S255607
[Google Scholar]
McDonald D.M., Bowden J.J., Baluk P., Bunnett N.W., . Neurogenic Inflammation. In: Zapata P., Eyzaguirre C., Torrance R.W., eds. Frontiers in Arterial Chemoreception. Advances in Experimental Medicine and Biology. Vol Vol. 410. Boston, MA: Springer US; 1996. p. :453-462. https://doi.org/10.1007/978-1-4615-5891-0_70
[Google Scholar]
Baluk P., Nadel J.A., McDonald D.M.. Substance P‐immunoreactive sensory axons in the rat respiratory tract: A quantitative study of their distribution and role in neurogenic inflammation. J. Comparative Neurology. 1992;319:586-598.
[Google Scholar]
Bhandarkar M.R., Khan A.. Antihepatotoxic effect of Nymphaea stellata willd., against carbon tetrachloride-induced hepatic damage in albino rats. J. Ethnopharmacol.. 2004;91:61-64. https://doi.org/10.1016/j.jep.2003.11.020
[Google Scholar]
Boros F.A., Vécsei L.. Immunomodulatory effects of genetic alterations affecting the kynurenine pathway. Front. Immunol.. 2019;10:2570. https://doi.org/10.3389/fimmu.2019.02570
[Google Scholar]
Buabeid M., Arafa E.A., Yaseen H.S., Umar M.I., Murtaza G.. Anti-inflammatory effect of simvastatin by impeding TNF-α and interleukin-1ß pathways: Antiangiogenic activity of simvastatin and simvastatin-loaded silver nanoparticles. Artif Cells Nanomed Biotechnol. 2022;50:208-217. https://doi.org/10.1080/21691401.2022.2098306
[Google Scholar]
Bussi C., Mangiarotti A., Vanhille-Campos C., Aylan B., Pellegrino E., Athanasiadi N., Fearns A., Rodgers A., Franzmann T.M., Šarić A., Dimova R., Gutierrez M.G.. Stress granules plug and stabilize damaged endolysosomal membranes. Nature. 2023;623:1062-1069. https://doi.org/10.1038/s41586-023-06726-w
[Google Scholar]
Carter C., Thornburg R.W.. Tobacco Nectarin I. J. Biol. Chem.. 2000;275:36726-36733. https://doi.org/10.1074/jbc.m006461200
[Google Scholar]
Chakravorty S., Malvi A., Chaturvedi A., Sonkusare K., Dave N.. Glutathione–the master antioxidant. Int. J. Med. Res. Pharm. Sci.. 2020;7:1-11.
[Google Scholar]
Chen T.S., Liou S.Y., Wu H.C., Tsai F.J., Tsai C.H., Huang C.Y., Chang Y.L.. New analytical method for investigating the antioxidant power of food extracts on the basis of their electron-donating ability: Comparison to the ferric reducing/antioxidant power (FRAP) assay. J. Agric. Food Chem.. 2010;58:8477-8480. https://doi.org/10.1021/jf9044292
[Google Scholar]
Chopade A.R., Somade P.M., Sayyad F.J.. Membrane stabilizing activity and protein denaturation: A possible mechanism of action for the anti-inflammatory activity of Phyllanthus amarus. J. Krishna Institute Med. Sci. Uni.. 2012;1:67-72.
[Google Scholar]
Clemente S.M., Martínez-Costa O.H., Monsalve M., Samhan-Arias A.K.. Targeting lipid peroxidation for cancer treatment. Molecules. 2020;25:5144. https://doi.org/10.3390/molecules25215144
[Google Scholar]
Draper H.H., Hadley M.. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol.. 1990;186:421-431. https://doi.org/10.1016/0076-6879(90)86135-i
[Google Scholar]
Ekechi H.O., Ikwuka A.O., Udeh F.C., Abraham J.C.. Effects of ethanol extract of Rauwolfia vomitoria leaf on lipid profile and cerebellar histology in cisplatin-induced oxidative stress. Brit. J. Med. Health Res.. 2023;10:16-39. http://dx.doi.org/10.5281/zenodo.8042521
[Google Scholar]
Engin A.B., Hayes A.W.. The impact of immune toxicity in evaluation of the nanomaterials safety. Toxicol. Res. Appl.. 2018;2:2397-2473. https://doi.org/10.1177/2397847318755579
[Google Scholar]
Ghaffar N., Javad S., Farrukh M.A., Shah A.A., Gatasheh M.K., Al-Munqedhi B.M.A., Chaudhry O.. Metal nanoparticles assisted revival of Streptomycin against MDRS Staphylococcus aureus. PLoS One. 2022;17:e0264588. https://doi.org/10.1371/journal.pone.0264588
[Google Scholar]
Ghaffar N., Javad S., Shah A.A., Ilyas S., Hashem A., Avila-Quezada G.D., Abd Allah E.F., Tariq A.. Restoration of antibacterial activity of inactive antibiotics via combined treatment with AgNPs. ACS Omega. 2024;9:13621-13635. https://doi.org/10.1021/acsomega.3c07000
[Google Scholar]
Gorinstein S., Jastrzebski Z., Leontowicz H., Leontowicz M., Namiesnik J., Najman K., Park Y.-S., Heo B.-G., Cho J.-Y., Bae J.-H.. Comparative control of the bioactivity of some frequently consumed vegetables subjected to different processing conditions. Food Control. 2009;20:407-413. https://doi.org/10.1016/j.foodcont.2008.07.008
[Google Scholar]
Haleem A., Javaid M., Singh R.P., Rab S., Suman R.. Applications of nanotechnology in medical field: A brief review. Global Health J.. 2023;7:70-77. https://doi.org/10.1016/j.glohj.2023.02.008
[Google Scholar]
Hayyan M., Hashim M.A., AlNashef I.M.. Superoxide Ion: Generation and chemical implications. Chem. Rev.. 2016;116:3029-3085. https://doi.org/10.1021/acs.chemrev.5b00407
[Google Scholar]
Ighodaro O.M., Akinloye O.A.. First line defense antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defense grid. Alexandria J. Med.. 2018;54:287-93. https://doi.org/10.1016/j.ajme.2017.09.001
[Google Scholar]
Jackie T., Haleagrahara N., Chakravarthi S.. Antioxidant effects of Etlingera elatior flower extract against lead acetate - induced perturbations in free radical scavenging enzymes and lipid peroxidation in rats. BMC Res. Notes. 2011;4:67. https://doi.org/10.1186/1756-0500-4-67
[Google Scholar]
Jadoon S., Malik A.A.. A review article on the formation, mechanism and biochemistry of MDA and MDA as a biomarker of oxidative stress. Int. J. Adv. Res.. 2017;5:811-818. https://doi.org/10.21474/IJAR01/6024
[Google Scholar]
Jaisankar A.I., Arivarasu L.. Free radical scavenging and anti-inflammatory activity of chlorogenic acid mediated silver nanoparticle. J. Pharm. Res. Int.. 2020;32:106-112. https://doi.org/10.9734/jpri/2020/v32i1930715
[Google Scholar]
Jyoti K., Baunthiyal M., Singh A.. Characterization of silver nanoparticles synthesized using Urtica dioica Linn. leaves and their synergistic effects with antibiotics. J. Radiat. Res. Appl. Sci.. 2016;9:217-227. https://doi.org/10.1016/j.jrras.2015.10.002
[Google Scholar]
Kaltalioglu K.. Prunus laurocerasus L. extracts prevent paracetamol induced nephrotoxicity by regulating antioxidant status: an experimental animal model. Hittite J. Sci. Eng.. 2022;9:275-280. http://dx.doi.org/10.17350/HJSE19030000280
[Google Scholar]
Kedi P.B.E., Etah B.N., Deli V., Gbamble A.P.. In-vitro and in-vivo anti-inflammatory activity of green synthesized silver nanoparticles from the aqueous bark extract of Mangifera indica Linn. (Anacardiaceae) Int. J. Green Herb. Chem.. 2020;9:345-360. http://dx.doi.org/10.24214/IJGHC/HC/9/3/34560
[Google Scholar]
Koleva L., Umar A., Yasin N.A., Shah A.A., Siddiqui M.H., Alamri S., Riaz L., Raza A., Javed T., Shabbir Z.. Iron oxide and silicon nanoparticles modulate mineral nutrient homeostasis and metabolism in cadmium-stressed Phaseolus vulgaris. Front Plant Sci. 2022;13:806781. https://doi.org/10.3389/fpls.2022.806781
[Google Scholar]
Kusar S., Saddiqe Z., Ali F., Bashir S., Zubairi T.. GCMS and HPLC profiling, antioxidant and anti-inflammatory activities of Crotalaria medicaginea Lamk. South African Journal of Botany. 2024;168:196-208. https://doi.org/10.1016/j.sajb.2024.03.014
[Google Scholar]
Mancipe J.C., Vargas-Pinto P., Rodríguez O.E., Borrego-Muñoz P., Castellanos Londoño I., Ramírez D., Piñeros L.G., Mejía M.C., Pombo L.M.. Anti-inflammatory effect of izalpinin derived from Chromolaena leivensis: λ-carrageenan-induced paw edema and in silico model. Molecules. 2023;28:3722. https://doi.org/10.3390/molecules28093722
[Google Scholar]
Menichetti A., Mavridi-Printezi A., Mordini D., Montalti M.. Effect of size, shape and surface functionalization on the antibacterial activity of silver nanoparticles. J. Funct. Biomater.. 2023;14:244. https://doi.org/10.3390/jfb14050244
[Google Scholar]
Mohd Zaid N.A., Sekar M., Bonam S.R., Gan S.H., Lum P.T., Begum M.Y., Mat Rani N.N.I., Vaijanathappa J., Wu Y.S., Subramaniyan V., Fuloria N.K., Fuloria S.. Promising natural products in new drug design, development, and therapy for skin disorders: An overview of scientific evidence and understanding their mechanism of action. Drug Des. Devel. Ther.. 2022;16:23-66. https://doi.org/10.2147/DDDT.S326332
[Google Scholar]
Moldovan B., Filip A., Clichici S., Suharoschi R., Bolfa P., David L.. Antioxidant activity of Cornelian cherry (Cornus mas l.) fruits extract and the in vivo evaluation of its anti-inflammatory effects. J. Funct. Foods. 2016;26:77-87. https://doi.org/10.1016/j.jff.2016.07.004
[Google Scholar]
Netam, aashish, Bhargava V., Singh R., Sharma P.. Physico-chemical characterization of ayurvedic swarna bhasma by using SEM, EDAX, XRD and PSA. J. Complement Med. Res.. 2021;12:204. https://doi.org/10.5455/jcmr.2021.12.02.23
[Google Scholar]
Nunes C.D.R., Barreto Arantes M., Menezes de Faria Pereira S., Leandro da Cruz L., de Souza Passos M., Pereira de Moraes L., Vieira I.J.C., Barros de Oliveira D.. Plants as sources of anti-inflammatory agents. Molecules. 2020;25:3726. https://doi.org/10.3390/molecules25163726
[Google Scholar]
Odabasoglu F., Halici Z., Aygun H., Halici M., Atalay F., Cakir A., Cadirci E., Bayir Y., Suleyman H.. α-Lipoic acid has anti-inflammatory and anti-oxidative properties: An experimental study in rats with carrageenan-induced acute and cotton pellet-induced chronic inflammations. Br. J. Nutr.. 2011;105:31-43. https://doi.org/10.1017/S0007114510003107
[Google Scholar]
Parvez S., Kaushik M., Ali M., Alam M.M., Ali J., Tabassum H., Kaushik P.. Dodging blood brain barrier with “nano” warriors: Novel strategy against ischemic stroke. Theranostics. 2022;12:689-719. https://doi.org/10.7150/thno.64806
[Google Scholar]
Psachoulia K., Chamberlain K.A., Heo D., Davis S.E., Paskus J.D., Nanescu S.E., Dupree J.L., Wynn T.A., Huang J.K.. IL4I1 augments CNS remyelination and axonal protection by modulating T cell driven inflammation. Brain. 2016;139:3121-3136. https://doi.org/10.1093/brain/aww254
[Google Scholar]
Sarma R.M., Subramanian B., Tamilmaran P., Ramakrishnan G.. Anti-inflammatory and anti-granuloma effect of the extract of the leaf of Terminalia arjuna (Roxb.) Wight Arn. J. Pharm. Sci. Res.. 2019;11:3579-3586.
[Google Scholar]
Shahrajabian M.H., Sun W.. Survey on Medicinal plants and herbs in traditional iranian medicine with anti-oxidant, anti-viral, anti-microbial, and anti-inflammation properties. LDDD. 2023;20:1707-1743. https://doi.org/10.2174/1570180819666220816115506
[Google Scholar]
Soliman S.M., Teaima M.H., Rashwan K.O., Ali B.M., Jasti B.R., El-Nabarawi M.A., Abd El-Halim S.M.. The deleterious effect of xylene-induced ear edema in rats: Protective role of dexketoprofen trometamol transdermal invasomes via inhibiting the oxidative stress/NF-κB/COX-2 pathway. Int. J. Pharm.. 2023;631:122525. https://doi.org/10.1016/j.ijpharm.2022.122525
[Google Scholar]
Sriramulu M., Sumathi S.. Photocatalytic, antioxidant, antibacterial and anti-inflammatory activity of silver nanoparticles synthesized using forest and edible mushroom. Adv. Nat. Sci: Nanosci. Nanotech.. 2017;8:45012. https://doi.org/10.1088/2043-6254/aa92b5
[Google Scholar]
Tijani A.A.. In-vitro and in-vivo determination of hydroxyl radical scavenging activity (hrsa) of fractions of aqueous extract of Moringa oleifera leaves (aemol) Eurasian J. Med. Oncol. 2018 https://doi.org/10.14744/ejmo.2018.46330
[Google Scholar]
Tu H., Rady P.L., Juelich T., Smith E.M., Tyring S.K., Hughes T.K.. Cytokine regulation of tryptophan metabolism in the hypothalamic-pituitary-adrenal (HPA) axis: Implications for protective and toxic consequences in neuroendocrine regulation. Cell. Mol. Neurobiol.. 2005;25:673-680. https://doi.org/10.1007/s10571-005-4007-1
[Google Scholar]
Varghese R.M., S A.K., Shanmugam R.. Comparative anti-inflammatory activity of silver and zinc oxide nanoparticles synthesized using Ocimum tenuiflorum and Ocimum gratissimum herbal formulations. Cureus. 2024;16:e52995. https://doi.org/10.7759/cureus.52995
[Google Scholar]
Zappavigna S., Cossu A.M., Grimaldi A., Bocchetti M., Ferraro G.A., Nicoletti G.F., Filosa R., Caraglia M.. Anti-inflammatory drugs as anticancer agents. Int. J. Mol. Sci.. 2020;21:2605. https://doi.org/10.3390/ijms21072605
[Google Scholar]
Zhang J., Chen R., Yu Z., Xue L.. Superoxide dismutase (SOD) and Catalase (CAT) activity assay protocols for Caenorhabditis elegans. Bio Protoc. 2017;7:e2505. https://doi.org/10.21769/BioProtoc.2505
[Google Scholar]

Show Sections

[1] Agbaje A., Fageyinbo M.S.. Evaluating anti-inflammatory activity of aqueous root extract of Strophanthus hispidus DC. (Apocynaceae) Int. J. Appl. Res. Nat. Prod.. 2012;4:7-14.
[Google Scholar]

[2] Ahmad I., Khan M.N., Hayat K., Ahmad T., Shams D.F., Khan W., Tirth V., Rehman G., Muhammad W., Elhadi M., Alotaibi A., Shah S.K.. Investigating the antibacterial and anti-inflammatory potential of polyol-synthesized silver nanoparticles. ACS Omega. 2024;9:13208-13216. https://doi.org/10.1021/acsomega.3c09851
[Google Scholar]

[3] Ahmad K., Noor R., Younus M., Chohan A., Habiba U., Asif H.M., Chishti A.W., Saifullah S.. Green synthesis and characterization of silver nanoparticles using crude extract of Crotalaria burhia. RADS J. Pharm. Pharm. Sci.. 2021;8 https://doi.org/10.37962/jpps.v8i4.464
[Google Scholar]

[4] Ahmed S., Saifullah, Ahmad M., Swami B.L., Ikram S.. Green synthesis of silver nanoparticles using Azadirachta indica aqueous leaf extract. J. Radiat. Res. Appl. Sci.. 2016;9:1-7. https://doi.org/10.1016/j.jrras.2015.06.006
[Google Scholar]

[5] Akhlaq M., Alum M.K., Alam M.M.. Anti-inflammatory potential of medicinal plants. Med. J. Pharm. Sci.. 2024;2:13-21. http://dx.doi.org/10.5281/zenodo.6399381
[Google Scholar]

[6] Albert H.M., Mendam K., Bansod P.G., Rao M.S.S., Asatkar A., Chakravarthi M.K., Mallesh M.P.. Biosynthesis, spectroscopic, and antibacterial investigations of silver nanoparticles. J. Fluoresc.. 2024;34:2009-2017. https://doi.org/10.1007/s10895-023-03398-7
[Google Scholar]

[7] Alkhathlan A.H., AL-Abdulkarim H.A., Khan M., Khan M., AlDobiy A., Alkholief M., Alshamsan A., Alkhathlan H.Z., Siddiqui M.R.H.. Ecofriendly synthesis of silver nanoparticles using aqueous extracts of Zingiber officinale (Ginger) and Nigella sativa L Seeds (Black Cumin) and comparison of their antibacterial potential. Sustainability. 2020;12:10523. https://doi.org/10.3390/su122410523
[Google Scholar]

[8] Anosike C.A., Obidoa O., Ezeanyika L.U.. Membrane stabilization as a mechanism of the anti-inflammatory activity of methanol extract of garden egg (Solanum aethiopicum) Daru. 2012;20:76. https://doi.org/10.1186/2008-2231-20-76
[Google Scholar]

[9] Ashraf A., Sarfraz R.A., Mahmood A., Din M., ud. Chemical composition and in vitro antioxidant and antitumor activities of Eucalyptus camaldulensis Dehn. leaves. Industrial Crops and Products. 2015;74:241-248. https://doi.org/10.1016/j.indcrop.2015.04.059
[Google Scholar]

[10] Atawodi S.E., Liman M.L., Onyike E.O.. Antioxidant effects of Tamarindus indica following acute and chronic carbon tetrachloride induced liver injury. Int. J. Agr. Biol. Sci.. 2013;15:60-65.
[Google Scholar]

[11] Azab A., Nassar A., Azab A.N.. Anti-inflammatory activity of natural products. Molecules. 2016;21:1321. https://doi.org/10.3390/molecules21101321
[Google Scholar]

[12] Aziz T.A., Kareem A.A., Othman H.H., Ahmed Z.A.. The anti-inflammatory effect of different doses of aliskiren in rat models of inflammation. Drug Des. Devel. Ther.. 2020;14:2841-2851. https://doi.org/10.2147/DDDT.S255607
[Google Scholar]

[13] McDonald D.M., Bowden J.J., Baluk P., Bunnett N.W., . Neurogenic Inflammation. In: Zapata P., Eyzaguirre C., Torrance R.W., eds. Frontiers in Arterial Chemoreception. Advances in Experimental Medicine and Biology. Vol Vol. 410. Boston, MA: Springer US; 1996. p. :453-462. https://doi.org/10.1007/978-1-4615-5891-0_70
[Google Scholar]

[14] Baluk P., Nadel J.A., McDonald D.M.. Substance P‐immunoreactive sensory axons in the rat respiratory tract: A quantitative study of their distribution and role in neurogenic inflammation. J. Comparative Neurology. 1992;319:586-598.
[Google Scholar]

[15] Bhandarkar M.R., Khan A.. Antihepatotoxic effect of Nymphaea stellata willd., against carbon tetrachloride-induced hepatic damage in albino rats. J. Ethnopharmacol.. 2004;91:61-64. https://doi.org/10.1016/j.jep.2003.11.020
[Google Scholar]

[16] Boros F.A., Vécsei L.. Immunomodulatory effects of genetic alterations affecting the kynurenine pathway. Front. Immunol.. 2019;10:2570. https://doi.org/10.3389/fimmu.2019.02570
[Google Scholar]

[17] Buabeid M., Arafa E.A., Yaseen H.S., Umar M.I., Murtaza G.. Anti-inflammatory effect of simvastatin by impeding TNF-α and interleukin-1ß pathways: Antiangiogenic activity of simvastatin and simvastatin-loaded silver nanoparticles. Artif Cells Nanomed Biotechnol. 2022;50:208-217. https://doi.org/10.1080/21691401.2022.2098306
[Google Scholar]

[18] Bussi C., Mangiarotti A., Vanhille-Campos C., Aylan B., Pellegrino E., Athanasiadi N., Fearns A., Rodgers A., Franzmann T.M., Šarić A., Dimova R., Gutierrez M.G.. Stress granules plug and stabilize damaged endolysosomal membranes. Nature. 2023;623:1062-1069. https://doi.org/10.1038/s41586-023-06726-w
[Google Scholar]

[19] Carter C., Thornburg R.W.. Tobacco Nectarin I. J. Biol. Chem.. 2000;275:36726-36733. https://doi.org/10.1074/jbc.m006461200
[Google Scholar]

[20] Chakravorty S., Malvi A., Chaturvedi A., Sonkusare K., Dave N.. Glutathione–the master antioxidant. Int. J. Med. Res. Pharm. Sci.. 2020;7:1-11.
[Google Scholar]

[21] Chen T.S., Liou S.Y., Wu H.C., Tsai F.J., Tsai C.H., Huang C.Y., Chang Y.L.. New analytical method for investigating the antioxidant power of food extracts on the basis of their electron-donating ability: Comparison to the ferric reducing/antioxidant power (FRAP) assay. J. Agric. Food Chem.. 2010;58:8477-8480. https://doi.org/10.1021/jf9044292
[Google Scholar]

[22] Chopade A.R., Somade P.M., Sayyad F.J.. Membrane stabilizing activity and protein denaturation: A possible mechanism of action for the anti-inflammatory activity of Phyllanthus amarus. J. Krishna Institute Med. Sci. Uni.. 2012;1:67-72.
[Google Scholar]

[23] Clemente S.M., Martínez-Costa O.H., Monsalve M., Samhan-Arias A.K.. Targeting lipid peroxidation for cancer treatment. Molecules. 2020;25:5144. https://doi.org/10.3390/molecules25215144
[Google Scholar]

[24] Draper H.H., Hadley M.. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol.. 1990;186:421-431. https://doi.org/10.1016/0076-6879(90)86135-i
[Google Scholar]

[25] Ekechi H.O., Ikwuka A.O., Udeh F.C., Abraham J.C.. Effects of ethanol extract of Rauwolfia vomitoria leaf on lipid profile and cerebellar histology in cisplatin-induced oxidative stress. Brit. J. Med. Health Res.. 2023;10:16-39. http://dx.doi.org/10.5281/zenodo.8042521
[Google Scholar]

[26] Engin A.B., Hayes A.W.. The impact of immune toxicity in evaluation of the nanomaterials safety. Toxicol. Res. Appl.. 2018;2:2397-2473. https://doi.org/10.1177/2397847318755579
[Google Scholar]

[27] Ghaffar N., Javad S., Farrukh M.A., Shah A.A., Gatasheh M.K., Al-Munqedhi B.M.A., Chaudhry O.. Metal nanoparticles assisted revival of Streptomycin against MDRS Staphylococcus aureus. PLoS One. 2022;17:e0264588. https://doi.org/10.1371/journal.pone.0264588
[Google Scholar]

[28] Ghaffar N., Javad S., Shah A.A., Ilyas S., Hashem A., Avila-Quezada G.D., Abd Allah E.F., Tariq A.. Restoration of antibacterial activity of inactive antibiotics via combined treatment with AgNPs. ACS Omega. 2024;9:13621-13635. https://doi.org/10.1021/acsomega.3c07000
[Google Scholar]

[29] Gorinstein S., Jastrzebski Z., Leontowicz H., Leontowicz M., Namiesnik J., Najman K., Park Y.-S., Heo B.-G., Cho J.-Y., Bae J.-H.. Comparative control of the bioactivity of some frequently consumed vegetables subjected to different processing conditions. Food Control. 2009;20:407-413. https://doi.org/10.1016/j.foodcont.2008.07.008
[Google Scholar]

[30] Haleem A., Javaid M., Singh R.P., Rab S., Suman R.. Applications of nanotechnology in medical field: A brief review. Global Health J.. 2023;7:70-77. https://doi.org/10.1016/j.glohj.2023.02.008
[Google Scholar]

[31] Hayyan M., Hashim M.A., AlNashef I.M.. Superoxide Ion: Generation and chemical implications. Chem. Rev.. 2016;116:3029-3085. https://doi.org/10.1021/acs.chemrev.5b00407
[Google Scholar]

[32] Ighodaro O.M., Akinloye O.A.. First line defense antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defense grid. Alexandria J. Med.. 2018;54:287-93. https://doi.org/10.1016/j.ajme.2017.09.001
[Google Scholar]

[33] Jackie T., Haleagrahara N., Chakravarthi S.. Antioxidant effects of Etlingera elatior flower extract against lead acetate - induced perturbations in free radical scavenging enzymes and lipid peroxidation in rats. BMC Res. Notes. 2011;4:67. https://doi.org/10.1186/1756-0500-4-67
[Google Scholar]

[34] Jadoon S., Malik A.A.. A review article on the formation, mechanism and biochemistry of MDA and MDA as a biomarker of oxidative stress. Int. J. Adv. Res.. 2017;5:811-818. https://doi.org/10.21474/IJAR01/6024
[Google Scholar]

[35] Jaisankar A.I., Arivarasu L.. Free radical scavenging and anti-inflammatory activity of chlorogenic acid mediated silver nanoparticle. J. Pharm. Res. Int.. 2020;32:106-112. https://doi.org/10.9734/jpri/2020/v32i1930715
[Google Scholar]

[36] Jyoti K., Baunthiyal M., Singh A.. Characterization of silver nanoparticles synthesized using Urtica dioica Linn. leaves and their synergistic effects with antibiotics. J. Radiat. Res. Appl. Sci.. 2016;9:217-227. https://doi.org/10.1016/j.jrras.2015.10.002
[Google Scholar]

[37] Kaltalioglu K.. Prunus laurocerasus L. extracts prevent paracetamol induced nephrotoxicity by regulating antioxidant status: an experimental animal model. Hittite J. Sci. Eng.. 2022;9:275-280. http://dx.doi.org/10.17350/HJSE19030000280
[Google Scholar]

[38] Kedi P.B.E., Etah B.N., Deli V., Gbamble A.P.. In-vitro and in-vivo anti-inflammatory activity of green synthesized silver nanoparticles from the aqueous bark extract of Mangifera indica Linn. (Anacardiaceae) Int. J. Green Herb. Chem.. 2020;9:345-360. http://dx.doi.org/10.24214/IJGHC/HC/9/3/34560
[Google Scholar]

[39] Koleva L., Umar A., Yasin N.A., Shah A.A., Siddiqui M.H., Alamri S., Riaz L., Raza A., Javed T., Shabbir Z.. Iron oxide and silicon nanoparticles modulate mineral nutrient homeostasis and metabolism in cadmium-stressed Phaseolus vulgaris. Front Plant Sci. 2022;13:806781. https://doi.org/10.3389/fpls.2022.806781
[Google Scholar]

[40] Kusar S., Saddiqe Z., Ali F., Bashir S., Zubairi T.. GCMS and HPLC profiling, antioxidant and anti-inflammatory activities of Crotalaria medicaginea Lamk. South African Journal of Botany. 2024;168:196-208. https://doi.org/10.1016/j.sajb.2024.03.014
[Google Scholar]

[41] Mancipe J.C., Vargas-Pinto P., Rodríguez O.E., Borrego-Muñoz P., Castellanos Londoño I., Ramírez D., Piñeros L.G., Mejía M.C., Pombo L.M.. Anti-inflammatory effect of izalpinin derived from Chromolaena leivensis: λ-carrageenan-induced paw edema and in silico model. Molecules. 2023;28:3722. https://doi.org/10.3390/molecules28093722
[Google Scholar]

[42] Menichetti A., Mavridi-Printezi A., Mordini D., Montalti M.. Effect of size, shape and surface functionalization on the antibacterial activity of silver nanoparticles. J. Funct. Biomater.. 2023;14:244. https://doi.org/10.3390/jfb14050244
[Google Scholar]

[43] Mohd Zaid N.A., Sekar M., Bonam S.R., Gan S.H., Lum P.T., Begum M.Y., Mat Rani N.N.I., Vaijanathappa J., Wu Y.S., Subramaniyan V., Fuloria N.K., Fuloria S.. Promising natural products in new drug design, development, and therapy for skin disorders: An overview of scientific evidence and understanding their mechanism of action. Drug Des. Devel. Ther.. 2022;16:23-66. https://doi.org/10.2147/DDDT.S326332
[Google Scholar]

[44] Moldovan B., Filip A., Clichici S., Suharoschi R., Bolfa P., David L.. Antioxidant activity of Cornelian cherry (Cornus mas l.) fruits extract and the in vivo evaluation of its anti-inflammatory effects. J. Funct. Foods. 2016;26:77-87. https://doi.org/10.1016/j.jff.2016.07.004
[Google Scholar]

[45] Netam, aashish, Bhargava V., Singh R., Sharma P.. Physico-chemical characterization of ayurvedic swarna bhasma by using SEM, EDAX, XRD and PSA. J. Complement Med. Res.. 2021;12:204. https://doi.org/10.5455/jcmr.2021.12.02.23
[Google Scholar]

[46] Nunes C.D.R., Barreto Arantes M., Menezes de Faria Pereira S., Leandro da Cruz L., de Souza Passos M., Pereira de Moraes L., Vieira I.J.C., Barros de Oliveira D.. Plants as sources of anti-inflammatory agents. Molecules. 2020;25:3726. https://doi.org/10.3390/molecules25163726
[Google Scholar]

[47] Odabasoglu F., Halici Z., Aygun H., Halici M., Atalay F., Cakir A., Cadirci E., Bayir Y., Suleyman H.. α-Lipoic acid has anti-inflammatory and anti-oxidative properties: An experimental study in rats with carrageenan-induced acute and cotton pellet-induced chronic inflammations. Br. J. Nutr.. 2011;105:31-43. https://doi.org/10.1017/S0007114510003107
[Google Scholar]

[48] Parvez S., Kaushik M., Ali M., Alam M.M., Ali J., Tabassum H., Kaushik P.. Dodging blood brain barrier with “nano” warriors: Novel strategy against ischemic stroke. Theranostics. 2022;12:689-719. https://doi.org/10.7150/thno.64806
[Google Scholar]

[49] Psachoulia K., Chamberlain K.A., Heo D., Davis S.E., Paskus J.D., Nanescu S.E., Dupree J.L., Wynn T.A., Huang J.K.. IL4I1 augments CNS remyelination and axonal protection by modulating T cell driven inflammation. Brain. 2016;139:3121-3136. https://doi.org/10.1093/brain/aww254
[Google Scholar]

[50] Sarma R.M., Subramanian B., Tamilmaran P., Ramakrishnan G.. Anti-inflammatory and anti-granuloma effect of the extract of the leaf of Terminalia arjuna (Roxb.) Wight Arn. J. Pharm. Sci. Res.. 2019;11:3579-3586.
[Google Scholar]

[51] Shahrajabian M.H., Sun W.. Survey on Medicinal plants and herbs in traditional iranian medicine with anti-oxidant, anti-viral, anti-microbial, and anti-inflammation properties. LDDD. 2023;20:1707-1743. https://doi.org/10.2174/1570180819666220816115506
[Google Scholar]

[52] Soliman S.M., Teaima M.H., Rashwan K.O., Ali B.M., Jasti B.R., El-Nabarawi M.A., Abd El-Halim S.M.. The deleterious effect of xylene-induced ear edema in rats: Protective role of dexketoprofen trometamol transdermal invasomes via inhibiting the oxidative stress/NF-κB/COX-2 pathway. Int. J. Pharm.. 2023;631:122525. https://doi.org/10.1016/j.ijpharm.2022.122525
[Google Scholar]

[53] Sriramulu M., Sumathi S.. Photocatalytic, antioxidant, antibacterial and anti-inflammatory activity of silver nanoparticles synthesized using forest and edible mushroom. Adv. Nat. Sci: Nanosci. Nanotech.. 2017;8:45012. https://doi.org/10.1088/2043-6254/aa92b5
[Google Scholar]

[54] Tijani A.A.. In-vitro and in-vivo determination of hydroxyl radical scavenging activity (hrsa) of fractions of aqueous extract of Moringa oleifera leaves (aemol) Eurasian J. Med. Oncol. 2018 https://doi.org/10.14744/ejmo.2018.46330
[Google Scholar]

[55] Tu H., Rady P.L., Juelich T., Smith E.M., Tyring S.K., Hughes T.K.. Cytokine regulation of tryptophan metabolism in the hypothalamic-pituitary-adrenal (HPA) axis: Implications for protective and toxic consequences in neuroendocrine regulation. Cell. Mol. Neurobiol.. 2005;25:673-680. https://doi.org/10.1007/s10571-005-4007-1
[Google Scholar]

[56] Varghese R.M., S A.K., Shanmugam R.. Comparative anti-inflammatory activity of silver and zinc oxide nanoparticles synthesized using Ocimum tenuiflorum and Ocimum gratissimum herbal formulations. Cureus. 2024;16:e52995. https://doi.org/10.7759/cureus.52995
[Google Scholar]

[57] Zappavigna S., Cossu A.M., Grimaldi A., Bocchetti M., Ferraro G.A., Nicoletti G.F., Filosa R., Caraglia M.. Anti-inflammatory drugs as anticancer agents. Int. J. Mol. Sci.. 2020;21:2605. https://doi.org/10.3390/ijms21072605
[Google Scholar]

[58] Zhang J., Chen R., Yu Z., Xue L.. Superoxide dismutase (SOD) and Catalase (CAT) activity assay protocols for Caenorhabditis elegans. Bio Protoc. 2017;7:e2505. https://doi.org/10.21769/BioProtoc.2505
[Google Scholar]

Effect of Crotalaria medicaginea-based silver nanoparticles on morphological and molecular inflammatory markers

Abstract

Keywords

Antioxidants

Nanoparticle synthesis

Natural anti-inflammatory agents

Plant secondary metabolites

Silver nanoparticles

1. Introduction

2. Materials and Methods

2.1 Materials and chemicals

2.2 Preparation of plant extract

2.3 Green synthesis of NPs

2.4 Characterizing C-AgNPs

2.4.1 Visual and UV-visible analysis

2.4.2 Fourier transform infrared spectroscopy (FT-IR)

2.4.3 Particle size analysis (PSA)

2.4.4 Scanning electron microscopy (SEM)

2.5 In vitro antioxidant activity

2.5.1 DPPH assay

2.5.2 Hydroxyl radical scavenging activity (HRSA)

2.5.3 FRAP Assay

2.5.4 ABTS radical scavenging assay

2.6 Anti-inflammatory action (in vitro)

2.6.1 Inhibition of heat-induced protein denaturation

2.6.2 Inhibition of hypotonicity-induced hemolysis

2.7 In-vivo assays

2.7.1 Pre-experimental animals’ conditioning

2.7.2 Animal grouping for in-vivo studies

2.7.3 In vivo antioxidant activity

2.7.3.1 Determination of SOD activity

2.7.3.2 Determination of CAT activity

2.7.3.3 Determination of GPx activity

2.7.3.4 Determination of lipid peroxidase activity (LPO)

2.7.4 Anti-inflammatory activity (In-vivo)

2.7.4.1 Carrageenan-induced paw edema assay (CPE)

2.7.4.2 Xylene-induced ear edema (XEE)

2.7.4.3 Cotton pellet-induced granuloma (CPG) in rats

2.8 Determination of mRNA expression for inflammatory markers using qPCR

2.9 Statistical analysis

3. Results and Discussion

3.1 Synthesizing and characterizing C-AgNPs

3.1.1 UV-Visible Spectroscopy

3.1.2 Fourier Transform Infrared Spectroscopy (FT-IR)

3.1.3 Particle Size Analysis (PSA)

3.1.4 SEM Analysis

3.1.5 EDX Analysis

3.2 In vitro antioxidant activities

3.3 In vitro anti-inflammatory activities

3.4 In vivo antioxidant activity

3.5 In vivo anti-inflammatory activity

3.6 C-AgNPs and inflammatory markers

4. Conclusion

Acknowledgment

CRediT authorship contribution statement

Declaration of competing interest

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

Data availability

Ethical approval and consent to participate

References

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