In-Vitro Bioactive Silver Nanoparticles Synthesized from Halodule uninervis Plant Extract for Multifunctional Drug Delivery

Abstract

The synthesis of AgNPs (silver nanoparticles) was performed using the aqueous extract of seagrass Halodule uninervis (HU) along with silver nitrate. The synthetic production of HU-AgNPs was confirmed by a color change and analyzed with a UV–vis spectrophotometer at 450 nm. The existence of functional groups was verified using Fourier transform infrared (FTIR) spectroscopy. The HU-AgNPs’ spherical shape was validated by scanning electron microscopy (SEM) tests. The crystal structure of HU-AgNPs was established by X-ray diffraction (XRD). The roughness and surface morphology were confirmed by atomic force microscopy (AFM). Significant antibacterial action was demonstrated by the HU-AgNPs against Pseudomonas aeruginosa and Escherichia coli, two Gram-negative pathogens. The antioxidant showed the highest inhibition of ABTs (2,2′-azino-bis­(3-ethylbenzothiazoline-6-sulfonic acid)) when compared to hydroxyl scavenging, 2,2′-diphenyl-2-picrylhydroxyl (DPPH), hydrogen peroxide (H₂O₂), nitric oxide (NO), and superoxide tests. By inhibiting α-amylase along with α-glycosidase, the HU-AgNPs’ antidiabetic effectiveness was evaluated. Therefore, to validate the findings and create an antibacterial, anti-inflammatory, antioxidant, and antidiabetic drug, a molecule-level in vivo study is required. Such synthesized nanoparticles have potential applications in multifunctional drug delivery applications.

1. Introduction

The field related to green nanotechnology includes biological procedures that enable the production, processing, and use of materials in the 1 to 100 nm range, offering simple and affordable methods. As an alternative technique for producing nanoparticles, the introduction of green synthesis in nanotechnology has generated a great deal of interest. Because of their unique characteristics, researchers have investigated the ecologically friendly and green production of metallic or metal oxide nanoparticles. Ion reduction, clustering, and subsequent nanoparticle development are the three main steps in the production process. The plant extract’s biomolecular components are essential reducing and stabilizing agents. Each step’s properties depend on the kind of reducing agents and how much of it is used.

An inventive and cost-effective technique is the green synthesis of nanoparticles using plant extracts. In this context, silver nanoparticles (AgNPs) exhibit exceptional antibacterial, chemical, structural, and optical capabilities compared with other metal nanoparticles. Furthermore, AgNPs produced by green methods exhibit various significant features, including antibacterial, antioxidant, medical diagnostic, therapeutic, and cytotoxic effects. Plants like Nerium indicum, Momordica charantia, Zingiber officinale, Catharanthus roseus, Taxus wallichiana, Eupatorium adenophorum, Consolida orientalis, and Linum usitatissimum have also been used to synthesize AgNPs, as shown in previous literature.

The oceans are a prolific source of diverse natural resources. Seagrasses are a type of marine angiosperm. Seagrasses represent one of the most prolific and active ecosystems. Moreover, they provide as a rich source of a diverse array of structurally distinct natural compounds recognized for their biomedical qualities. During stressful conditions, seagrass functions as a defense mechanism by creating secondary metabolites. Recently, numerous researchers have documented various biosynthetic nanoparticles derived from marine sources such as seaweeds, mangroves, sponges, corals as well as coastal plants of medicinal value. According to the review of related literature study, seagrasses have not been fully studied for their potential to produce nanoparticles. − Halodule uninervisis a marine angiosperm and seagrass species that is a member of the Cymodoceaceae family. It is abundant in phenolic and phenylpropanoid derivatives, including vanillic acid, p-hydroxybenzoic acid, caffeic acid, and p-coumaric acid. It is known that seagrasses use the production of distinct secondary metabolites primarily as a form of defense. Halodule uninervis serves as a significant food source for the green sea turtle in addition to the dugong, and it is also traditionally utilized for treating wounds. Accordingly, the purpose of this study was to create silver nanoparticles (AgNPs) for the first time using an aqueous extract of the whole Halodule uninervis (HU) plant, define the biosynthesized HU-AgNPs, and investigate their antibacterial, anti-inflammatory, antidiabetic, and antioxidant qualities.

2. Materials and Methods

2.1. Collection of Plant Samples

Halodule uninervis (Forsskål) Asch. was obtained from the Biosphere Reserve of the Gulf of Mannar in Mandapam, Tamil Nadu, and identified using the taxonomic key of Kuo and Hartog. The voucher specimen of H. uninervis (EPH: 376) was submitted to the Herbarium of the Ethnopharmacology Unit at the Research Department of Botany, V.O. Chidambaram College, Tuticorin, Tamil Nadu, India.

To create a uniform and seamless fracture, the collected samples were cut into tiny pieces and dried in the shade. The active components in the plant material were extracted by pulverizing or granulating the dehydrated material in a blender and then sieving the resulting homogeneous powder.

2.2. Preparation of Extract for Phytochemical Screening

The necessary amount of powder was measured and then transferred to a Stoppard flask. Additionally, it was treated individually using an aqueous solution. This process continued until the powder was completely submerged. During the initial 6 h period, the flask was agitated hourly. Whatman No. 1 filter paper was used to filter the extract. Qualitative tests were conducted on the extract according to conventional protocols for determining the existence of various phytochemical elements.

2.3. Green Synthesis of Silver Nanoparticles

2.3.1. Preparation of Precursor

Silver nitrate (AgNO₃) precursors were purchased from Hi-Media Chemicals, India. Using twice-distilled water, 1 mM silver nitrate, the precursor for creating silver nanoparticles, was employed.

2.3.2. Synthesis of Silver Nanoparticles

With continuous stirring for 20 min, 10 mL of an aqueous seagrass HU extract was deliberately mixed with 20 mL of a 1 mM silver nitrate solution. The mixture was then left at room temperature for 24 h. The initial indication of silver nanoparticles (AgNPs) formation was observed through a color change from pale yellow to reddish-brown. The change signified the reduction of Ag⁺ to metallic silver (Ag). The synthesized nanoparticles were separated by centrifugation at 9000 rpm for 30 min, a step repeated three times to remove unbound silver. Finally, the collected HU-AgNPs were dried in an oven at 60 °C.

2.4. Characterization of the Synthesized Silver Nanoparticles

2.4.1. UV–Vis Spectroscopy

A Shimadzu V-650 UV–vis spectrophotometer was used to characterize the silver nanoparticles. The samples were scanned at wavelengths between 300 and 700 nm. The blank orientation was double-distilled water.

2.4.2. Fourier Transform Infrared Spectroscopy (FTIR)

A Fourier transform infrared spectrophotometer (FTIR Thermo Scientific iS5) was used to evaluate the nanoparticles. 100 mg of potassium bromide (KBr) was mixed with 2 mg of the substance. A 3 mm irregular diameter was achieved by further condensing the salt disc. In its sample holder, the disc was held in place. FTIR spectra were allowed in the absorption range of 400–4000 cm^–1.

2.4.3. Scanning Electron Microscope (SEM)

A scanning electron microscope (SEM) (Model: Carl Zeiss) was used to examine the morphology of the AgNPs that were created. It was then prepared with a 15–30 kV acceleration voltage.

2.4.4. X-ray Diffraction (XRD) and Dynamic Light Scattering (DLS) Analysis

Using XRD, the silver nanoparticles’ size and properties were ascertained. Using CuKα radiation at a 2θ angle and the Shimadzu XRD-6000/6100 model, this operation was carried out at 30 kV and 30 mA. X-ray diffraction is a systematic and effective process. This is mostly used to classify a crystalline substance’s phases. Additionally, this can provide data about the dimensions of the unit cells. The substance under study is carefully ground. It is devoted to the average bulk composition. Debye–Scherrer’s equation, as shown in eq , was used to calculate the silver nanoparticles’ particle or grain size.

$$D=0.94\lambda /B\mathrm{cos\theta }$$ 1

The particle size distribution of AgNPs and their sizes in colloidal solutions were examined by DLS. The sample was resuspended in Millipore water and subjected to DLS measurement using the Anton Paar Litesizer DLS.

2.4.5. Atomic Force Microscopy (AFM) Analysis

Through investigation using 1 μm x 1 μm Atomic Force Microscopy (AFM Nanosurf 2), the surface topology of the generated AgNPs was ascertained. Using an ultrasonicator, 0.01 g of amalgamated nanoparticles was mixed with 20 mL of acetone and sonicated for 5–10 min. A sterilized glass slide was coated with the solution. The material was left to dry until the acetone had evaporated. Atomic force microscopy was used in a noncontact mode to inspect the glass slide. XEI software was used to process the captured image.

2.5. Antioxidant Activity

2.5.1. DPPH Radical Scavenging Assay

The method outlined by Shen et al. has been used to assess the (2,2-diphenyl-1-picrylhydrazyl) (DPPH) radical scavenging capacity of both the synthesized HU-AgNPs and conventional ascorbic acid. Methanol was used to make a 0.1 mM DPPH solution. 3 mL of HU-AgNPs at varying concentrations (25, 50, 75, 100, and 125 μg/mL) was mixed with 1 mL of the resultant solution. The mixture was then vigorously stirred and allowed to rest. A UV–vis spectrophotometer (Genesys 10S UV: Thermo Electron Corporation) was used to measure absorbance at 517 nm after 30 min, using ascorbic acid as the standard. A solution with higher free radical scavenging activity has lower absorbance values. The computation was accomplished by using the following formula.

$$\mathrm{DPPH\; scavenging\; effect}(\mathrm{\%\; inhibition})=\{(A_0{-A}_1){/A}_0)\mathrm{\times \; 100}\}$$ 2

where A ₀ is the absorbance of the control reaction, and A ₁ is the absorbance of HU-AgNPs and reference.

2.5.2. Hydrogen Peroxide (H₂O₂) Radial Scavenging Activity

The approach of Sousa et al. was used to evaluate the synthetic AgNPs’ and standard ascorbic acid’s ability to scavenge H₂O₂. A buffered phosphate solution with a pH of 7.4 was used to create 40 mM hydrogen peroxide. To a solution of 0.6 mL of H₂O₂ (40 mM), different doses of HU-AgNPs and ascorbic acid (25, 50, 75, 100, and 125 μg/mL) were added in 3.4 mL of phosphate buffer. The mean ± standard deviation was used to compute the absorbance of the reaction mixture and determine the IC₅₀ value at 230 nm. As previously mentioned, eq was utilized to determine the percentage of H₂O₂ scavenging activity.

2.5.3. Nitric Oxide (NO) Scavenging Activity

With a few modifications, the Kumar and Hemalatha technique was used to measure the nitric oxide scavenging activity. Sodium nitroprusside produced nitric oxide radicals (NO). Different concentrations of the test chemicals (HU-AgNPs and ascorbic acid) were mixed with 1 mL of sodium nitroprusside (10 mM) and 1.5 mL of phosphate-buffered saline (0.2 M, pH 7.4). After 150 min of incubation at 25 °C, 1 mL of the reaction mixture was treated with 0.1% naphthyl ethylenediamine dihydrochloride, 1 mL of Griess reagent (1% sulfanilamide), and 2% H₃PO₄. The compounds’ absorption capacity was measured at 546 nm, and the mean ± standard deviation was used to get the IC₅₀ value. As mentioned before, eq was used to calculate the percentage of nitric oxide scavenging.

2.5.4. Antioxidant Activity by Radical Cation (ABTs+)

The procedure was carried out using a slightly revised methodology of Huang et al. To create the ABTs radical cation, this approach used a 7 mM ABTs solution, along with 2.45 mM potassium persulfate. The reaction mixture was kept in the dark for 12–16 h at room temperature. The solution was diluted with ethanol, yielding an absorbance value of 0.70 ± 0.02 at 734 nm. After 6 min, 3.9 mL of the diluted ABTs+ solution was added to the sample extract and used to measure absorbance at 734 nm with the Genesys 10S UV–vis (Thermo Scientific). The IC₅₀ value was derived with ascorbic acid to be the standard and expressed as mean ± SD. The scavenging activity percentage of ABTs was evaluated using eq as previously stated.

2.5.5. Superoxide Radical Scavenging Activity

Synthesized HU-AgNPs and regular ascorbic acid exhibited controlled action, according to Keshari et al. The oxidation process of nicotinamide adenine dinucleotide hydrogen (NADH) in the phenazine methosulfate (PMS) system produced superoxide radicals. The Nitro Blue Tetrazolium (NBT) reduction process was then used to assess it. 1 mL of HU-AgNPs/ascorbic acid in methanol, 1 mL of Tris-HCl buffer (16 mM, pH 8), 1 mL of NBT (50 μM), 1 mL of NADH (78 μM), and 1 mL of PMS (10 μM) were all included in the combination. The mixture was incubated for 5 min at 25 °C, and a UV–vis spectrophotometer was used to measure the absorbance at 560 nm. The inhibition percentage of superoxide formation was computed using eq , and the IC₅₀ value was derived using the mean ± SD.

2.5.6. Hydroxyl Radical Scavenging Activity

The modified method of Halliwell et al. was used to carry out the hydroxyl radical scavenging experiment. Ascorbic acid (1 mM), ethylenediaminetetraacetic acid (EDTA) (1 mM), H₂O₂ (10 mM), FeCl₃ (10 mM), and deoxyribose (10 mM) were the stock solutions utilized in this process. Distilled deionized water was used to formulate each solution. 0.1 mL of EDTA, 0.01 mL of FeCl₃, 0.1 mL of H₂O₂, 0.36 mL of deoxyribose, 1.0 mL of HU-AgNPs at different concentrations, 0.33 mL of phosphate buffer (50 mM, pH 7.9), and 0.1 mL of ascorbic acid were added one after the other to perform the hydroxyl radical scavenging activity. For 1 h, the mixture was kept at 37 °C. After the incubation period, the pink chromogen was created by mixing 1.0 mL of the mixture with 1.0 mL of 10% trichloroacetic acid (TCA) and 1.0 mL of 0.5% thiobarbituric acid (TBA) (dissolved in 0.025 M NaOH containing 0.025% butylated hydroxyanisole (BHA)). At 532 nm, the measurement was made. Using the previously mentioned formula, the percentage of inhibition was determined by comparing the test findings with those of the control group.

2.6. In Vitro Anti-inflammatory Activity of AgNPs

2.6.1. Inhibition of Albumin Denaturation

0.2 mL portion of 1% bovine albumin, 4.78 mL of phosphate-buffered saline (pH 6.4), and 0.2 mL of manufactured HU-AgNPs at different concentrations made up the reaction mixture (5 mL). After mixing, the mixture was kept for 15 min at 37 °C in a water bath. After that, the reaction mixture was heated for 5 min at 51 °C. After cooling, a Genesys 10s UV–vis spectrophotometer was used to measure the turbidity at 660 nm. The control was a phosphate buffer solution. The reference drug used in this study was aspirin. When calculating absorbance, this was considered as such. Three runs of the experiment were carried out. , Equation was used to determine the percentage inhibition of protein denaturation.

$$\mathrm{\%\; inhibition\; of\; denaturation}=\frac{A\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-A\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{A\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$ 3

Here the absorbance of the solution without AgNPs is “A control,” while the absorbance of the solution with HU-AgNPs/standard is “A sample.”

2.6.2. Proteinase Inhibitory Activity

According to Sakat et al.’s methodology, the HU-AgNPs generated demonstrated a proteinase inhibitory activity. Gunathilake et al. made this adjustment. 1 mL portion of 20 mM Tris-HCl buffer (pH 7.4), 0.06 mg of trypsin, and 1 mL of HU-AgNPs in various doses made up the reaction mixture (2 mL). After that, the entire mixture was incubated at 37 °C for 5 min. Additionally, 1 mL of 0.8% (w/v) casein was added. After another 20 min of incubation, the liquid was combined with 2 mL of 70% perchloric acid to stop the reaction. The cloudy suspension was then centrifuged, and the absorbance of the supernatant at 210 nm was measured by using the buffer as a control. Aspirin served as the standard medication. The IC₅₀ value was determined using the mean ± standard deviation (SD). As previously mentioned, eq was used to determine the percentage of inhibition of the proteinase inhibitory activity.

2.6.3. Membrane Stabilization Assay

The erythrocyte suspension was prepared following the guidelines given by Giridasappa et al., and the red blood cell (RBC) suspension was prepared using recent blood samples drawn from a healthy volunteer. 1 mL of a 10% RBC suspension and 1 mL of heat-induced hemolysis prevention HU-AgNPs at various dosages made up the 2 mL test solution. Phosphate buffer solution was utilized in place of HU-AgNPs in the control sample. Aspirin was used as a common prescription drug. It should be mentioned that the tabs had been kept at 56 °C for 30 min before being submerged in a water bath, and the specimens were carefully mixed by gently inverting them. Following the incubation period, the reaction mixture was centrifuged at 2500 rpm and 37 °C. A UV–visible spectrophotometer was used to measure the absorbance of the supernatant at 560 nm after it had been collected. The mean ± standard deviation (SD) was used to calculate the IC₅₀ value. As previously mentioned, eq was used to calculate the existing membrane stabilization activity.

2.7. Antilipoxygenase Activity

Using lipoxidase as the enzyme and linoleic acid as the substrate, the antilipoxygenase activity was investigated as done by Shinde et al. A 0.25 mL portion of 2 M borate buffer at pH 9.0 was used to dissolve the test materials. After adding 0.25 mL of lipoxidase enzyme solution (20,000 U/mL), the mixture was incubated at 25 °C for 5 min. A 1.0 mL solution of 0.6 mM linoleic acid was then added and carefully mixed. At 234 nm, the absorbance was measured. The reference norm used was indomethacin. The aforementioned equation (eq ) was used to get the inhibition percentage. The mean ± standard deviation (SD) was used to determine the IC₅₀ value.

2.8. In Vitro Antidiabetic Assay

2.8.1. α-Amylase Inhibition Assay

The 3,5-dinitrosalicylic acid (DNSA) method described by Wickramarctne et al. was used to perform the α-amylase inhibition assay. The assay mixture included 500 μL of α-amylase solution (1 U/mL) and 0.02 M sodium phosphate buffer (pH 6.9), to which different concentrations of HU-AgNPs (25–125 μg/mL) were added. For 20 min, the test mixture was preincubated at 37 °C. After incubation, the tubes were filled with 250 μL of a 1% starch solution in the designated buffer, and they were incubated for 15 min at 37 °C. 1 mL of dinitrosalicylic acid reagent was added to complete the catalytic reaction, and the mixture was then incubated for 10 min at 90 ± 5 °C in a boiling water bath. After the tubes were brought up to room temperature, UV–visible spectrophotometry was used to measure the absorbance at 540 nm (Genesys 10s UV–vis, Thermo Scientific). Apart from the test sample, the reference sample included all of the other reagents and enzymes. The mean ± SD was used to calculate the IC₅₀ value, with acarbose serving as a positive control. Equation was used to calculate the inhibition percentage of the α-amylase inhibitory activity.

$$\%\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\alpha \mathrm{‐}\mathrm{a}\mathrm{m}\mathrm{y}\mathrm{l}\mathrm{a}\mathrm{s}\mathrm{e}=\frac{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}\times 100$$ 4

2.8.2. α-Glucosidase Inhibition Assay

A modified method as outlined by Kim et al. for the α-glucosidase inhibition experiment was used in the current experiment. A range of HU-AgNP dosages, from 25 to 125 μg/mL, was added to the assay mixture, which contained 150 μL of 0.1 M sodium phosphate buffer (with 6 mM NaCl, pH 6.9) and 0.1 U of α-glucosidase. After that, the assay mixture was preincubated for 10 min at 37 °C. Then, to start the reaction, 50 μL of 2 mM paranitrophenyl α-d-glucopyranoside in 0.1 M sodium phosphate buffer was added to the mixture and left to incubate. 50 μL of 0.1 M Na₂CO₃ was added to stop the reaction after it had been incubated for 20 min at 37 °C. Next, using a UV–visible spectrophotometer (Genesys 10S UV–vis, Thermo Scientific), the absorbance was measured at 405 nm. While the antidiabetic drug acarbose was used as a positive control, the tube containing α-glucosidase without AgNPs served as a control with 100% enzyme activity. Equation was utilized to determine the α-glucosidase inhibition proportion.

2.9. Antibacterial Activity

The antibacterial activity of the synthesized nanoparticles (HU-NPs) was determined using the disc diffusion approach, as described by Bauer et al. The bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli were acquired. They were obtained from the Research Laboratory, Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu. The freshly made Muller–Hinton agar plates were covered with the bacterial cultures that had been cultured for the entire night. At the center, there was a 6 mm sterile disc (HiMedia), on which different concentrations of the generated nanoparticles (40, 80, and 100 in 1 mL of solution each) were applied. After that, they were placed on the platter. The aqueous extract, AgNP solution without extract, and a tetracycline disc (reference or positive control) were also kept. The plates were then incubated for 24 h at 37 °C. The inhibitory zone was identified after incubation.

2.10. Statistical Analysis

There were three copies of each experiment (n = 3). The Prism program (GraphPad Software Inc., USA), version 5.01, was used to analyze the data. Dunnett’s test was used to determine the significance of differences between the treatment and a control group, and a two-way analysis of variance (ANOVA) was used to determine the significance of treatments. **p < 0.001, *** p < 0.0001, and *p < 0.005.

3. Results and Discussion

3.1. Phytochemical Analysis

Aqueous extract of HU was subjected to phytochemical examination, which revealed the presence of coumarins, catechins, flavonoids, phenols, saponins, glycosides, tannins, and xanthoproteins. In addition to helping to reduce Ag⁺ to Ag⁰, these phytoconstituents are anticipated to act as stabilizing and capping agents for the green synthesis of HU-AgNPs.

3.1.1. Green Synthesis of HU-AgNPs

The AgNO₃ aqueous solution was mixed with the HU aqueous extract. Ag⁺ to Ag⁰ quickly decreased, as seen by the solution’s rapid color change from a light brown to an even darker reddish-brown after 24 h (Figure ). By stimulating the surface plasmon resonance of the produced HU-AgNPs, the active phytoconstituents in the aqueous extract of HU aided in the reduction of silver metal to HU-AgNPs. The color transition of the reactive solution from light brown to dark reddish-brown, utilizing leaf extracts of Avicennia marina and Cymodocea rotundata, was corroborated by Soni et al. and Darshinidevi et al., respectively.

1.

Synthesis of silver nanoparticles from HU (a) Plant extract, (b) AgNO₃, and (c) AgNPs.

3.2. Characterization of Green-Synthesized HU-AgNPs

3.2.1. UV–Vis Spectroscopy

Utilizing UV–vis spectroscopy, the optical properties of the generated HU-AgNPs were investigated. The presence of HU-AgNPs was verified by the UV–vis absorbance spectrum’s peak of absorption at 405 nm (Figure ). AgNPs’ surface plasmon resonance (SPR) was responsible for identifying the absorption peak. For HU-AgNPs, surface plasmon activation produced a noticeable absorption peak in the visible spectrum. SPR was responsible for the mixture’s initial color shift from a light brown to darker reddish-brown, which indicated the reduction of AgNO3 to AgNPs. Previous research has similarly observed the absorption maxima of AgNPs at 411, 420, 425, and 428 nm utilizing leaf extracts of Momordica charantia, Avicennia marina, Cymodocea rotundata, Knoxia sumatrensis, and Calophyllum tomentosum, respectively.

2.

UV–visible spectrum of HU-AgNPs (gray line) and HU extract (green line).

3.2.2. FTIR Analysis

With the use of FTIR analysis, the biomolecules and functional groups that stabilize the HU-AgNPs that are currently being developed were identified. The aqueous extract of HU served as both the capping agent and the reducing agent in the environmentally friendly synthesis of HU-AgNPs. As shown by FTIR examination of the aqueous extract of HU and the synthesized HU-AgNPs (Figure a,b), this can be attributed to the presence of certain functional groups. O–H stretching linked to hydrogen-bonded alcohols and phenols was indicated by peaks at 3454 cm^–1 in the FTIR spectra of the HU aqueous extract. Additional peaks were observed at 2366 cm^–1 (NH₄ stretch of tertiary amine salt), 1637 cm^–1 (>N–H bending of secondary amine), 1421 cm^–1 (C–C stretch of aromatics), 1325 cm^–1 (N–O symmetric stretch of nitro compounds), 1253 cm^–1 (C–O stretch of esters and ethers), 1026 cm^–1 (C–N stretch of aliphatic amines), and 663 and 584 cm^–1 (C–Br stretch of alkyl halides). The pronounced wide absorbance at 3454 cm^–1 is indicative of the hydroxyl functional group present in alcohols and phenolic compounds. Peaks at 3448 cm^–1 (O–H stretch, hydrogen-bonded alcohols/phenols) and 1637 cm^–1 (>N–H bending of secondary amines) were visible in the FTIR spectrum of HU-AgNPs. Alkane C–H rocking is 1384 cm^–1, aliphatic amine C–N stretching is 1114 cm^– 1, and alkyl halide C–Br stretching is 612 cm^– 1. The hydroxyl (O–H) and amine (−NH) groups in the leaf extract are crucially involved in the synthesis of AgNPs, according to Kumar et al. The existence of HU-AgNPs was suggested by the fundamental functional groups, which functioned as stabilizing, capping, and dipping agents in the AgNPs and included alcohols/phenols, alkanes, aliphatic amines, and halides. The decrease in Ag⁺ to Ag⁰ may be actively caused by several physiologically significant active components, as the HU extract has shown. A few transmittance peaks had shifted or disappeared in the case of AgNPs, which could be attributed to the interaction between the phytochemicals in the HU extract and the extracted silver nanoparticles, corresponding to the biosynthesis and functionalization of the AgNPs.

3.

FTIR spectrum of aqueous HU (gray line) and HU-AgNPs (red line).

3.2.3. SEM Analysis

The SEM pictures of the synthesized HU-AgNPs are shown in Figure . The surface morphology of HU-AgNPs made from HU’s aqueous extract clearly shows that they are spherical and evenly distributed. Analogous results were also reported by Adelere et al. A number of variables, such as extract concentration, contact time, pH, and silver salt content, affect the form of nanoparticles.

4.

SEM Image of HU-AgNPs at different magnification images: (a) 5 μm, (b) 10 μm, (c) 20 μm, (d) 30 μm.

3.2.4. XRD Analysis

Using X-ray diffraction, we examined the crystalline structure of the green-produced HU-AgNPs. The XRD pattern of the synthesized HU-AgNPs is shown in Figure , where the planes (110), (111), (200), (222), (220), and (311) are represented by the peaks at 28.5, 32.6, 46.4, 57.5, 62.2, and 76.4, respectively. The XRD pattern of the synthesized HU-AgNPs shows a face-centered cubic structure typical of metallic silver in AgNPs, which is in good agreement with the Joint Committee on Powder Diffraction Standards (JCPDS file no: 04–0783). The crystalline nature of the nanoscale-produced nanoparticles is evident from the comparison of the prominent peaks in the obtained data from the present study with standard references, which is consistent with previous results. The average particle size is determined by the Debye–Scherrer formula, and the average crystalline size of HU-AgNPs is roughly 23.48 nm.

5.

XRD peaks of HU-AgNPs.

The particle size of the synthesized AgNPs measured through DLS analysis was 34.45 nm. The synthesized AgNPs were confirmed to have nanosized particles with enhanced surface reactivity and functional properties (Figure ).

6.

Particle size of the AgNPs from DLS analysis.

3.2.5. AFM Analysis

The usual AFM image obtained for green-produced HU-AgNPs is shown in Figure . The distribution of the particle morphology was uniform. The material that was synthesized showed uniform dispersion. The generated HU-AgNPs’ surface roughness showed a folded form.

7.

AFM image of HU-AgNPs (a) 2D image and (b) 3D Profile.

3.3. Antioxidant Activity

3.3.1. DPPH Radical Scavenging Activity

The percentage of inhibition of the DPPH radical scavenging activity is shown in Figure a. In this study, the DPPH activity showed a dose-dependent relationship. AgNPs made with Knoxia sumatrensis leaf extract showed a similar dose-dependent activity. With an IC₅₀ value of 92.40 μg/mL, the synthesized HU-AgNPs’ DPPH radical scavenging activity showed the highest inhibition of 62.18% at a dose of 125 μg/mL as well as the lowest inhibition of 10.26% at 25 μg/mL. By contrast, the conventional ascorbic acid showed an IC₅₀ value of 78.10 μg/mL and an inhibition of 66.34% at 125 μg/mL. The IC₅₀ values of AgNPs biosynthesized from Catharanthus roseus and Azadirachta indica were 36 and 35 μg/mL, respectively. Another work indicated that AgNPs produced from Zingiber officinale had an IC₅₀ value of 68 μg/mL.

8.

Antioxidant activity of synthesized HU-AgNPs (a) DPPH, (b) H₂O₂, (c) NO, (d) ABTs, (e) Superoxide, and (f) Hydroxyl radical scavenging activity.

3.3.2. H₂O₂ Radical Scavenging Activity

Figure b shows how the generated HU-AgNPs scavenged the H₂O₂ radical in a concentration-dependent manner. At 125 μg/mL, the greatest inhibition percentage of biosynthesized HU-AgNPs is 61.28%, whereas at 25 μg/mL, the minimum inhibition percentage is 10.86%. Conventional ascorbic acid has an IC₅₀ value of 71.14 μg/mL, whereas biosynthesized HU-AgNPs have an IC₅₀ value of 88.16 μg/mL. AgNPs synthesized from olive fruit extract showed a 55.4% H₂O₂ radical scavenging inhibition in a recent study. In a separate investigation, AgNPs derived from Withania somnifera exhibited an IC₅₀ value of 102.16 μg/mL.

3.3.3. NO Radical Scavenging Activity

The percentage of suppression of the nitric oxide radical scavenging activity is shown in Figure c. The scavenging of NO radicals in this study was dose-dependent. AgNPs showed a similar dose-dependent impact, utilizing Brassica oleracea leaf extract. Various concentrations of biosynthesized HU-AgNPs demonstrate a maximum inhibition percentage of 61.46% at 125 μg/mL and a minimum inhibition percentage of 11.24% at 25 μg/mL. The IC₅₀ value for radical scavenging of HU-AgNPs is 92.66 μg/mL, while that of conventional ascorbic acid is 70.18 μg/mL. A recent study indicated that AgNPs produced from Brassica oleracea exhibited a NO radical scavenging inhibition of 81% at a dosage of 200 μg/mL. The AgNPs produced from Viscum orientale exhibited an IC₅₀ value of 56.01 μg/mL.

3.3.4. ABTs Radical Cation Scavenging Activity

Using ABTs, the biosynthesized HU-AgNPs’ free radical scavenging activity shows a low inhibition percentage of 11.84% at 25 μg/mL and a high inhibition percentage of 64.24% at 125 μg/mL (Figure d). Biosynthesized HU-AgNPs have an IC₅₀ value of 94.12 μg/mL, while conventional ascorbic acid has an IC₅₀ value of 79.04 μg/mL. The results of this study showed that the inhibition value and mean concentration increased in a dose-dependent manner. Comparable results were observed with biosynthesized AgNPs from Asphodelus aestivus, demonstrating significant ABTs radical scavenging activity (79.94%). Recent data indicate that Alnus nitida leaf extract exhibits 78.81% radical scavenging efficacy against ABTs, with an IC₅₀ value of 16.46 μg/mL.

3.3.5. Superoxide Radical Scavenging Activity

There was a dose-dependent interaction between the biosynthesized HU-AgNPs. A 63.52% suppression of superoxide radical scavenging activity was noted at the highest dosage of 125 μg/mL, while the lowest inhibition percentage of 11.78% was recorded at a dose of 25 μg/mL (Figure e). Biosynthesized HU-AgNPs have an IC₅₀ value of 94.36 μg/mL, while conventional ascorbic acid has an IC₅₀ value of 72.40 μg/mL. The IC₅₀ value of biosynthesized AgNPs utilizing Sargassum wightii is 59.67 μg/mL. A separate investigation indicated that AgNPs produced from Brassica oleracea leaf extract had a superoxide radical scavenging inhibition of 70%.

3.3.6. Hydroxyl Radical Scavenging Activity

The biosynthesized HU-AgNPs demonstrated a concentration-dependent hydroxyl radical scavenging activity of 58.30% (Figure f) at a higher concentration of 125 μg/mL, which was less than that of standard ascorbic acid (67.18%). Conventional ascorbic acid has an IC₅₀ value of 72.42 μg/mL, whereas biosynthesized HU-AgNPs have an IC₅₀ value of 88.56 μg/mL. The latest work indicated that the aerial extract of Lippia nudiflora-mediated AgNPs demonstrated a 69% inhibition of hydroxyl radicals at a dosage of 200 μg/mL, whereas AgNPs manufactured from Brassica oleracea leaf extract revealed a 71% inhibition of hydroxyl radicals.

3.4. Anti-inflammatory Activity

3.4.1. Protein (Albumin) Denaturation

According to Figure a, the protein denaturation activity was most suppressed at 125 μg/mL (63.28%) and least inhibited at 25 μg/mL (11.28%). Conventional aspirin has an IC₅₀ value of 53.10 μg/mL, whereas biosynthesized HU-AgNPs have an IC₅₀ value of 76.28 μg/mL. AgNPs derived from Anoectochilus elatus show the highest inhibition of protein denaturation (67%) at a concentration of 100 μg/mL, according to Kumari et al. In a similar vein, Ejaz et al. showed that AgNPs derived from Thymus vulgaris have the highest percentage of protein denaturation inhibition (69%) at 200 μg/mL. At a dosage of 500 μg/mL, AgNPs made from Aconitum lycoctonum demonstrated the highest percentage of protein denaturation inhibition (91.78%) in a different study.

9.

Anti-inflammatory activity of synthesized HU-AgNPs (a) Albumin denaturation, (b) Proteinase inhibition action, (c) Membrane stabilization test, (d) Lipoxygenase inhibition assay.

3.4.2. Proteinase Inhibition Action

The proteinase inhibitory action of HU-AgNPs is contrasted with that of the common medication aspirin in Figure b. The greatest proteinase inhibition capacities were recorded by HU-AgNPs and the common medication aspirin at a concentration of 125 μg/mL, with 57.18% and 66.20%, respectively. Proteinase inhibition by HU-AgNPs has an IC₅₀ value of 77.46 μg/mL, whereas aspirin, a common medication, has an IC₅₀ value of 52.58 μg/mL. Anwar et al. (2021) reported that AgNPs synthesized from Tamarix articulata leaves exhibit a 70.196% inhibition of proteinase activity at a concentration of 600 μg/mL. In a separate report, Galaxaure elongata, Turbinaria ornata, and Enteromorpha flexuosa synthesized AgNPs demonstrated proteinase inhibition activities of 59.78%, 44.40%, and 47.38%, respectively, with IC₅₀ values of 90.8, 127.01, and 125.24 μg/mL, respectively.

3.4.3. Membrane Stabilization Test

The protective efficacy of HU-AgNPs and the standard medicine aspirin against erythrocyte membrane lysis generated by heat was assessed to elucidate the anti-inflammatory mechanism of HU-AgNPs. In the current investigation, HU-AgNPs at varying concentrations demonstrated the significant stability of the erythrocyte membrane. The protective efficacy of HU-AgNPs and aspirin was greater at a concentration of 125 μg/mL, measuring 59.64% and 64.30%, respectively (Figure c). The IC₅₀ value for membrane stabilization of HU-AgNPs is 73.86 μg/mL, while the IC₅₀ value for the reference medication aspirin is 50.10 μg/mL. Anwar et al. indicated that AgNPs produced from Tamarix articulata exhibit membrane stability of 74.16 % at a concentration of 600 μg/mL. A separate investigation indicated that AgNPs produced from Anoecto chiluselatus demonstrated 74.39 % membrane stability at a concentration of 100 μg/mL.

3.4.4. Lipoxygenase Inhibition Activity

At dosages between 25 and 125 μg/mL, biosynthesized HU-AgNPs showed notable lipoxygenase inhibitory action. At 125 μg/mL, the percentage of lipoxygenase inhibition by HU-AgNPs and indomethacin was higher, at 69.34% and 71.10%, respectively (Figure d). The IC₅₀ result for the reference drug indomethacin is 48.14 μg/mL, but the IC₅₀ value for lipoxygenase inhibition by HU-AgNPs is 79.10 μg/mL. Ongtanasup et al. indicated that Zingiber officinale rhizome extract demonstrated 57.53% suppression of lipoxygenase activity. Corciova et al. indicated that Lythrum salicaria aerial extract exhibited 98.09% lipoxygenase inhibition at a dosage of 5 mg/mL.

3.5. Antidiabetic Activity

3.5.1. α-Amylase Inhibition Activity

Figure a shows the inhibitory impact of HU-AgNPs on α-amylase. The results showed that, when compared to the reference material acarbose, the biosynthesized HU-AgNPs showed significant inhibition. HU-AgNPs’ inhibitory effects increased in a dose-dependent fashion. At 125 μg/mL, the greatest α-amylase inhibition was 57.24%, whereas at 25 μg/mL, the lowest inhibition was 12.10%. The standard acarbose has an IC₅₀ value of 27.34 μg/mL, while the α-amylase inhibition IC₅₀ value is 43.26 μg/mL. According to a recent study, AgNPs made from Azadirachta indica seed extract inhibited α-amylase by 73.85%. Silver nanoparticles (AgNPs) produced from Strobilanthes cordifolia exhibited significant inhibition at a concentration of 14.51 μg/mL. The HU-AgNPs reduced the amylase level, which facilitates the breakdown of complex carbs into simpler carbohydrates, hence promoting glucose use. The suppression of the amylase enzyme is specifically implicated in the management of insulin-independent diabetes, as it attenuates the release of glucose into the bloodstream. The results indicate that α-amylase was significantly inhibited in a dose-dependent manner following incubation with varying concentrations of AgNPs.

10.

Antidiabetic activity of synthesized HU-AgNPs (a) α-Amylase and (b) Glucosidase.

3.5.2. α-Glucosidase Inhibitory Activity

The aqueous extract of HU was used to create silver nanoparticles, which showed concentration-dependent α-glucosidase inhibitory activity. Green-manufactured AgNPs made from Cassia auriculata leaves showed comparable outcomes. The inhibitory activity of α-glucosidase showed low inhibition at 25 μg/mL, recording 13.26%, and peak inhibition at 125 μg/mL, reaching 60.28%. The conventional acarbose has an IC₅₀ value of 23.10 μg/mL, while the α-glucosidase inhibition IC₅₀ value is 48.32 μg/mL. The antidiabetic effectiveness of AgNPs was reported by Balan et al., who showed an IC₅₀ value of 37.86 μg/mL for α-glucosidase. Saratale et al. synthesized AgNPs from Punica granatum leaves, demonstrating an IC₅₀ value of 53.8 μg/mL for α-glucosidase inhibitory action. Balu et al., in another research, produced AgNPs using Rosa indica flower extract, which demonstrated an IC₅₀ value of 75 μg/mL for α-glucosidase inhibitory action. The enzymes α-amylase and α-glucosidase aid in the breakdown of carbohydrates and the absorption of glucose. Inhibiting either of these enzymes used for digestion slows the flow of glucose into the bloodstream and prevents starch from breaking down.

3.6. Antibacterial Activity

The HU-AgNPs demonstrated an inhibitory zone against the Gram-negative bacteria Escherichia coli (17.80 ± 0.14 mm) and Pseudomonas aeruginosa (18.20 ± 0.12 mm). The bacteria exhibited the greatest zone of inhibition at a dose of 100 μg/mL (Table ). Donga and Chanda discovered that the seed extract of Moringa indica-mediated AgNPs exhibits greater antibacterial efficacy against Gram-negative bacteria compared to Gram-positive bacteria. The antibacterial efficacy of AgNPs produced from Tamarix articulata leaf extract and olive fruit extract was greater against Gram-negative bacteria than Gram-positive bacteria. , Gram-negative bacteria were generally more susceptible to the antimicrobial effects of NPs than Gram-positive bacteria. Gram-positive and Gram-negative bacteria differ in their cell wall construction, which gives rise to this dichotomy. Gram-positive bacteria have a thick layer of peptidoglycans in their cell walls, while Gram-negative bacteria have a thin layer.

1. Antibacterial Activity of HU-AgNPs.

				Different Concentration of HU-AgNPs (μg)
Name of the Bacteria	Tetracycline 30 μg/disc	HU Extract 100 μg/mL	AgNO 3 100 μg/mL	40	80	100
Bacillus subtilis	22.10 ± 0.11	3.80 ± 0.03	7.80 ± 0.06	9.60 ± 0.06	13.20 ± 0.08	14.80 ± 0.05
Staphylococcus aureus	22.50 ± 0.08	4.10 ± 0.06	8.20 ± 0.07	9.80 ± 0.07	13.40 ± 0.11	13.90 ± 0.06
Escherichia coli	23.50 ± 0.15	3.30 ± 0.04	8.40 ± 0.05	10.20 ± 0.04	13.60 ± 0.04	17.80 ± 0.14
Pseudomonans aeruginosa	23.70 ± 0.21	3.60 ± 0.02	8.20 ± 0.03	11.30 ± 0.02	14.60 ± 0.10	18.20 ± 0.12

4. Conclusions

The current study demonstrated that the green-produced HU-AgNPs, utilizing the extract of seagrass H. uninervis, exhibit diverse biological activities. The absorption peak of the biosynthesized HU-AgNPs was observed at 405 nm. Functional groups such as alcohols, phenols, alkanes, secondary amines, aliphatic amines, and alkyl halides are present in HU-AgNPs, according to FTIR research. By SEM, the spherical form was confirmed. A crystalline structure was shown by the XRD spectrum with a standard crystalline size of 23.48 nm. AFM techniques corroborated the surface morphological and topographical roughness of the generated HU-AgNPs. The HU-AgNPs exhibited significant antibacterial efficacy against Gram-negative bacteria such as Escherichia coli as well as Pseudomonas aeruginosa. Significant antioxidant activity was shown by the biosynthesized HU-AgNPs in tests using DPPH, H₂O₂, NO, ABTs, superoxide, and hydroxyl. Albumin denaturation, proteinase inhibition, HRBC membrane stability, and lipoxygenase inhibition demonstrate anti-inflammatory efficacy, while α-amylase and α-glucosidase inhibition assesses antidiabetic effects. In conclusion, the current study shows that AgNPs made from the extract of H. uninervis have a variety of biological uses.

The authors declare no competing financial interest.