The properties of materials at the nanoscale are distinct from those of bulk materials. Nanoparticles, which range in size from 1 to 100 nanometers, have recently attracted a lot of attention due to their unique electrical, physical, magnetic, chemical, and optical features as compared to bulk materials[1]. The majority of researchers employ metal oxide nanoparticles because of their special qualities, which include stability, hydrophobicity, and photocatalysis. Coatings, catalysts, antibacterials, medical sciences, sensors, semiconductors, capacitors, and batteries are just a few of the various uses for them[2]. The special qualities of nanomaterials have piqued academics' interest over the past ten years in creating more straightforward and affordable methods for creating nanostructures with significant technological applications. Due to their potential application as operational components for nano electronics, optoelectronics, and sensing devices, metal oxide nanoparticles with large surface area and porosity have garnered a lot of interest for scientific research. Magnesium oxide is an inorganic element with incredible heat resistance, high chemical and alkali resistance, thermal stability, and high surface reactivity. The magnesium is 2nd. Oxygen is a group element with atomic number 8 and a group element with atomic number 12. The chemical magnesium oxide has melting and boiling temperatures of 2852ºC and 3600ºC, respectively[3]. Magnesium oxide is a white, hygroscopic solid mineral that is commonly referred to as periclase (from the Greek term periklao, peri "around," klao "to cut"). Its empirical formula is MgO, and its lattice is made up of Mg2+ and O2- ions joined by an ionic link. Usually, magnesium hydroxide (Mg(OH)2) or magnesium carbonate (MgCO3) are calcined to generate magnesium oxide. Thermal treatment, which is applied during the calcination process, has an impact on the final reactivity of the magnesium oxide as well as its surface area and pore size. Caustic calcined magnesium oxide is formed between 700 and 1000 degrees Celsius; lower chemical activity magnesium oxide is formed between 1000 and 1500 degrees Celsius; and reduced chemical activity refractory magnesium oxid, which is mainly used for electrical and refractory applications, is formed above 1500 degrees Celsius[4].

Magnesium oxide is used in many different industry sectors. Its refractory qualities make it an important component of building materials for fireproofing. Additionally, in industries like nuclear, chemical, and superalloy where corrosion is unacceptable. It is employed in medicine [5], where MgO is used as an antacid, a magnesium supplement, a temporary laxative, and to relieve sour stomach and heartburn. Additional uses include protective coatings, water treatment, insulators, fertilizers[6], and more. The usage of nanoscale fillers is currently popular.The creation of functional structures between 0.1 and 100 nm using a variety of physical or chemical techniques is generally referred to as nanotechnology[7]. This also holds true for magnesium oxide. Magnesium oxide at the nanoscale could be produced using the hydrothermal method or the sol-gel method. MgO has the potential to be used as a filler in electrical applications, such high-voltage insulation. Especially because of the high volume resistivity (1017 Wm) and large band gap (7.8 eV). Among commonly used nanoscale oxides, it has the highest volume resistivity value. It has also been utilized as a transition layer for the creation of many thin film materials due to its very large band gap, superior thermal stability, low dielectric constant, and refractive index[8]. MgO nanostructures were frequently produced by coprecipitation and thermal evaporation of various magnesium salts, or by dehydrating Mg(OH)2.

While there are several metal oxides, including ZnO, CuO, MgO, TiO, CdO, etc., magnesium oxide has special qualities in comparison to other metal oxides, including optical, electrical, thermal, mechanical, and chemical properties (Diachenko et al., 2016). According to Abdoul et al. (2020) and Fatiqin et al. (2021), it has a high melting point, a wide energy bandgap, strong reactivity, and a low heat capacity, making it a very stable and safe material for usage in various industries[9].

These oxide materials can be made using a variety of synthesis methods, including solution combustion, coprecipitation, natural reducing and stabilizing agents, Sol-Gel, hydrothermal, Solvothermal, microwave assisted Sol-Gel, and green synthesis. One of the best methods for producing nanoparticles without agglomeration in the yield is co-precipitation with a natural reducing and stabilizing agent; size may be readily regulated. UV-visible spectroscopy and X-ray diffraction (XRD) are used to characterize the produced nanoparticles. FTIR spectroscopy and SEMEDX. This chapter discusses the interpretation or analysis of the data and confirms that the particles are made of MgO nanoparticles using a natural reducing and stabilizing agent technique and co-precipitation[4].

Aim: Green Synthesis and Characterization of Magnesium Oxide (MgO) Nanoparticles as Biological Agent. 

Co-precipitation method[10]: The block diagram of the entire process is displayed below, along with the chemical reaction that took place during the synthesis-

Mg (NO3)2 .6H2O + 2 NaOH → Mg(OH)2 + 2NaNO3 

Δ 

Mg(OH)2 → 2 MgO + H2O

There are several steps involved in the manufacture of MgO nanoparticles, including mixing, stirring, filtering, drying, and grinding. First, two grams of magnesium NO3.6H2O were added to a beaker to create a 0.2 M solution in distilled water. Next, 100 milliliters of distilled water were used to dissolve 0.8 grams (0.5 M) of NaOH. A magnetic stirrer was used to continuously mix the magnesium nitrate solution for 30 minutes. Then, while continuously stirring the produced magnesium nitrate (Mg NO3.6H2O) solution, 0.5 M NaOH solution was added drop by drop using a dropper. A milky white precipitate of magnesium hydroxide formed in the beaker after 30 minutes. The pH paper indicated that the solution's pH was 12.5. After filtering the precipitate through filter paper and washing it with methanol three or four times to get rid of ionic contaminants, the white powder sample was dried in the air and then annealed for two hours at 300 and 500 degrees Celsius. A deadly pestle is then used to break the dried powder into an extremely fine powder[10]. 

Magnesium oxide nanoparticles were synthesized using a tannic-acid. Firstly, 1 g of tannic acid was dissolved in 50 mL of distilled water to obtain a homogeneous phenolic solution. In a separate beaker, 2 g of magnesium nitrate hexahydrate (Mg(NO₃)₂·6H₂O) was dissolved in 50 mL of distilled water. The two solutions were mixed and stirred continuously for 2–3 hours to promote chelation between Mg²⁺ ions and tannic acid, leading to the formation of a stable tannic acid–magnesium complex[11].

The reaction mixture was then heated on a water bath to evaporate the solvent until a solid Mg–tannic acid precursor was obtained. This precursor was carefully collected, dried, and subsequently calcined in a muffle furnace at 400–500 °C for 2 hours. During calcination, the organic matrix decomposed, leaving behind crystalline magnesium oxide nanoparticles. The final product appeared as a fine white powder, confirming successful formation of MgO nanoparticles[11]. 

i. Formation of Precipitate: A milky white precipitate of magnesium hydroxide (Mg(OH)₂) appeared instantly when 0.5 M NaOH was dropwise added to the 0.2 M magnesium nitrate solution. The pH of the solution rose to 12.5, indicating that all of the Mg2+ ions had precipitated.

ii. Filtration and Washing: Filtration was used for obtaining the white precipitate. Ionic contaminants were eliminated by washing with methanol three to four times, producing a clearer, brighter white solid.

iii. Drying and Annealing: The precipitate turned into a dry solid mass that was easily ground into powder after air drying. Annealing at: 300 °C → Light white powder, 500 °C → finer, more brilliant white MgO powder.

iv. Physical Appearance of Final Product: Colour- White; Texture: Fine, smooth powder; Flow property: Non-sticky, non-hygroscopic; Grinding: Easy to powder using mortar & pestle.

i. Solution Behaviour

ii. Heating and Precursor Formation

iii. Drying and Calcination

iv. Physical Characteristics

v. Final Experimental Outcome

Characterization of nanoparticles is an important step for determining their physicochemical properties such as structure, crystallinity, purity, functional groups, and optical behavior. Various analytical techniques are commonly employed to confirm the successful synthesis of magnesium oxide (MgO) nanoparticles and to evaluate their structural and chemical properties.

The crystalline structure and phase purity of nanoparticles are frequently ascertained by X-ray diffraction (XRD) investigation. The diffraction pattern helps in identifying the crystal planes and confirms the formation of crystalline MgO nanoparticles.

Fourier Transform Infrared Spectroscopy (FTIR) is used to identify the functional groups and biomolecules present on the surface of nanoparticles. FTIR analysis also helps in understanding the role of phytochemicals involved in the reduction and stabilization of MgO nanoparticles during green synthesis[12].

UV–Visible spectroscopy is an important analytical technique used to study the optical properties of nanoparticles. The absorption spectrum obtained from UV analysis confirms the formation of MgO nanoparticles and provides information regarding their optical behavior and nanoscale characteristics[13].

These characterization techniques collectively help in confirming the successful synthesis and stability of MgO nanoparticles for further biological and pharmaceutical applications.

Assessing the effectiveness of Antioxidant by the Hydrogen Peroxide (H2O2): As a standard, 100, 200, and 300 µg/ml solutions of magnesium oxide (MgO) nanoparticles and ascorbic acid were prepared using distilled water. A 40 mM hydrogen peroxide (H₂O₂) solution was prepared in phosphate buffer (pH 7.4). To the aforementioned solutions, 1 ml of H₂O₂ solution was added to 3 ml of each sample solution. After mixing, the reaction mixture was allowed to stand for 10 minutes at room temperature (≈25–26°C).

Using phosphate buffer as a blank, the absorbance was measured at 230 nm using a UV–Visible spectrophotometer. As a negative control, a mixture of 3 ml phosphate buffer and 1 ml H₂O₂ solution without sample or standard was used. The mean value was calculated after performing the experiment in triplicate.

The following formula was used to determine the percentage scavenging of the samples and the standard:

% Scavenging = [Acontrol – Asample/Acontrol × 100]

where

Asample = absorbance of the test sample

Acontrol = absorbance of the control

Antioxidant Activity by the Scavenging Method: Distilled water was used to create solutions containing 100, 200, and 300 µg/ml of magnesium oxide (MgO) nanoparticles and the standard (ascorbic acid). Phosphate buffer (pH 7.4) was used to create a 40 mM hydrogen peroxide (H2O₂) solution.

To each sample solution (3 ml), 1 ml of H₂O₂ solution was added. The reaction mixtures were mixed thoroughly and incubated at room temperature for 10 minutes. The absorbance of the resulting solution was measured at 230 nm using a UV–Visible spectrophotometer, with phosphate buffer used as a blank.

A control was prepared by mixing 3 ml of phosphate buffer with 1 ml of H₂O₂ solution without the addition of sample or standard. All experiments were performed in triplicate, and the mean values were calculated[14].

The following formula was used to determine the % scavenging activity of both the MgO nanoparticles and the standard (ascorbic acid):

% Scavenging = [Acontrol – Asample/Acontrol × 100]

where

Asample = absorbance of the test sample

Acontrol = absorbance of the control

Sample Preparation: Distilled water was used to create solutions containing 100, 200, and 300 µg/ml of magnesium oxide (MgO) nanoparticles and standard (ascorbic acid or diclofenac sodium). Two milliliters of phosphate-buffered saline (PBS, pH 6.4) and 0.3 milliliters of egg albumin were added to each of the produced solutions.

After 20 minutes of incubation at 37°C, the reaction mixtures were heated for six minutes at 70°C. A UV-visible spectrophotometer was used to measure the absorbance at 660 nm after it had cooled to room temperature, using DMSO as a blank.

A control was prepared by replacing the sample with distilled water while maintaining all other components under identical conditions. All experiments were carried out in triplicate, and the mean values were recorded[14, 15].

The percentage inhibition of protein denaturation was calculated using the following equation:

% Inhibition = 100[Vsample/Vcontrol – 1]

Where,

Vsample = absorbance of the test sample and

Vcontro  l= absorbance of the control.

Standard Sample Preparation: Using distilled water, diclofenac sodium solutions were made at 100, 200, and 300 µg/ml. 0.3 ml of egg albumin and 2.9 ml of phosphate-buffered saline (PBS, pH 6.4) were added to each of the produced solutions.

The reaction mixtures were incubated at 37°C for 20 minutes, followed by heating at 70°C for 6 minutes. After cooling to room temperature, the absorbance was measured at 660 nm using a UV–Visible spectrophotometer, with DMSO used as a blank.

A control was prepared by replacing diclofenac sodium with distilled water while keeping all other components constant. All experiments were performed in triplicate, and the mean values were calculated.

The percentage inhibition of protein denaturation was determined using the following equation:

% Inhibition = 100[Vsample/Vcontrol – 1]

Where,

Vsample = absorbance of the test sample and

Vcontrol = absorbance of the control.

i. UV- Visible Absorption Sperctroscopy 

The most popular analytical method for describing the electronic structure of the optical band gap of the nanomaterial is UV-Vis absorption spectroscopy. To characterize the size of nanoparticles in the range of 5–100 nm, the UV-Vis spectra of metal nanoparticles were recorded in the wavelength range of 200 nm to 800 nm. In the absorption spectra of MgO nanoparticles, distilled water was employed as a solvent. The charge transfer transition between energy levels causes a prominent band in the UV portion of the spectrum [Fig. 5]. First, as the wavelength increases close to the band edge (250 nm), the absorbance rapidly drops, indicating the size of MgO nanoparticles[16].

The creation of nanosized MgO particles was confirmed by the UV-Vis spectrum, which showed a strong peak at 265.2 nm. The band gap energy of the synthesized MgO Nps was determined from the UV Vis spectrum using the formula Eg = h², where Eg, h, and Ψ stand for optical band gap energy, plank's constant, and frequency, respectively. The calculated band gap energy of MgO Nps was found to be 4.2 eV, in contrast to bulk MgO, which had a band gap energy of 7.8 eV. While the bulk materials include 6-coordinated surface anions, the presence of 4-coordinated surface anions at the margins of the MgO Nps is unquestionably responsible for the decreased band gap energy. This finding is consistent with the earlier research conducted by Berger et al.[17]. At addition to bulk materials, the band gap energy will also be influenced by MgO with varying particle sizes at the nanoscale. According to Moon and his colleagues, MgO Nps with an average size of 20 nm have a band gap of 5.6 eV. It is evident that the band gap energy increases as the particle size decreases because the band gap of our MgO Nps (average particle size ~28 nm, which will be covered in the XRD section) is 4.2 eV. Because the energy levels in bulk materials are tightly separated, they create quasi-continuous bands. Discrete energy levels are seen as the energy level separation rises in the nano-regime[18].

ii. Fourier Transform Infrared (FTIR) Spectroscopy: 

FTIR spectroscopy was used to determine the vibrational frequency of the molecules' stretching and bending modes as well as possible biomolecules responsible for the reduction and capping of MgO NPs. The MgO nanoparticle spectra and the analysis carried out in the 400-4000 cm-1 band are shown in the [Fig. 6]. The stretching vibration of the O-H group is shown by the peaks at 3415 cm-1 and 2925 cm-1. The stretching vibration of an aromatic C=C bond is shown by the peak at 1633 cm-1. MgO-NP formation is shown by the peak seen at 434 cm-1[16].

iii. X-ray diffraction 

The X-ray diffraction (XRD) analysis of magnesium oxide (MgO) nanoparticles was carried out to determine their crystalline structure and phase purity. The diffractogram shows several sharp and intense peaks, indicating the highly crystalline nature of the synthesized MgO nanoparticles[14, 19].

The prominent diffraction peaks were observed in the mid-to-high 2θ range, particularly around 30°, 43°, and 62°, which are characteristic of MgO. These peaks correspond well with the standard diffraction pattern of cubic MgO, confirming successful formation of magnesium oxide nanoparticles[14].

Among these, the most intense peak at approximately 43° corresponds to the (200) plane, while the peak near 62° is attributed to the (220) plane of MgO. The presence of these well-defined peaks confirms the crystalline cubic phase of MgO nanoparticles.

The absence of additional impurity peaks in the diffractogram suggests that the synthesized nanoparticles are pure and free from secondary phases[19]. The sharpness and intensity of the peaks further indicate good crystallinity and nanoscale particle formation.

Overall, the XRD examination verifies that the produced MgO nanoparticles have a highly pure crystalline cubic structure, which qualifies them for additional biological and medicinal uses[14].

iv. Antioxidant activity 

The antioxidant potential of magnesium oxide (MgO) nanoparticles was evaluated using the hydrogen peroxide scavenging assay and compared with the standard ascorbic acid. The results revealed that MgO nanoparticles exhibited a concentration-dependent increase in free radical scavenging activity.

Ascorbic acid

MgO Nanoparticles 

The percentage scavenging activity of MgO nanoparticles gradually increased at concentrations of 100, 200, and 300 µg/ml. This suggests that the nanoparticles' ability to neutralize hydrogen peroxide improves with concentration.

The observed antioxidant activity of MgO nanoparticles can be attributed to their high surface area and active surface sites, which facilitate interaction with reactive oxygen species (ROS). These nanoparticles donate electrons to hydrogen peroxide, thereby converting it into harmless products such as water and oxygen.

Although the scavenging activity of MgO nanoparticles is lower than that of ascorbic acid, they still demonstrate moderate antioxidant potential, suggesting their possible application in reducing oxidative stress-related damage.

v. Anti-inflammatory activity:

The anti-inflammatory activity of MgO nanoparticles was assessed using the protein denaturation method and compared with the standard drug diclofenac sodium. The anti-inflammatory activity of MgO nanoparticles was assessed using the protein denaturation method and compared with the standard drug diclofenac sodium.

Diclofenac Sodium 

MgO Nanoparticles

The results demonstrated that MgO nanoparticles showed a significant inhibition of protein denaturation, which increased with concentration (100, 200, and 300 µg/ml). This confirms that MgO nanoparticles possess the ability to stabilize protein structures under stress conditions.

Protein denaturation is a well-known cause of inflammation, and substances that prevent denaturation can act as anti-inflammatory agents. MgO nanoparticles likely interact with protein molecules through surface binding and stabilization mechanisms, thereby preventing structural alteration.

When compared with diclofenac sodium, MgO nanoparticles exhibited comparable inhibitory activity, indicating their potential as an alternative or supportive anti-inflammatory agent. 

Tannic acid was used as a natural reducing and stabilizing agent in the effective production of magnesium oxide (MgO) nanoparticles using both chemical co-precipitation and green synthesis techniques. The synthesized nanoparticles were characterized using UV–Visible spectroscopy, FTIR, and XRD analysis, which confirmed the successful formation, crystalline nature, purity, and nanoscale properties of MgO nanoparticles. UV analysis showed a characteristic absorption peak around 265.2 nm, while FTIR confirmed the presence of Mg–O functional groups and biomolecules involved in stabilization. The nanoparticles exhibited significant antioxidant and anti-inflammatory activities in a concentration-dependent manner when compared with standard drugs such as ascorbic acid and diclofenac sodium. The green synthesis method produced smoother, more uniform, and environmentally friendly nanoparticles with better stability. The study demonstrates that MgO nanoparticles possess promising biological and pharmaceutical potential for future biomedical, antioxidant, antimicrobial, and drug delivery applications.