System for the synthesis of ZnO nanoparticles using Annona muricata leaf extract for its antibacterial and antifungal activities

Abstract

A system for the synthesis of zinc oxide nanoparticles (ZnO-NPs) comprising the steps of preparing an aqueous extract from Annona muricata leaves, adding the extract to an aqueous solution of zinc acetate dihydrate, adjusting the pH to approximately 8 with sodium hydroxide, stirring and heating the mixture at approximately 70 °C, and collecting and drying the resulting ZnO nanoparticles.

Description

Technical field of expertise:

[0001] The present invention relates to a green synthesis system for the production of zinc oxide nanoparticles (ZnO-NPs) using Annona muricata (Soursop) leaf extract and their application in antibacterial and antifungal activities. Background of the invention:

[0002] The present invention offers a novel system for the synthesis of zinc oxide nanoparticles (ZnO-NPs) using aqueous extracts from Annona muricata leaves and furthermore demonstrates their strong antibacterial and antifungal activities.

[0003] The invention discloses an environmentally friendly, cost-effective, and reproducible methodology that eliminates the use of toxic chemicals typically required in the conventional synthesis of nanoparticles. The system involves preparing fresh *A. muricata* leaves, washing and drying them under controlled conditions, and extracting them in distilled water to obtain a bioactive solution rich in phytochemicals. This extract is then combined with an aqueous zinc acetate dihydrate solution, which serves as the starting salt for nanoparticle formation. By carefully adjusting the pH to approximately 8 with sodium hydroxide and applying stirring and heating to 70 °C, nanoparticles are formed through the reduction and stabilization effects of the plant extract. The moist nanoparticles are collected, dried, and characterized to confirm their structural and functional properties.

[0004] The invention demonstrates that the synthesized ZnO nanoparticles exhibit a sharp absorption band at 380 nm in ultraviolet-visible spectroscopy, confirming their optical properties at the nanoscale. X-ray diffraction patterns reveal highly crystalline structures belonging to the hexagonal wurtzite phase, while Fourier-transform infrared (FTIR) spectroscopy shows the involvement of hydroxyl, carboxyl, and amino groups from the phytochemicals of A. muricata, which stabilize the nanoparticles and prevent agglomeration. These results together confirm the success of the green synthesis process and the efficiency of A. muricata leaves in mediating nanoparticle formation.

[0005] A key advantage of the invention lies in the demonstrated antimicrobial activity of the synthesized ZnO nanoparticles. Antibacterial tests against the gram-positive Staphylococcus aureus and the gram-negative Escherichia coli showed significant zones of inhibition, with greater efficacy observed against E. coli, likely due to differences in cell wall structure. Antifungal tests against Candida albicans and Aspergillus niger also showed pronounced zones of inhibition, confirming broad activity against clinically relevant fungi. These results validate the synthesized nanoparticles as potential alternatives to conventional antimicrobial agents, particularly in an era of increasing antibiotic resistance.

[0006] In addition to inhibiting planktonic cells, the system also exhibits antibiofilm activity, which is crucial for combating persistent infections. Biofilm assays showed that the ZnO-NPs effectively inhibited biofilm formation in a dose-dependent manner in both Gram-positive and Gram-negative bacteria, with the inhibition being stronger in Gram-negative strains. This property significantly increases the biomedical relevance of the invention, as biofilm-based infections are among the most difficult to treat with traditional antibiotics. Furthermore, studies on the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) confirmed the dose-dependent antibacterial efficacy, with MIC values ​​ranging from 200 to 400 µg / mL and showing remarkable differences in activity between bacterial species.

[0007] To assess the safety profile of the synthesized nanoparticles, the invention also discloses cytotoxicity assessments conducted using MTT tests in mammalian L929 fibroblast cells. The results showed a moderate decrease in cell viability with increasing concentrations of ZnO-NPs, indicating dose-dependent cytotoxicity, while acceptable biocompatibility is maintained at lower concentrations. This finding underscores the potential of the nanoparticles for biomedical applications such as wound dressings, drug delivery systems, or antimicrobial coatings, where controlled concentrations can provide therapeutic effects without excessive toxicity.

[0008] The invention thus establishes a comprehensive system that begins with the environmentally friendly synthesis of nanoparticles and extends through detailed physicochemical characterization, antimicrobial evaluation, antibiofilm testing, and cytotoxicity analyses. This integrated approach ensures not only the reproducibility of the synthesis but also the validation of biological efficacy and safety. The reliance on Annona muricata leaf extract offers added value by utilizing a widely available, cost-effective, and renewable natural resource. Compared to conventional chemical and physical synthesis methods, the disclosed system offers advantages in terms of simplicity, scalability, environmental compatibility, and biomedical relevance.

[0009] In conclusion, the invention offers a sustainable and effective system for the synthesis of zinc oxide nanoparticles using Annona muricata leaf extract and demonstrates their broad spectrum of antibacterial, antifungal, and antibiofilm properties, as well as acceptable cytotoxicity profiles. The nanoparticles produced by this system can be used in biomedical, pharmaceutical, and clinical applications as alternatives to or complements to conventional antimicrobial therapies. By bridging the gap between green synthesis and biomedical application, the invention significantly advances the field of nanomedicine and offers a promising solution to global challenges such as antibiotic resistance and environmental sustainability. Detailed description of the invention:

[0010] The present invention provides a system for the green synthesis of zinc oxide nanoparticles using Annona muricata leaf extract and further demonstrates their antibacterial, antifungal, antibiofilm, and cytotoxic activities. This invention integrates the use of a natural plant source rich in bioactive phytochemicals as a reducing and stabilizing agent with an optimized synthesis process that yields stable, crystalline, and biologically active zinc oxide nanoparticles. In contrast to traditional chemical synthesis methods that rely on toxic reducing agents, high-temperature calcination, and generate environmentally harmful byproducts, the system presented here offers an environmentally friendly, cost-effective, and sustainable route to the production of nanoparticles.

[0011] The invention begins with the preparation of the plant material. Fresh leaves of Annona muricata are thoroughly washed with distilled water to remove dust, surface contaminants, or microbial growth that could interfere with the synthesis of nanoparticles. The leaves are then air-dried at room temperature in the shade to prevent the degradation of sensitive phytochemicals. Once sufficiently dry, the leaves are processed to obtain an aqueous extract. The extract is produced by boiling or soaking the powdered or cut leaves in distilled water, which releases a complex mixture of secondary metabolites into solution.The resulting extract contains alkaloids, flavonoids, polyphenols and acetogenins, which are known to possess reducing and antioxidant properties and can act as natural mediators in the synthesis of nanoparticles.

[0012] The next phase of the system involves preparing the zinc precursor solution. Zinc acetate dihydrate is dissolved in double-distilled water to form a clear solution with a pH close to 6. This solution serves as a source of zinc ions, which are subsequently reduced to zinc oxide nanoparticles. The Annona muricata leaf extract is then introduced into this zinc acetate solution. The extract interacts with the dissolved zinc ions, initiating reduction reactions facilitated by the phytochemicals. The reaction environment is made alkaline by the careful addition of sodium hydroxide solution, added dropwise with continuous stirring until the pH reaches approximately 8. The alkaline medium plays a crucial role in the hydrolysis of the zinc precursor, enabling the conversion of zinc acetate to zinc hydroxide intermediates.

[0013] The mixture is heated to approximately 70 degrees Celsius and held at this temperature for a specific duration while being continuously stirred. This gentle heating accelerates the nucleation and growth of zinc oxide nanoparticles while maintaining the structural integrity of the phytochemicals that stabilize the nanoparticles. During this phase, nanoparticle formation can often be visually indicated by a color change in the reaction mixture. Once formed, the moist nanoparticles are separated from the reaction medium, repeatedly washed with distilled water and ethanol to remove unreacted chemicals and excess plant extract, and finally dried under controlled conditions. The dried powder represents the stabilized zinc oxide nanoparticles synthesized through this environmentally friendly approach.

[0014] Characterization of the synthesized nanoparticles demonstrates their successful formation and stability. Ultraviolet-visible spectroscopy is used to confirm their optical properties, revealing a strong absorption peak at approximately 380 nanometers. This absorption peak is characteristic of zinc oxide nanoparticles and corresponds to their intrinsic electronic band gap. The sharpness and intensity of this peak indicate that the nanoparticles are of high purity, free from major impurities, and were successfully synthesized at the nanoscale.

[0015] X-ray diffraction studies further confirm the crystalline nature of the nanoparticles. The diffraction spectrum shows pronounced and sharp peaks corresponding to the planes of the hexagonal wurtzite crystal structure of zinc oxide. The absence of additional peaks indicates the phase purity of the synthesized nanoparticles, while the narrowness of the peaks highlights their high degree of crystallinity. The presence of such crystalline structures is important to ensure consistent electronic and antimicrobial performance of the nanoparticles.

[0016] Fourier-transform infrared spectroscopy provides further evidence for the role of phytochemicals in the synthesis process. The spectrum shows broad and intense bands corresponding to hydroxyl groups, CO vibrations associated with flavonoids, and CN bonds linked to alkaloids. These functional groups originate from compounds present in the Annona muricata extract and indicate their active participation in the reduction of the zinc precursor and the stabilization of the nanoparticle surface. The phytochemicals essentially act as both reducing and capping agents, ensuring that the nanoparticles do not aggregate and remain well dispersed.

[0017] The biological activity of the synthesized nanoparticles is a central aspect of the invention. Antibacterial tests show that the nanoparticles exhibit strong inhibitory activity against both Gram-negative and Gram-positive bacteria. In tests against Escherichia coli, a common Gram-negative pathogen, the nanoparticles generate significant zones of inhibition, indicating strong bactericidal or bacteristatic effects. Similarly, inhibition is observed in tests against Staphylococcus aureus, a Gram-positive pathogen, but to a somewhat lesser degree. This differing activity can be attributed to structural differences in bacterial cell walls. The thin peptidoglycan layer and outer membrane of Gram-negative bacteria may facilitate greater penetration of reactive oxygen species generated by the nanoparticles, resulting in stronger antibacterial activity compared to Gram-positive strains.

[0018] The antifungal potential of the nanoparticles is also demonstrated using pathogenic fungi such as Candida albicans and Aspergillus niger. The nanoparticles show clear zones of inhibition against both species, with more pronounced activity against Aspergillus niger. The ability of zinc oxide nanoparticles to act as antifungals is attributed to their interaction with fungal cell walls and membranes, the generation of oxidative stress, and the disruption of essential intracellular processes.

[0019] A key feature of the invention is the demonstration of its antibiofilm activity. Biofilm formation by bacteria and fungi poses a significant clinical challenge, as biofilm-associated microorganisms are resistant to antibiotics and immune responses. The nanoparticles synthesized in this system exhibit dose-dependent inhibition of biofilm formation. In Gram-positive organisms, inhibition is observed in a range from minimal at lower concentrations to significant at higher concentrations. In Gram-negative organisms, the inhibition is more pronounced, indicating greater efficacy against this group of pathogens. By disrupting initial adhesion and subsequent biofilm formation, the nanoparticles prevent the establishment of resistant microbial communities, thus highlighting their usefulness in preventing chronic infections.

[0020] The antimicrobial potential of the nanoparticles is further quantified by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCC) tests. The nanoparticles exhibit inhibitory effects against bacterial growth at concentrations of 200 to 400 micrograms per milliliter. At higher concentrations, near 600 micrograms per milliliter, bacterial growth is almost completely inhibited, indicating strong bactericidal activity. These values ​​confirm that the nanoparticles synthesized using this green approach exhibit strong antimicrobial activity comparable to that of chemically synthesized zinc oxide nanoparticles.

[0021] To assess the biocompatibility and potential cytotoxic effects of the nanoparticles, cytotoxicity studies are conducted using mammalian fibroblast cell lines. The MTT test shows that cell viability gradually decreases as the nanoparticle concentration increases. At lower concentrations, cell viability remains above 90 percent, indicating minimal toxicity. However, with increasing concentration, viability gradually decreases, reaching approximately 70 percent at the highest tested concentration. This dose-dependent cytotoxicity suggests that the nanoparticles are moderately cytotoxic at higher concentrations but safe to use at lower concentrations, where they retain their antimicrobial efficacy.This balance between activity and safety highlights the suitability of nanoparticles for biomedical applications such as wound dressings, antimicrobial coatings, or drug systems, where controlled dosage ensures therapeutic benefits without unacceptable toxicity.

[0022] The invention thus describes a system that integrates green synthesis, physicochemical characterization, and biological evaluation into a single framework. By using Annona muricata leaf extract, which is inexpensive, widely available, and renewable, the synthesis is scalable and environmentally friendly. The process eliminates the need for hazardous chemicals, minimizes energy consumption by operating at relatively low temperatures, and produces nanoparticles that are stable, crystalline, and biologically active. The nanoparticles' antimicrobial and antifungal activities make them suitable for addressing urgent healthcare challenges, particularly those associated with multidrug-resistant bacteria and opportunistic fungal infections. Their antibiofilm-active properties further enhance their clinical potential by targeting one of the most resilient survival strategies of microbes.Finally, the moderate cytotoxicity profile ensures that the nanoparticles can be used safely in biomedical contexts when applied at appropriate concentrations.

[0023] In summary, the detailed description of the invention presents a sustainable and efficient method for the synthesis of zinc oxide nanoparticles using Annona muricata leaf extract. The invention describes each phase, from plant preparation and extract formation to the synthesis, characterization, and biological testing of the nanoparticles, in a reproducible and environmentally friendly manner. The nanoparticles produced by this method combine high crystalline quality with strong antimicrobial and antifungal efficacy, antibiofilm potential, and acceptable cytotoxicity. This invention therefore makes a versatile and effective contribution to the fields of green nanotechnology and biomedical sciences by offering solutions to the challenges of antibiotic resistance, fungal infections, and the sustainable production of nanomaterials.

Claims

[1] A system for the synthesis of zinc oxide nanoparticles (ZnO-NPs) comprising the steps of producing an aqueous extract from Annona muricata leaves, adding the extract to an aqueous solution of zinc acetate dihydrate, adjusting the pH to about 8 with sodium hydroxide, stirring and heating the mixture at about 70 °C, and collecting and drying the resulting ZnO nanoparticles. [2] System according to claim 1, wherein the phytochemicals present in the Annona muricata extract, including alkaloids, flavonoids and polyphenols, act as reducing and stabilizing agents for the formation of nanoparticles. [3] System according to claim 1, wherein the synthesized ZnO nanoparticles exhibit an ultraviolet-visible absorption peak at approximately 380 nanometers, confirming the optical properties on the nanometer scale. [4] System according to claim 1, wherein the nanoparticles exhibit antibacterial activity against Escherichia coli and Staphylococcus aureus and produce inhibition zones of up to 8 millimeters at a concentration of 1 milligram per milliliter. [5] System according to claim 1, wherein the nanoparticles exhibit antifungal activity against Candida albicans and Aspergillus niger and produce inhibition zones of up to 8 millimeters at a concentration of 1 milligram per milliliter. [6] System according to claim 1, wherein the nanoparticles exhibit dose-dependent antibiofilm inhibition and moderate cytotoxicity in mammalian fibroblasts, enabling their use in biomedical treatments and antimicrobial coatings.